U.S. patent application number 10/107532 was filed with the patent office on 2004-01-01 for nucleic acid and corresponding protein entitled 158p3d2 useful in treatment and detection of cancer.
Invention is credited to Afar, Daniel E. H., Challita-Eid, Pia M., Faris, Mary, Ge, Wangmao, Hubert, Rene S., Jakobovits, Aya, Morrison, Karen Jane Meyrick, Morrison, Robert Kendall, Raitano, Arthur B..
Application Number | 20040003418 10/107532 |
Document ID | / |
Family ID | 26961881 |
Filed Date | 2004-01-01 |
United States Patent
Application |
20040003418 |
Kind Code |
A1 |
Jakobovits, Aya ; et
al. |
January 1, 2004 |
Nucleic acid and corresponding protein entitled 158P3D2 useful in
treatment and detection of cancer
Abstract
A novel gene (designated 158P3D2) and its encoded protein, and
variants thereof, are described wherein 158P3D2 exhibits tissue
specific expression in normal adult tissue, and is aberrantly
expressed in the cancers listed in Table I. Consequently, 158P3D2
provides a diagnostic, prognostic, prophylactic and/or therapeutic
target for cancer. The 158P3D2 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 158P3D2 can be used in active or passive
immunization.
Inventors: |
Jakobovits, Aya; (Beverly
Hills, CA) ; Faris, Mary; (Los Angeles, CA) ;
Morrison, Karen Jane Meyrick; (Santa Monica, CA) ;
Morrison, Robert Kendall; (Santa Monica, CA) ;
Hubert, Rene S.; (Los Angeles, CA) ; Afar, Daniel E.
H.; (Brisbane, CA) ; Ge, Wangmao; (Culver
City, CA) ; Raitano, Arthur B.; (Los Angeles, CA)
; Challita-Eid, Pia M.; (Encino, CA) |
Correspondence
Address: |
Kate H. Murashige
Morrison & Foerster LLP
Suite 500
3811 Valley Centre Drive
San Diego
CA
92130-2332
US
|
Family ID: |
26961881 |
Appl. No.: |
10/107532 |
Filed: |
March 25, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60283112 |
Apr 10, 2001 |
|
|
|
60286630 |
Apr 25, 2001 |
|
|
|
Current U.S.
Class: |
800/3 ;
424/146.1; 435/326; 514/19.3; 514/19.6; 514/19.8; 514/44R;
800/8 |
Current CPC
Class: |
Y10T 436/143333
20150115; A61P 37/04 20180101; C07K 14/4748 20130101; A61P 35/00
20180101; Y02A 50/466 20180101; Y02A 50/30 20180101; A61K 39/00
20130101 |
Class at
Publication: |
800/3 ; 514/12;
800/8; 424/146.1; 435/326; 514/44 |
International
Class: |
A01K 067/00; A61K
039/395; C12N 005/06; A61K 048/00; A61K 038/17 |
Claims
1. A composition comprising: a substance that a) modulates the
status of 158P3D2, or b) a molecule that is modulated by 158P3D2
whereby the status of a cell that expresses 158P3D2 is
modulated.
2. A composition of claim 1, further comprising a physiologically
acceptable carrier.
3. A pharmaceutical composition that comprises the composition of
claim 1 in a human unit dose form.
4. A composition of claim 1 wherein the substance comprises an
antibody or fragment thereof that specifically binds to a
158P3D2-related protein.
5. An antibody or fragment thereof of claim 4, which is
monoclonal.
6. An antibody of claim 4, which is a human antibody, a humanized
antibody or a chimeric antibody.
7. A non-human transgenic animal that produces an antibody of claim
4.
8. A hybridoma that produces an antibody of claim 5.
9. A method of delivering a cytotoxic agent or a diagnostic agent
to a cell that expresses 158P3D2, 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.
10. A composition of claim 1 wherein the substance comprises a
polynucleotide that encodes an antibody or fragment thereof either
of which immunospecifically bind to a 158P3D2-related protein.
11. A composition of claim 1 wherein the substance comprises a
158P3D2-related protein.
12. A protein of claim 11 that is at least 90% homologous to an
entire amino acid sequence shown in FIG. 2 (SEQ ID NOS:
______).
13. A composition of claim 1 wherein the substance comprises a
peptide of eight, nine, ten, or eleven contiguous amino acids of
FIG. 2 or Tables V to XIX, or an analog thereof (SEQ ID NOS:
______).
14. A composition of claim 1 wherein the substance comprises a CTL
polypeptide of the amino acid sequence of FIG. 2 (SEQ ID NOS:
______).
15. A composition of claim 14 further limited by a proviso that the
epitope is not an entire amino acid sequence of FIG. 2 (SEQ ID
NOS:).
16. A composition of claim 14 wherein the substance comprises a CTL
polypeptide set forth in Tables V to XIX (SEQ ID NOS: ______).
17. A composition of claim 16 further limited by a proviso that the
polypeptide is not an entire amino acid sequence of FIG. 2 (SEQ ID
NOS: ______).
18. A composition of claim 1 wherein the substance comprises an
antibody polypeptide epitope of the amino acid sequence of FIG. 2
(SEQ ID NOS: ______).
19. A composition of claim 18 further limited by a proviso that the
epitope is not an entire amino acid sequence of FIG. 2 (SEQ ID NOS:
______).
20. A composition of claim 18 wherein the antibody epitope
comprises a peptide region of at least 5 amino acids of FIG. 2 (SEQ
ID NOS: ______) in any whole number increment up to 328 that
includes an amino acid position selected from: an amino acid
position having a value greater than 0.5 in the Hydrophilicity
profile of FIG. 5, an amino acid position having a value less than
0.5 in the Hydropathicity profile of FIG. 6; an amino acid position
having a value greater than 0.5 in the Percent Accessible Residues
profile of FIG. 7; an amino acid position having a value greater
than 0.5 in the Average Flexibility profile on FIG. 8; or an amino
acid position having a value greater than 0.5 in the Beta-turn
profile of FIG. 9.
21. A composition of claim 20 further limited by a proviso that the
epitope is not an entire amino acid sequence of FIG. 2 (SEQ ID NOS:
______, ______, ______, ______, ______, ______, ______, ______,
______, and ______).
22. A polynucleotide that encodes a protein of claim 11.
23. A polynucleotide of claim 22 that comprises a nucleic acid
molecule set forth in FIG. 2.
24. A polynucleotide of claim 22 further limited by a proviso that
the encoded protein is not an entire amino acid sequence of FIG. 2
(SEQ ID NOS: ______).
25. A polynucleotide of claim 22 wherein T is substituted with
U.
26. A composition of claim 1 wherein the substance comprises a
polynucleotide comprising a coding sequence of a nucleic acid
sequence of FIG. 2 (SEQ ID NOS: ______).
27. A polynucleotide of claim 24 further comprising a
polynucleotide that encodes a 158P3D2-related protein that is at
least 90% homologous to an entire amino acid sequence shown in FIG.
2 (SEQ ID NOS: ______).
28. A composition comprising a polynucleotide that is fully
complementary to a polynucleotide of claim 22.
29. A composition comprising a polynucleotide that is fully
complementary to a polynucleotide of claim 25.
30. A composition comprising a polynucleotide that is fully
complementary to a polynucleotide of claim 27.
31. A composition of claim 1 wherein the substance comprises a) a
ribozyme that cleaves a polynucleotide having 158P3D2 coding
sequence, or b) a nucleic acid molecule that encodes the ribozyme;
and, a physiologically acceptable carrier.
32. A composition comprising the composition of claim 1 wherein the
substance comprises human T cells, wherein said T cells
specifically recognize a 158P3D2 peptide sequence in the context of
a particular HLA molecule.
33. A method of inhibiting growth of cancer cells that expresses
158P3D2, the method comprising: administering to the cells the
composition of claim 1.
34. A method of claim 33 of inhibiting growth of cancer cells that
express 158P3D2, the method comprising steps of: administering to
said cells an antibody or fragment thereof, either of which
specifically bind to a 158P3D2-related protein.
35. A method of claim 33 of inhibiting growth of cancer cells that
express 158P3D2, the method comprising steps of: administering to
said cells a 158P3D2-related protein.
36. A method of claim 33 of inhibiting growth of cancer cells that
express 158P3D2, the method comprising steps of: administering to
said cells a polynucleotide comprising a 158P3D2-related protein
coding sequence or a polynucleotide complementary to a
polynucleotide having a 158P3D2 coding sequence.
37. A method of claim 33 of inhibiting growth of cancer cells that
express 158P3D2, the method comprising steps of: administering to
said cells a ribozyme that cleaves a polynucleotide having 158P3D2
coding sequence.
38. A method of claim 33 of inhibiting growth of cancer cells that
express 158P3D2 and a particular HLA molecule, the method
comprising steps of: administering to said cells human T cells,
wherein said T cells specifically recognize a 158P3D2 peptide
subsequence in the context of the particular HLA molecule.
39. A method of claim 33, the method comprising steps of:
administering a vector that delivers a single chain monoclonal
antibody coding sequence, whereby the encoded single chain antibody
is expressed intracellularly within cancer cells that express
158P3D2.
40. A method of generating a mammalian immune response directed to
158P3D2, the method comprising: exposing cells of the mammal's
immune system to a portion of a) a 158P3D2-related protein and/or
b) a nucleotide sequence that encodes said protein, whereby an
immune response is generated to 158P3D2.
41. A method of generating an immune response of claim 40, said
method comprising: providing a 158P3D2-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 induced.
42. A method of claim 41 wherein the immune system cell is a B
cell, whereby the induced B cell generates antibodies that
specifically bind to the 158P3D2-related protein.
43. A method of claim 41 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 158P3D2-related protein.
44. A method of claim 41 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.
45. A method for detecting the presence of a 158P3D2-related
protein or polynucleotide in a sample comprising steps of:
contacting the sample with a substance of claim 1 that specifically
binds to the 158P3D2-related protein or polynucleotide,
respectively; and, determining that there is a complex of the
substance and 158P3D2-related protein or the substance and
158P3D2-related polynucleotide, respectively.
46. A method of claim 45 for detecting the presence of a
158P3D2-related protein in a sample comprising steps of: contacting
the sample with an antibody or fragment thereof either of which
specifically bind to the 158P3D2-related protein; and, determining
that there is a complex of the antibody or fragment thereof and
158P3D2-related protein.
47. A method of claim 45 further comprising a step of: taking the
sample from a patient who has or who is suspected of having
cancer.
48. A method of claim 45 for detecting the presence of 158P3D2 mRNA
in a sample comprising: producing cDNA from the sample by reverse
transcription using at least one primer; amplifying the cDNA so
produced using 158P3D2 polynucleotides as sense and antisense
primers, wherein the 158P3D2 polynucleotides used as the sense and
antisense primers serve to amplify 158P3D2 cDNA; and, detecting the
presence of the amplified 158P3D2 cDNA.
49. A method of claim 45 for monitoring 158P3D2 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
158P3D2 gene products expressed by cells in a tissue sample from an
individual; comparing the status so determined to the status of
158P3D2 gene products in a corresponding normal sample; and,
identifying the presence of aberrant 158P3D2 gene products in the
sample relative to the normal sample.
50. A method of monitoring the presence of cancer in an individual
comprising: performing the method of claim 49 whereby the presence
of elevated gene products 158P3D2 mRNA or 158P3D2 protein in the
test sample relative to the normal tissue sample indicates the
presence or status of a cancer.
51. A method of claim 50 wherein the cancer occurs in a tissue set
forth in Table I.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Serial No.
60/283,112 filed Apr. 10, 2001, and U.S. Serial No. 60/286,630,
filed Apr. 25, 2001. The contents of these applications are hereby
incorporated by reference herein in their entirety.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0002] Not applicable.
FIELD OF THE INVENTION
[0003] The invention described herein relates to a gene and its
encoded protein, termed 158P3D2 expressed in certain cancers, and
to diagnostic and therapeutic methods and compositions useful in
the management of cancers that express 158P3D2.
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. September
1996 2 (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. Natl. Acad. Sci. USA 95:
1735).
[0009] While previously identified markers such as PSA, PSM, PCTA
and PSCA have facilitated efforts to diagnose and treat prostate
cancer, there is need for the identification of additional markers
and therapeutic targets for prostate and related cancers in order
to further improve diagnosis and therapy.
[0010] Renal cell carcinoma (RCC) accounts for approximately 3
percent of adult malignancies. Once adenomas reach a diameter of 2
to 3 cm, malignant potential exists. In the adult, the two
principal malignant renal tumors are renal cell adenocarcinoma and
transitional cell carcinoma of the renal pelvis or ureter. The
incidence of renal cell adenocarcinoma is estimated at more than
29,000 cases in the United States, and more than 11,600 patients
died of this disease in 1998. Transitional cell carcinoma is less
frequent, with an incidence of approximately 500 cases per year in
the United States.
[0011] Surgery has been the primary therapy for renal cell
adenocarcinoma for many decades. Until recently, metastatic disease
has been refractory to any systemic therapy. With recent
developments in systemic therapies, particularly immunotherapies,
metastatic renal cell carcinoma may be approached aggressively in
appropriate patients with a possibility of durable responses.
Nevertheless, there is a remaining need for effective therapies for
these patients.
[0012] Of all new cases of cancer in the United States, bladder
cancer represents approximately 5 percent in men (fifth most common
neoplasm) and 3 percent in women (eighth most common neoplasm). The
incidence is increasing slowly, concurrent with an increasing older
population. In 1998, there was an estimated 54,500 cases, including
39,500 in men and 15,000 in women. The age-adjusted incidence in
the United States is 32 per 100,000 for men and 8 per 100,000 in
women. The historic male/female ratio of 3:1 may be decreasing
related to smoking patterns in women. There were an estimated
11,000 deaths from bladder cancer in 1998 (7,800 in men and 3,900
in women). Bladder cancer incidence and mortality strongly increase
with age and will be an increasing problem as the population
becomes more elderly.
[0013] Most bladder cancers recur in the bladder. Bladder cancer is
managed with a combination of transurethral resection of the
bladder (TUR) and intravesical chemotherapy or immunotherapy. The
multifocal and recurrent nature of bladder cancer points out the
limitations of TUR. Most muscle-invasive cancers are not cured by
TUR alone. Radical cystectomy and urinary diversion is the most
effective means to eliminate the cancer but carry an undeniable
impact on urinary and sexual function. There continues to be a
significant need for treatment modalities that are beneficial for
bladder cancer patients.
[0014] An estimated 130,200 cases of colorectal cancer occurred in
2000 in the United States, including 93,800 cases of colon cancer
and 36,400 of rectal cancer. Colorectal cancers are the third most
common cancers in men and women. Incidence rates declined
significantly during 1992-1996 (-2.1% per year). Research suggests
that these declines have been due to increased screening and polyp
removal, preventing progression of polyps to invasive cancers.
There were an estimated 56,300 deaths (47,700 from colon cancer,
8,600 from rectal cancer) in 2000, accounting for about 11% of all
U.S. cancer deaths.
[0015] At present, surgery is the most common form of therapy for
colorectal cancer, and for cancers that have not spread, it is
frequently curative. Chemotherapy, or chemotherapy plus radiation,
is given before or after surgery to most patients whose cancer has
deeply perforated the bowel wall or has spread to the lymph nodes.
A permanent colostomy (creation of an abdominal opening for
elimination of body wastes) is occasionally needed for colon cancer
and is infrequently required for rectal cancer. There continues to
be a need for effective diagnostic and treatment modalities for
colorectal cancer.
[0016] There were an estimated 164,100 new cases of lung and
bronchial cancer in 2000, accounting for 14% of all U.S. cancer
diagnoses. The incidence rate of lung and bronchial cancer is
declining significantly in men, from a high of 86.5 per 100,000 in
1984 to 70.0 in 1996. In the 1990s, the rate of increase among
women began to slow. In 1996, the incidence rate in women was 42.3
per 100,000.
[0017] Lung and bronchial cancer caused an estimated 156,900 deaths
in 2000, accounting for 28% of all cancer deaths. During 1992-1996,
mortality from lung cancer declined significantly among men (-1.7%
per year) while rates for women were still significantly increasing
(0.9% per year). Since 1987, more women have died each year of lung
cancer than breast cancer, which, for over 40 years, was the major
cause of cancer death in women. Decreasing lung cancer incidence
and mortality rates most likely resulted from decreased smoking
rates over the previous 30 years; however, decreasing smoking
patterns among women lag behind those of men. Of concern, although
the declines in adult tobacco use have slowed, tobacco use in youth
is increasing again.
[0018] Treatment options for lung and bronchial cancer are
determined by the type and stage of the cancer and include surgery,
radiation therapy, and chemotherapy. For many localized cancers,
surgery is usually the treatment of choice. Because the disease has
usually spread by the time it is discovered, radiation therapy and
chemotherapy are often needed in combination with surgery.
Chemotherapy alone or combined with radiation is the treatment of
choice for small cell lung cancer; on this regimen, a large
percentage of patients experience remission, which in some cases is
long lasting. There is however, an ongoing need for effective
treatment and diagnostic approaches for lung and bronchial
cancers.
[0019] An estimated 182,800 new invasive cases of breast cancer
were expected to occur among women in the United States during
2000. Additionally, about 1,400 new cases of breast cancer were
expected to be diagnosed in men in 2000. After increasing about 4%
per year in the 1980s, breast cancer incidence rates in women have
leveled off in the 1990s to about 110.6 cases per 100,000.
[0020] In the U.S. alone, there were an estimated 41,200 deaths
(40,800 women, 400 men) in 2000 due to breast cancer. Breast cancer
ranks second among cancer deaths in women. According to the most
recent data, mortality rates declined significantly during
1992-1996 with the largest decreases in younger women, both white
and black. These decreases were probably the result of earlier
detection and improved treatment.
[0021] Taking into account the medical circumstances and the
patient's preferences, treatment of breast cancer may involve
lumpectomy (local removal of the tumor) and removal of the lymph
nodes under the arm; mastectomy (surgical removal of the breast)
and removal of the lymph nodes under the arm; radiation therapy;
chemotherapy; or hormone therapy. Often, two or more methods are
used in combination. Numerous studies have shown that, for early
stage disease, long-term survival rates after lumpectomy plus
radiotherapy are similar to survival rates after modified radical
mastectomy. Significant advances in reconstruction techniques
provide several options for breast reconstruction after mastectomy.
Recently, such reconstruction has been done at the same time as the
mastectomy.
[0022] Local excision of ductal carcinoma in situ (DCIS) with
adequate amounts of surrounding normal breast tissue may prevent
the local recurrence of the DCIS. Radiation to the breast and/or
tamoxifen may reduce the chance of DCIS occurring in the remaining
breast tissue. This is important because DCIS, if left untreated,
may develop into invasive breast cancer. Nevertheless, there are
serious side effects or sequelae to these treatments. There is,
therefore, a need for efficacious breast cancer treatments.
[0023] There were an estimated 23,100 new cases of ovarian cancer
in the United States in 2000. It accounts for 4% of all cancers
among women and ranks second among gynecologic cancers. During
1992-1996, ovarian cancer incidence rates were significantly
declining. Consequent to ovarian cancer, there were an estimated
14,000 deaths in 2000. Ovarian cancer causes more deaths than any
other cancer of the female reproductive system.
[0024] Surgery, radiation therapy, and chemotherapy are treatment
options for ovarian cancer. Surgery usually includes the removal of
one or both ovaries, the fallopian tubes (salpingo-oophorectomy),
and the uterus (hysterectomy). In some very early tumors, only the
involved ovary will be removed, especially in young women who wish
to have children. In advanced disease, an attempt is made to remove
all intra-abdominal disease to enhance the effect of chemotherapy.
There continues to be an important need for effective treatment
options for ovarian cancer.
[0025] There were an estimated 28,300 new cases of pancreatic
cancer in the United States in 2000. Over the past 20 years, rates
of pancreatic cancer have declined in men. Rates among women have
remained approximately constant but may be beginning to decline.
Pancreatic cancer caused an estimated 28,200 deaths in 2000 in the
United States. Over the past 20 years, there has been a slight but
significant decrease in mortality rates among men (about -0.9% per
year) while rates have increased slightly among women.
[0026] Surgery, radiation therapy, and chemotherapy are treatment
options for pancreatic cancer. These treatment options can extend
survival and/or relieve symptoms in many patients but are not
likely to produce a cure for most. There is a significant need for
additional therapeutic and diagnostic options for pancreatic
cancer.
SUMMARY OF THE INVENTION
[0027] The present invention relates to a gene, designated 158P3D2,
that has now been found to be overexpressed in the cancer(s) listed
in Table I. Northern blot expression analysis of 158P3D2 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 158P3D2 are provided. The tissue-related
profile of 158P3D2 in normal adult tissues, combined with the
over-expression observed in the tumors listed in Table I, shows
that 158P3D2 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 158P3D2 genes, mRNAs, and/or
coding sequences, preferably in isolated form, including
polynucleotides encoding 158P3D2-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, 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, 328 or more than
328 contiguous amino acids of a 158P3D2-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 158P3D2
genes or mRNA sequences or parts thereof, and polynucleotides or
oligonucleotides that hybridize to the 158P3D2 genes, mRNAs, or to
158P3D2-encoding polynucleotides. Also provided are means for
isolating cDNAs and the genes encoding 158P3D2. Recombinant DNA
molecules containing 158P3D2 polynucleotides, cells transformed or
transduced with such molecules, and host-vector systems for the
expression of 158P3D2 gene products are also provided. The
invention further provides antibodies that bind to 158P3D2 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 158P3D2 polynucleotides and proteins in
various biological samples, as well as methods for identifying
cells that express 158P3D2. A typical embodiment of this invention
provides methods for monitoring 158P3D2 gene products in a tissue
or hematology sample having or suspected of having some form of
growth dysregulation such as cancer.
[0030] The invention further provides various immunogenic or
therapeutic compositions and strategies for treating cancers that
express 158P3D2 such as cancers of tissues listed in Table I,
including therapies aimed at inhibiting the transcription,
translation, processing or function of 158P3D2 as well as cancer
vaccines. In one aspect, the invention provides compositions, and
methods comprising them, for treating a cancer that expresses
158P3D2 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 158P3D2.
Preferably, the carrier is a uniquely human carrier. In another
aspect of the invention, the agent is a moiety that is
immunoreactive with 158P3D2 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 158P3D2 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 158P3D2 as
described above. The one or more than one nucleic acid molecule may
also be, or encodes, a molecule that inhibits production of
158P3D2. Non-limiting examples of such molecules include, but are
not limited to, those complementary to a nucleotide sequence
essential for production of 158P3D2 (e.g. antisense sequences or
molecules that form a triple helix with a nucleotide double helix
essential for 158P3D2 production) or a ribozyme effective to lyse
158P3D2 mRNA.
BRIEF DESCRIPTION OF THE FIGURES
[0032] FIG. 1. The 158P3D2 SSH sequence of 312 nucleotides.
[0033] FIG. 2. The cDNA (SEQ ID. NO.: ______) and amino acid
sequence (SEQ ID. NO.: ______) of 158P3D2 variant 1 clone
158P3D2-BCP1 (also called "158P3D2 v.1" or "158P3D2 variant 1" or
"158P3D2 var1") is shown in FIG. 2A. The start methionine is
underlined. The open reading frame extends from nucleic acid
849-1835 including the stop codon. The cDNA (SEQ ID. NO.: ______)
and amino acid sequence (SEQ ID. NO.: ______) of 158P3D2 variant 2a
(also called "158P3D2 var2a" or "158P3D2 v.2a") is shown in FIG.
2B. The codon for the start methionine is underlined. The open
reading frame extends from nucleic acid 117 to 827 including the
stop codon. The cDNA (SEQ ID. NO. : ______) and amino acid sequence
(SEQ ID. NO.: ______) of 158P3D2 variant 2b (also called "158P3D2
var2b" or "158P3D2 v.2b") is shown in FIG. 2C. The codon for the
start methionine is underlined. The open reading frame extends from
nucleic acid 2249-2794 including the stop codon. The cDNA (SEQ ID.
NO.: ______) and amino acid sequence (SEQ ID. NO.: ______) of
158P3D2 variant 3 (also called "158P3D2 var3" or "158P3D2 v.3") is
shown in FIG. 2D. The codon for the start methionine is underlined.
The open reading frame extends from nucleic acid 849-1835 including
the stop codon. The cDNA (SEQ ID. NO.: ______) and amino acid
sequence (SEQ ID. NO.: ______) of 158P3D2 variant 4 (also called
"158P3D2 var4" or "158P3D2 v.4") is shown in FIG. 2E. The codon for
the start methionine is underlined. The open reading frame extends
from nucleic acid 849-1835 including the stop codon. The cDNA (SEQ
ID. NO.: ______) and amino acid sequence (SEQ ID. NO.: ______) of
158P3D2 variant 5a clone 158P3D2-BCP2 (also called "158P3D2 variant
Sa" or "158P3D2 varSa" or "158P3D2 v.5a") is shown in FIG. 2F. The
codon for the start methionine is underlined. The open reading
frame extends from nucleic acid 849-1385 including the stop codon.
The cDNA (SEQ ID. NO.: ______) and amino acid sequence (SEQ ID.
NO.: ______) of 158P3D2 variant 5b clone 158P3D2-BCP2 (also called
"158P3D2 variant Sb" or "158P3D2 var5b" or "158P3D2 v.Sb") is shown
in FIG. 2G. The codon for the start methionine is underlined. The
open reading frame extends from nucleic acid 1289-1834 including
the stop codon. The cDNA (SEQ ID. NO.: ______) and amino acid
sequence (SEQ ID. NO.: ______) of 158P3D2 variant 6 (also called
"158P3D2 var6" or "158P3D2 v.6") is shown in FIG. 2H. The codon for
the start methionine is underlined. The open reading frame extends
from nucleic acid 849-1835 including the stop codon. The cDNA (SEQ
ID. NO.: ______) and amino acid sequence (SEQ ID. NO.: ______) of
158P3D2 variant 7 (also called "158P3D2 var7" or "158P3D2 v.7") is
shown in FIG. 2I. The codon for the start methionine is underlined.
The open reading frame extends from nucleic acid 849-1835 including
the stop codon. The cDNA (SEQ ID. NO.: ______) and amino acid
sequence (SEQ ID. NO.: ______) of 158P3D2 variant 8 (also called
"158P3D2 var8" or "158P3D2 v.8") is shown in FIG. 2J. The codon for
the start methionine is underlined. The open reading frame extends
from nucleic acid 849-1835 including the stop codon. As used
herein, a reference to 158P3D2 includes all variants thereof,
including those shown in FIG. 10.
[0034] FIG. 3. Amino acid sequence of 158P3D2 var1 (SEQ ID. NO.:
______) is shown in FIG. 3A; it has 328 amino acids. The amino acid
sequence of 158P3D2 var2a (SEQ ID. NO.: ______) is shown in FIG.
3B; it has 236 amino acids. The amino acid sequence of 158P3D2
var2b (SEQ ID. NO.: ______) is shown in FIG. 3C; it has 181 amino
acids. The amino acid sequence of 158P3D2 var3 (SEQ ID. NO.:
______) is shown in FIG. 3D; it has 328 amino acids. The amino acid
sequence of 158P3D2 var4 (SEQ ID. NO.: ______) is shown in FIG. 3E;
it has 328 amino acids. The amino acid sequence of 158P3D2 var5a
(SEQ ID. NO.: ______) is shown in FIG. 3F; it has 178 amino acids.
The amino acid sequence of 158P3D2 var5b (SEQ ID. NO.: ______) is
shown in FIG. 3G; it has 181 amino acids. As used herein, a
reference to 158P3D2 includes all variants thereof, including those
shown in FIG. 11.
[0035] FIG. 4. The nucleic acid sequence alignment of 158P3D2 var1
to fer-1-like 4 (C.elegans) (FER1L4) mRNA is shown in FIG. 4A. The
amino acid sequence alignment of 158P3D2 var1 to dJ47704.1.1
(AL121586), a novel protein similar to otoferlin and dysferlin,
isoform 1 is shown in FIG. 4B. The amino acid sequence alignment
with human brain otoferlin long isoform is shown in FIG. 4C. The
amino acid sequence alignment with mouse otoferlin is shown in FIG.
4D. The amino acid sequence alignments of 158P3D2 protein var1, 2a,
2b, 3, 4, 5a, and 5b are shown in FIG. 4E.
[0036] FIG. 5. Hydrophilicity amino acid profile of A) 158P3D2
var1, B) 158P3D2 var2a and C) 158P3D2 var5a, 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
(www.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular
biology server.
[0037] FIG. 6. Hydropathicity amino acid profile of A) 158P3D2
var1, B) 158P3D2 var2a and C) 158P3D2 var5a, determined by computer
algorithm sequence analysis using the method of Kyte and Doolittle
(Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132)
accessed on the ProtScale website
(www.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular
biology server.
[0038] FIG. 7. Percent accessible residues amino acid profile of A)
158P3D2 var1, B) 158P3D2 var2a and C) 158P3D2 var5a, determined by
computer algorithm sequence analysis using the method of Janin
(Janin J., 1979 Nature 277:491-492) accessed on the ProtScale
website (www.expasy.ch/cgi-bin/protscale.pl) through the ExPasy
molecular biology server.
[0039] FIG. 8. Average flexibility amino acid profile of A) 158P3D2
var1, B) 158P3D2 var2a and C) 158P3D2 var5a, 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
(www.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular
biology server.
[0040] FIG. 9. Beta-turn amino acid profile of A) 158P3D2 var1, B)
158P3D2 var2a and C) 158P3D2 var5a, 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 (www.expasy.ch/cgi-bin/protscale.pl)
through the ExPasy molecular biology server.
[0041] FIG. 10. Schematic display of nucleotide variants of
158P3D2. Variant 158P3D2 v.2 is an alternative transcript. Others
are Single Nucleotide Polymorphism (also called "SNP") variants,
which could also occur in any alternative transcript. The numbers
in "( )" underneath the box correspond to those of 158P3D2 var1.
`-` indicate single nucleotide deletion. Variants 158P3D2 v.3
through v.8 are variants with single nucleotide variations. The
black boxes show the same sequence as 158P3D2 var1. SNPs are
indicated above the box.
[0042] FIG. 11. Schematic display of protein variants of 158P3D2.
Nucleotide variant 158P3D2 var2 and 158P3D2 v.5 in FIG. 10
potentially code for two different proteins, designated as variants
158P3D2 var2a and 158P3D2 var2b, 158P3D2 v.5a and 158P3D2 v.5b,
respectively. Variant 158P3D2 v.5b shares the same amino acid
sequence as variant 158P3D2 var2b. Variants 158P3D2 v.3 and v.4 are
variants with single amino acid variations. The black boxes show
the same sequence as 158P3D2 var1. The numbers in "( )" underneath
the box correspond to those of 158P3D2 var1. Single amino acid
differences are indicated above the box.
[0043] FIG. 12. Secondary structure prediction of 158P3D2 var1
(FIG. 12A), var2a (FIG. 12B) and var5a (FIG. 12C); and
transmembrane predictions for 158P3D2 var1 (FIGS. 12D and E). The
secondary structure of 158P3D2 proteins were predicted using the
HNN--Hierarchial 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
(http://www.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 given.
[0044] A schematic representation of the probability of existence
of transmembrane regions and orientation based on the TMpred
algorithm which utilizes TMBASE is shown in FIG. 12D (K. Hofmann,
W. Stoffel. TMBASE--A database of membrane spanning protein
segments Biol. Chem. Hoppe-Seyler 374:166, 1993). A schematic
representation of the probability of the existence of transmembrane
regions and the extracellular and intracellular orientation based
on the TMHMM algorithm is shown in FIG. 12E (Erik L. L. Sonnhammer,
Gunnar von Heijne, and Anders Krogh: A hidden Markov model for
predicting transmembrane helices in protein sequences. In Proc. of
Sixth Int. Conf. on Intelligent Systems for Molecular Biology, p
175-182 Ed J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D.
Sankoff, and C. Sensen Menlo Park, Calif.: AAAI Press, 1998). The
TMpred and TMHMM algorithms are accessed from the ExPasy molecular
biology server (http://www.expasy.ch/tools/). The results of the
transmembrane prediction programs depict 158P3D2 var1 as containing
1 transmembrane domain.
[0045] FIG. 13. Exon compositions of transcript variants of
158P3D2. Variant 158P3D2 var2 is an alternative transcript.
Compared with 158P3D2 var1, it has six additional exons to the 5'
end, an exon 7 longer than exon 1 of 158P3D2 var1 and an exon 10
shorter than exon 4 of 158P3D2 var. Exons 2, 3, 5, 6 and 7 of
158P3D2 var1 are the same as exons 8, 9, 11, 12 and 13 of 158P3D2
var2, respectively. The numbers in "( )" underneath the box
correspond to those of 158P3D2 var1. The black boxes show the same
sequence as 158P3D2 var1. The length of the introns are not
proportional.
[0046] FIG. 14. Expression of 158P3D2 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 from 2 different patients, prostate cancer pool, bladder
cancer pool, kidney cancer pool, colon cancer pool, lung cancer
pool, ovary cancer pool, breast cancer pool, cancer metastasis
pool, and pancreas cancer pool. Normalization was performed by PCR
using primers to actin and GAPDH. Semi-quantitative PCR, using
primers to 158P3D2, was performed at 26 and 30 cycles of
amplification. Results show strong expression of 158P3D2 in bladder
cancer pool, kidney cancer pool and cancer metastasis pool.
Expression of 158P3D2 is also detected in colon cancer pool, lung
cancer pool, ovary cancer pool, breast cancer pool, pancreas cancer
pool and prostate metastases to lymph node, and vital pool 2, but
not vital pool 1.
[0047] FIG. 15. Expression of 158P3D2 in normal tissues. Two
multiple tissue northern blots (Clontech) both with 2 ug of
mRNA/lane were probed with the 158P3D2 SSH fragment. Size standards
in kilobases (kb) are indicated on the side. Results show
restricted expression of an approximately 8 kb 158P3D2 transcript
in normal placenta.
[0048] FIG. 16. Expression of 158P3D2 in Multiple Normal Tissues.
An mRNA dot blot containing 76 different samples from human tissues
was analyzed using a 158P3D2 probe. Expression was detected in
placenta and stomach.
[0049] FIG. 17. Expression of 158P3D2 in Patient Cancer Specimens
and Normal Tissues. RNA was extracted from a pool of three bladder
cancers, as well as from normal prostate (NP), normal bladder (NB),
normal kidney (NK), normal colon (NC), normal lung (NL) and normal
breast (NBr). Northern blot with 10 10 .mu.g of total RNA/lane was
probed with 158P3D2 sequence. Size standards in kilobases (kb) are
indicated on the side. The results show expression of 158P3D2 in
the bladder cancer pool but not in the normal tissues tested.
[0050] FIG. 18. Expression of 158P3D2 in bladder cancer patient
tissues. RNA was extracted from normal bladder (N), bladder cancer
cell lines (UM-UC-3, J82, SCaBER), bladder cancer patient tumors
(T) and their normal adjacent tissues (NAT). Northern blots with 10
ug of total RNA were probed with the 158P3D2 SSH fragment. Size
standards in kilobases are on the side. Results show strong
expression of 158P3D2 in tumor tissues. The expression observed in
normal adjacent tissue (isolated from diseased tissues) but not in
normal tissue, isolated from healthy donors, may indicate that
these tissues are not fully normal and that 158P3D2 may be
expressed in early stage tumors.
[0051] FIG. 19. 158P3D2 Expression in 293T Cells Following
Transfection. 293T cells were transfected with either
158P3D2.pcDNA3.1/mychis or pcDNA3.1/mychis vector control. Forty
hours later, cell lysates were collected. Samples were run on an
SDS-PAGE acrylamide gel, blotted and stained with anti-his
antibody. The blot was developed using the ECL chemiluminescence
kit and visualized by autoradiography. Results show expression of
158P3D2 clones of 158P3D2.pcDNA3.1/mychis in the lysates of 158P3D2
.pcDNA3. 1/mychis transfected cells.
DETAILED DESCRIPTION OF THE INVENTION
[0052] Outline of Sections
[0053] I.) Definitions
[0054] II.) 158P3D2 Polynucleotides
[0055] II.A.) Uses of 158P3D2 Polynucleotides
[0056] II.A.1.) Monitoring of Genetic Abnormalities
[0057] II.A.2.) Antisense Embodiments
[0058] II.A.3.) Primers and Primer Pairs
[0059] II.A.4.) Isolation of 158P3D2-Encoding Nucleic Acid
Molecules
[0060] II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector
Systems
[0061] III.) 158P3D2-Related Proteins
[0062] III.A.) Motif-bearing Protein Embodiments
[0063] III.B.) Expression of 158P3D2-Related Proteins
[0064] III.C.) Modifications of 158P3D2-Related Proteins
[0065] III.D.) Uses of 158P3D2-Related Proteins
[0066] IV.) 158P3D2 Antibodies
[0067] V.) 158P3D2 Cellular Immune Responses
[0068] VI.) 158P3D2 Transgenic Animals
[0069] VII.) Methods for the Detection of 158P3D2
[0070] VIII.) Methods for Monitoring the Status of 158P3D2-Related
Genes and Their Products
[0071] IX.) Identification of Molecules that Interact with
158P3D2
[0072] X.) Therapeutic Methods and Compositions
[0073] X.A.) Anti-Cancer Vaccines
[0074] X.B.) 158P3D2 as a Target for Antibody-Based Therapy
[0075] X.C.) 158P3D2 as a Target for Cellular Immune Responses
[0076] X.C.1. Minigene Vaccines
[0077] X.C.2. Combinations of CTL Peptides with Helper Peptides
[0078] X.C.3. Combinations of CTL Peptides with T Cell Priming
Agents
[0079] X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL
and/or HTL Peptides
[0080] X.D.) Adoptive Immunotherapy
[0081] X.E.) Administration of Vaccines for Therapeutic or
Prophylactic Purposes
[0082] XI.) Diagnostic and Prognostic Embodiments of 158P3D2.
[0083] XII.) Inhibition of 158P3D2 Protein Function
[0084] XII.A.) Inhibition of 158P3D2 with Intracellular
Antibodies
[0085] XII.B.) Inhibition of 158P3D2 with Recombinant Proteins
[0086] XII.C.) Inhibition of 158P3D2 Transcription or
Translation
[0087] XII.D.) General Considerations for Therapeutic
Strategies
[0088] XIII.) KITS
[0089] I.) Definitions:
[0090] 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.
[0091] 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.
[0092] "Altering the native glycosylation pattern" is intended for
purposes herein to mean deleting one or more carbohydrate moieties
found in native sequence 158P3D2 (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 158P3D2. 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.
[0093] The term "analog" refers to a molecule which is structurally
similar or shares similar or corresponding attributes with another
molecule (e.g. a 158P3D2-related protein). For example an analog of
a 158P3D2 protein can be specifically bound by an antibody or T
cell that specifically binds to 158P3D2.
[0094] 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-158P3D2 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.
[0095] 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-158P3D2 antibodies and clones
thereof (including agonist, antagonist and neutralizing antibodies)
and anti-1 58P3D2 antibody compositions with polyepitopic
specificity.
[0096] 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."
[0097] 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 maytansinoids, yttrium, bismuth, ricin, ricin
A-chain, doxorubicin, daunorubicin, taxol, 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, P.sup.32 and radioactive isotopes of Lu.
Antibodies may also be conjugated to an anti-cancer pro-drug
activating enzyme capable of converting the pro-drug to its active
form.
[0098] 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.
[0099] "Human Leukocyte Antigen" or "HLA" is a human class I or
class II Major Histocompatibility Complex (MHC) protein (see, e.g.,
Stites, et al., Immunology, 8.sup.TH ED., Lange Publishing, Los
Altos, Calif. (1994).
[0100] 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.
[0101] 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 158P3D2 genes or that encode
polypeptides other than 158P3D2 gene product or fragments thereof.
A skilled artisan can readily employ nucleic acid isolation
procedures to obtain an isolated 158P3D2 polynucleotide. A protein
is said to be "isolated," for example, when physical, mechanical or
chemical methods are employed to remove the 158P3D2 proteins from
cellular constituents that are normally associated with the
protein. A skilled artisan can readily employ standard purification
methods to obtain an isolated 158P3D2 protein. Alternatively, an
isolated protein can be prepared by chemical means.
[0102] 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.
[0103] 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 TxNxM+ 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.
[0104] 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.
[0105] A "motif", as in biological motif of an 158P3D2-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.
[0106] 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.
[0107] "Pharmaceutically acceptable" refers to a non-toxic, inert,
and/or composition that is physiologically compatible with humans
or other mammals.
[0108] The term "polynucleotide" means a polymeric form of
nucleotides of at least 10 bases or base pairs in length, either
ribonucleotides or deoxynucleotides or a modified form of either
type of nucleotide, and is meant to include single and double
stranded forms of DNA and/or RNA. In the art, this term if often
used interchangeably with "oligonucleotide". A polynucleotide can
comprise a nucleotide sequence disclosed herein wherein thymidine
(T), as shown for example in FIG. 2, can also be uracil (U); this
definition pertains to the differences between the chemical
structures of DNA and RNA, in particular the observation that one
of the four major bases in RNA is uracil (U) instead of thymidine
(T).
[0109] 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".
[0110] An HLA "primary anchor residue" is an amino acid at a
specific position along a peptide sequence which is understood to
provide a contact point between the immunogenic peptide and the HLA
molecule. One to three, usually two, primary anchor residues within
a peptide of defined length generally defines a "motif" for an
immunogenic peptide. These residues are understood to fit in close
contact with peptide binding groove of an HLA molecule, with their
side chains buried in specific pockets of the binding groove. In
one embodiment, for example, the primary anchor residues for an HLA
class I molecule are located at position 2 (from the amino terminal
position) and at the carboxyl terminal position of a 8, 9, 10, 11,
or 12 residue peptide epitope in accordance with the invention. In
another embodiment, for example, the primary anchor residues of a
peptide that will bind an HLA class II molecule are spaced relative
to each other, rather than to the termini of a peptide, where the
peptide is generally of at least 9 amino acids in length. The
primary anchor positions for each motif and supermotif are set
forth in Table IV. For example, analog peptides can be created by
altering the presence or absence of particular residues in the
primary and/or secondary anchor positions shown in Table IV. Such
analogs are used to modulate the binding affinity and/or population
coverage of a peptide comprising a particular HLA motif or
supermotif.
[0111] A "recombinant" DNA or RNA molecule is a DNA or RNA molecule
that has been subjected to molecular manipulation in vitro.
[0112] Non-limiting examples of small molecules include compounds
that bind or interact with 158P3D2, ligands including hormones,
neuropeptides, chemokines, odorants, phospholipids, and functional
equivalents thereof that bind and preferably inhibit 158P3D2
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, 158P3D2 protein; are not found in naturally occurring
metabolic pathways; and/or are more soluble in aqueous than
non-aqueous solutions
[0113] "Stringency" of hybridization reactions is readily
determinable by one of ordinary skill in the art, and generally is
an empirical calculation dependent upon probe length, washing
temperature, and salt concentration. In general, longer probes
require higher temperatures for proper annealing, while shorter
probes need lower temperatures. Hybridization generally depends on
the ability of denatured nucleic acid sequences to reanneal when
complementary strands are present in an environment below their
melting temperature. The higher the degree of desired homology
between the probe and hybridizable sequence, the higher the
relative temperature that can be used. As a result, it follows that
higher relative temperatures would tend to make the reaction
conditions more stringent, while lower temperatures less so. For
additional details and explanation of stringency of hybridization
reactions, see Ausubel et al., Current Protocols in Molecular
Biology, Wiley Interscience Publishers, (1995).
[0114] "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 1 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.
[0115] An HLA "supermotif" is a peptide binding specificity shared
by HLA molecules encoded by two or more HLA alleles.
[0116] 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.
[0117] 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.
[0118] As used herein, an HLA or cellular immune response "vaccine"
is a composition that contains or encodes one or more peptides of
the invention. There are numerous embodiments of such vaccines,
such as a cocktail of one or more individual peptides; one or more
peptides of the invention comprised by a polyepitopic peptide; or
nucleic acids that encode such individual peptides or polypeptides,
e.g., a minigene that encodes a polyepitopic peptide. The "one or
more peptides" can include any whole unit integer from 1-328 or
more, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115,
120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180,
185, 190, 195, 200, 225, 250, 275, 300, 325, or 328 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.
[0119] 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 158P3D2
protein shown in FIG. 2 or FIG. 3. An analog is an example of a
variant protein. Splice isoforms and SNPs are further examples of
variants.
[0120] The "158P3D2-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 158P3D2 proteins or fragments thereof, as well as fusion
proteins of a 158P3D2 protein and a heterologous polypeptide are
also included. Such 158P3D2 proteins are collectively referred to
as the 158P3D2-related proteins, the proteins of the invention, or
158P3D2. The term "158P3D2-related protein" refers to a polypeptide
fragment or an 158P3D2 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, 328 or more than 328 amino acids.
[0121] II.) 158P3D2 Polynucleotides
[0122] One aspect of the invention provides polynucleotides
corresponding or complementary to all or part of an 158P3D2 gene,
mRNA, and/or coding sequence, preferably in isolated form,
including polynucleotides encoding an 158P3D2-related protein and
fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules,
polynucleotides or oligonucleotides complementary to an 158P3D2
gene or mRNA sequence or a part thereof, and polynucleotides or
oligonucleotides that hybridize to an 158P3D2 gene, mRNA, or to an
158P3D2 encoding polynucleotide (collectively, "158P3D2
polynucleotides"). In all instances when referred to in this
section, T can also be U in FIG. 2.
[0123] Embodiments of a 158P3D2 polynucleotide include: a 158P3D2
polynucleotide having the sequence shown in FIG. 2, the nucleotide
sequence of 158P3D2 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 158P3D2 nucleotides comprise, without
limitation:
[0124] (I) a polynucleotide comprising, consisting essentially of,
or consisting of a sequence as shown in FIG. 2A (SEQ ID NO:
______), wherein T can also be U;
[0125] (II) a polynucleotide comprising, consisting essentially of,
or consisting of the sequence as shown in FIG. 2A (SEQ ID NO:
______), from nucleotide residue number 849 through nucleotide
residue number 1835, including the stop codon, wherein T can also
be U;
[0126] (III) a polynucleotide comprising, consisting essentially
of, or consisting of the sequence as shown in FIG. 2B (SEQ ID NO:
______), from nucleotide residue number 117 through nucleotide
residue number 827, including the stop codon, wherein T can also be
U;
[0127] (IV) a polynucleotide comprising, consisting essentially of,
or consisting of the sequence as shown in FIG. 2C (SEQ ID NO:
______), from nucleotide residue number 2249 through nucleotide
residue number 2794, including the a stop codon, wherein T can also
be U;
[0128] (V) a polynucleotide comprising, consisting essentially of,
or consisting of the sequence as shown in FIG. 2D (SEQ ID NO:
______), from nucleotide residue number 849 through nucleotide
residue number 1835, including the stop codon, wherein T can also
be U;
[0129] (VI) a polynucleotide comprising, consisting essentially of,
or consisting of the sequence as shown in FIG. 2E (SEQ ID NO:
______), from nucleotide residue number 849 through nucleotide
residue number 1835, including the stop codon, wherein T can also
be U;
[0130] (VII) a polynucleotide comprising, consisting essentially
of, or consisting of the sequence as shown in FIG. 2F (SEQ ID NO:
______), from nucleotide residue number 849 through nucleotide
residue number 1385, including the stop codon, wherein T can also
be U;
[0131] (VIII) a polynucleotide comprising, consisting essentially
of, or consisting of the sequence as shown in FIG. 2G (SEQ ID NO:
______), from nucleotide residue number 1289 through nucleotide
residue number 1834, including the stop codon, wherein T can also
be U;
[0132] (IX) a polynucleotide comprising, consisting essentially of,
or consisting of the sequence as shown in FIG. 2H (SEQ ID NO:
______), from nucleotide residue number 849 through nucleotide
residue number 1835, including the stop codon, wherein T can also
be U;
[0133] (X) a polynucleotide comprising, consisting essentially of,
or consisting of the sequence as shown in FIG. 2I (SEQ ID NO:
______), from nucleotide residue number 849 through nucleotide
residue number 1835, including the stop codon, wherein T can also
be U;
[0134] (XI) a polynucleotide comprising, consisting essentially of,
or consisting of the sequence as shown in FIG. 2J (SEQ ID NO:
______), from nucleotide residue number 849 through nucleotide
residue number 1835, including the stop codon, wherein T can also
be U;
[0135] (XII) a polynucleotide that encodes an 158P3D2-related
protein that is at least 90% homologous to an entire amino acid
sequence shown in FIGS. 2A-I (SEQ ID NO: ______);
[0136] (XIII) a polynucleotide that encodes an 158P3D2-related
protein that is at least 90% identical to an entire amino acid
sequence shown in FIGS. 2A-I (SEQ ID NO: ______);
[0137] (XIV) a polynucleotide that encodes at least one peptide set
forth in Tables V-XIX;
[0138] (XV) a polynucleotide that encodes a peptide region of at
least 5 amino acids of a peptide of FIG. 3A in any whole number
increment up to 328 that includes an amino acid position having a
value greater than 0.5 in the Hydrophilicity profile of FIG. 5A; or
of FIG. 3B in any whole number increment up to 236 that includes an
amino acid position having a value greater than 0.5 in the
Hydrophilicity profile of FIG. 5B; or FIG. 3F in any whole number
increment up to 178 that includes an amino acid position having a
value greater than 0.5 in the Hydrophilicity profile of FIG.
5C;
[0139] (XVI) a polynucleotide that encodes a peptide region of at
least 5 amino acids of a peptide of FIG. 3A in any whole number
increment up to 328 that includes an amino acid position having a
value less than 0.5 in the Hydropathicity profile of FIG. 6A; or of
FIG. 3B in any whole number increment up to 236, that includes an
amino acid position having a value less than 0.5 in the
Hydropathicity profile of FIG. 6B; or FIG. 3F in any whole number
increment up to 178 that includes an amino acid position having a
value greater than 0.5 in the Hydropathicity profile of FIG.
6C;
[0140] (XVII) a polynucleotide that encodes a peptide region of at
least 5 amino acids of a peptide of FIG. 3A in any whole number
increment up to 328 that includes an amino acid position having a
value greater than 0.5 in the Percent Accessible Residues profile
of FIG. 7A; or of FIG. 3B in any whole number increment up to 236,
that includes an amino acid position having a value greater than
0.5 in the Percent Accessible Residues profile of FIG. 7B; or FIG.
3F in any whole number increment up to 178 that includes an amino
acid position having a value greater than 0.5 in the Percent
Accessible Residues profile of FIG. 7C;
[0141] (XVIII) a polynucleotide that encodes a peptide region of at
least 5 amino acids of a peptide of FIG. 3A in any whole number
increment up to 328 that includes an amino acid position having a
value greater than 0.5 in the Average Flexibility profile on FIG.
8A; or of FIG. 3B in any whole number increment up to 236, that
includes an amino acid position having a value greater than 0.5 in
the Average Flexibility profile on FIG. 8B; or FIG. 3F in any whole
number increment up to 178 that includes an amino acid position
having a value greater than 0.5 in the Average Flexibility profile
of FIG. 8C;
[0142] (XIX) a polynucleotide that encodes a peptide region of at
least 5 amino acids of a peptide of FIG. 3A in any whole number
increment up to 328 that includes an amino acid position having a
value greater than 0.5 in the Beta-turn profile of FIG. 9A; or of
FIG. 3B in any whole number increment up to 236, that includes an
amino acid position having a value greater than 0.5 in the
Beta-turn profile of FIG. 9B; or FIG. 3F in any whole number
increment up to 178 that includes an amino acid position having a
value greater than 0.5 in the Beta-turn profile of FIG. 9C;
[0143] (XX) a polynucleotide that is fully complementary to a
polynucleotide of any one of (I)-(XIX).
[0144] (XXI) a peptide that is encoded by any of (I)-(XX); and
[0145] (XXII) a polynucleotide of any of (I)-(XX) or peptide of
(XXI) together with a pharmaceutical excipient and/or in a human
unit dose form.
[0146] As used herein, a range is understood to specifically
disclose all whole unit positions thereof.
[0147] Typical embodiments of the invention disclosed herein
include 158P3D2 polynucleotides that encode specific portions of
158P3D2 mRNA sequences (and those which are complementary to such
sequences) such as those that encode the proteins and/or fragments
thereof, for example:
[0148] (a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,
80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145,
150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250,
275, 300, 325, 328 or more than 328 contiguous amino acids of
158P3D2.
[0149] 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 158P3D2 protein shown in FIG. 2 or FIG. 3,
polynucleotides encoding about amino acid 10 to about amino acid 20
of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides
encoding about amino acid 20 to a bout amino acid 30 of the 158P3D2
protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about
amino acid 30 to about amino acid 40 of the 158P3D2 protein shown
in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 40
to about amino acid 50 of the 158P3D2 protein shown in FIG. 2 or
FIG. 3, polynucleotides encoding about amino acid 50 to about amino
acid 60 of the 158P3D2 protein shown in FIG. 2 or FIG. 3,
polynucleotides encoding about amino acid 60 to about amino acid 70
of the 158P3D2 protein shown in FIG. 2 or FIG. 3, polynucleotides
encoding about amino acid 70 to about amino acid 80 of the 158P3D2
protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about
amino acid 80 to about amino acid 90 of the 158P3D2 protein shown
in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 90
to about amino acid 100 of the 158P3D2 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 158P3D2 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.
[0150] Polynucleotides encoding relatively long portions of a
158P3D2 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 158P3D2 protein 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 158P3D2 sequence as shown
in FIG. 2.
[0151] Additional illustrative embodiments of the invention
disclosed herein include 158P3D2 polynucleotide fragments encoding
one or more of the biological motifs contained within a 158P3D2
protein sequence, including one or more of the motif-bearing
subsequences of a 158P3D2 protein set forth in Tables V-XIX. In
another embodiment, typical polynucleotide fragments of the
invention encode one or more of the regions of 158P3D2 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 158P3D2 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.
[0152] II.A.) Uses of 158P3D2 Polynucleotides
[0153] II.A.I.) Monitoring of Genetic Abnormalities
[0154] The polynucleotides of the preceding paragraphs have a
number of different specific uses. The human 158P3D2 gene maps to
the chromosomal location set forth in Example 3. For example,
because the 158P3D2 gene maps to this chromosome, polynucleotides
that encode different regions of the 158P3D2 proteins are used to
characterize cytogenetic abnormalities of this chromosomal locale,
such as abnormalities that are identified as being associated with
various cancers. In certain genes, a variety of chromosomal
abnormalities including rearrangements have been identified as
frequent cytogenetic abnormalities in a number of different cancers
(see e.g. Krajinovic et al., Mutat. Res. 382(3-4): 81-83 (1998);
Johansson et al., Blood 86(10): 3905-3914 (1995) and Finger et al.,
P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotides encoding
specific regions of the 158P3D2 proteins provide new tools that can
be used to delineate, with greater precision than previously
possible, cytogenetic abnormalities in the chromosomal region that
encodes 158P3D2 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)).
[0155] Furthermore, as 158P3D2 was shown to be highly expressed in
bladder and other cancers, 158P3D2 polynucleotides are used in
methods assessing the status of 158P3D2 gene products in normal
versus cancerous tissues. Typically, polynucleotides that encode
specific regions of the 158P3D2 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 158P3D2 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.
[0156] II.A.2.) Antisense Embodiments
[0157] Other specifically contemplated nucleic acid related
embodiments of the invention disclosed herein are genomic DNA,
cDNAs, ribozymes, and antisense molecules, as well as nucleic acid
molecules based on an alternative backbone, or including
alternative bases, whether derived from natural sources or
synthesized, and include molecules capable of inhibiting the RNA or
protein expression of 158P3D2. 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 158P3D2 polynucleotides and polynucleotide
sequences disclosed herein.
[0158] 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., 158P3D2. See for example, Jack Cohen, Oligodeoxynucleotides,
Antisense Inhibitors of Gene Expression, CRC Press, 1989; and
Synthesis 1:1-5 (1988). The 158P3D2 antisense oligonucleotides of
the present invention include derivatives such as
S-oligonucleotides (phosphorothioate derivatives or S-oligos, see,
Jack Cohen, supra), which exhibit enhanced cancer cell growth
inhibitory action. S-oligos (nucleoside phosphorothioates) are
isoelectronic analogs of an oligonucleotide (O-oligo) in which a
nonbridging oxygen atom of the phosphate group is replaced by a
sulfur atom. The S-oligos of the present invention can be prepared
by treatment of the corresponding O-oligos with
3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transfer
reagent. See, e.g., Iyer, R. P. et al., J. Org. Chem. 55:4693-4698
(1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254
(1990). Additional 158P3D2 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).
[0159] The 158P3D2 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 158P3D2 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 158P3D2 mRNA and not to mRNA specifying other regulatory
subunits of protein kinase. In one embodiment, 158P3D2 antisense
oligonucleotides of the present invention are 15 to 30-mer
fragments of the antisense DNA molecule that have a sequence that
hybridizes to 158P3D2 mRNA. Optionally, 158P3D2 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
158P3D2. Alternatively, the antisense molecules are modified to
employ ribozymes in the inhibition of 158P3D2 expression, see,
e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet 12:
510-515 (1996).
[0160] II.A.3.) Primers and Primer Pairs
[0161] Further specific embodiments of this 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 158P3D2 polynucleotide in a sample and as a means for
detecting a cell expressing a 158P3D2 protein.
[0162] Examples of such probes include polypeptides comprising all
or part of the human 158P3D2 cDNA sequence shown in FIG. 2.
Examples of primer pairs capable of specifically amplifying 158P3D2
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 158P3D2 mRNA.
[0163] The 158P3D2 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
158P3D2 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 158P3D2
polypeptides; as tools for modulating or inhibiting the expression
of the 158P3D2 gene(s) and/or translation of the 158P3D2
transcript(s); and as therapeutic agents.
[0164] The present invention includes the use of any probe as
described herein to identify and isolate a 158P3D2 or 158P3D2
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.
[0165] II.A.4.) Isolation of 158P3D2-Encoding Nucleic Acid
Molecules
[0166] The 158P3D2 cDNA sequences described herein enable the
isolation of other polynucleotides encoding 158P3D2 gene
product(s), as well as the isolation of polynucleotides encoding
158P3D2 gene product homologs, alternatively spliced isoforms,
allelic variants, and mutant forms of a 158P3D2 gene product as
well as polynucleotides that encode analogs of 158P3D2-related
proteins. Various molecular cloning methods that can be employed to
isolate full length cDNAs encoding an 158P3D2 gene are well known
(see, for example, Sambrook, J. et al., Molecular Cloning: A
Laboratory Manual, 2d edition, Cold Spring Harbor Press, New York,
1989; Current Protocols in Molecular Biology. Ausubel et al., Eds.,
Wiley and Sons, 1995). For example, lambda phage cloning
methodologies can be conveniently employed, using commercially
available cloning systems (e.g., Lambda ZAP Express, Stratagene).
Phage clones containing 158P3D2 gene cDNAs can be identified by
probing with a labeled 158P3D2 cDNA or a fragment thereof. For
example, in one embodiment, a 158P3D2 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 158P3D2 gene.
A 158P3D2 gene itself can be isolated by screening genomic DNA
libraries, bacterial artificial chromosome libraries (BACs), yeast
artificial chromosome libraries (YACs), and the like, with 158P3D2
DNA probes or primers.
[0167] II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector
Systems
[0168] The invention also provides recombinant DNA or RNA molecules
containing an 158P3D2 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).
[0169] The invention further provides a host-vector system
comprising a recombinant DNA molecule containing a 158P3D2
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 158P3D2 or a fragment, analog or homolog thereof can be
used to generate 158P3D2 proteins or fragments thereof using any
number of host-vector systems routinely used and widely known in
the art.
[0170] A wide range of host-vector systems suitable for the
expression of 158P3D2 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 pSRottkneo (Muller et al.,
1991, MCB 11:1785). Using these expression vectors, 158P3D2 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 158P3D2 protein or fragment thereof. Such host-vector systems
can be employed to study the functional properties of 158P3D2 and
158P3D2 mutations or analogs.
[0171] Recombinant human 158P3D2 protein or an analog or homolog or
fragment thereof can be produced by mammalian cells transfected
with a construct encoding a 158P3D2-related nucleotide. For
example, 293T cells can be transfected with an expression plasmid
encoding 158P3D2 or fragment, analog or homolog thereof, a
158P3D2-related protein is expressed in the 293T cells, and the
recombinant 158P3D2 protein is isolated using standard purification
methods (e.g., affinity purification using anti-158P3D2
antibodies). In another embodiment, a 158P3D2 coding sequence is
subcloned into the retroviral vector pSR.alpha.MSVtkneo and used to
infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293
and rat-1 in order to establish 158P3D2 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 158P3D2 coding sequence can be used for the generation
of a secreted form of recombinant 158P3D2 protein.
[0172] As discussed herein, redundancy in the genetic code permits
variation in 158P3D2 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
www.dna.affrc.gojp/.about.nakamura/codon.html.
[0173] Additional sequence modifications are known to enhance
protein expression in a cellular host. These include elimination of
sequences encoding spurious polyadenylation signals, exon/intron
splice site signals, transposon-like repeats, and/or other such
well-characterized sequences that are deleterious to gene
expression. The GC content of the sequence is adjusted to levels
average for a given cellular host, as calculated by reference to
known genes expressed in the host cell. Where possible, the
sequence is modified to avoid predicted hairpin secondary mRNA
structures. Other useful modifications include the addition of a
translational initiation consensus sequence at the start of the
open reading frame, as described in Kozak, Mol. Cell Biol.,
9:5073-5080 (1989). Skilled artisans understand that the general
rule that eukaryotic ribosomes initiate translation exclusively at
the 5' proximal AUG codon is abrogated only under rare conditions
(see, e.g., Kozak PNAS 92(7): 2662-2666, (1995) and Kozak NAR
15(20): 8125-8148 (1987)).
[0174] II.) 158P3D2-Related Proteins
[0175] Another aspect of the present invention provides
158P3D2-related proteins. Specific embodiments of 158P3D2 proteins
comprise a polypeptide having all or part of the amino acid
sequence of human 158P3D2 as shown in FIG. 2 or FIG. 3.
Alternatively, embodiments of 158P3D2 proteins comprise variant,
homolog or analog polypeptides that have alterations in the amino
acid sequence of 158P3D2 shown in FIG. 2 or FIG. 3.
[0176] In general, naturally occurring allelic variants of human
158P3D2 share a high degree of structural identity and homology
(e.g., 90% or more homology). Typically, allelic variants of a
158P3D2 protein contain conservative amino acid substitutions
within the 158P3D2 sequences described herein or contain a
substitution of an amino acid from a corresponding position in a
homologue of 158P3D2. One class of 158P3D2 allelic variants are
proteins that share a high degree of homology with at least a small
region of a particular 158P3D2 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.
[0177] 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
(O) 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).
[0178] Embodiments of the invention disclosed herein include a wide
variety of art-accepted variants or analogs of 158P3D2 proteins
such as polypeptides having amino acid insertions, deletions and
substitutions. 158P3D2 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
158P3D2 variant DNA.
[0179] 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.
[0180] As defined herein, 158P3D2 variants, analogs or homologs,
have the distinguishing attribute of having at least one epitope
that is "cross reactive" with a 158P3D2 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 an
158P3D2 variant also specifically binds to a 158P3D2 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 158P3D2 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.
[0181] Other classes of 158P3D2-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 158P3D2
protein variants or analogs comprise one or more of the 158P3D2
biological motifs described herein or presently known in the art.
Thus, encompassed by the present invention are analogs of 158P3D2
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.
[0182] As discussed herein, embodiments of the claimed invention
include polypeptides containing less than the full amino acid
sequence of a 158P3D2 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 158P3D2 protein shown in
FIG. 2 or FIG. 3.
[0183] Moreover, representative embodiments of the invention
disclosed herein include polypeptides consisting of about amino
acid I to about amino acid 10 of a 158P3D2 protein shown in FIG. 2
or FIG. 3, polypeptides consisting of about amino acid 10 to about
amino acid 20 of a 158P3D2 protein shown in FIG. 2 or FIG. 3,
polypeptides consisting of about amino acid 20 to about amino acid
30 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides
consisting of about amino acid 30 to about amino acid 40 of a
158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting
of about amino acid 40 to about amino acid 50 of a 158P3D2 protein
shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino
acid 50 to about amino acid 60 of a 158P3D2 protein shown in FIG. 2
or FIG. 3, polypeptides consisting of about amino acid 60 to about
amino acid 70 of a 158P3D2 protein shown in FIG. 2 or FIG. 3,
polypeptides consisting of about amino acid 70 to about amino acid
80 of a 158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides
consisting of about amino acid 80 to about amino acid 90 of a
158P3D2 protein shown in FIG. 2 or FIG. 3, polypeptides consisting
of about amino acid 90 to about amino acid 100 of a 158P3D2 protein
shown in FIG. 2 or FIG. 3, etc. throughout the entirety of a
158P3D2 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 158P3D2 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.
[0184] 158P3D2-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
158P3D2-related protein. In one embodiment, nucleic acid molecules
provide a means to generate defined fragments of a 158P3D2 protein
(or variants, homologs or analogs thereof).
[0185] III.A.) Motif-bearing Protein Embodiments
[0186] Additional illustrative embodiments of the invention
disclosed herein include 158P3D2 polypeptides comprising the amino
acid residues of one or more of the biological motifs contained
within a 158P3D2 polypeptide sequence set forth in FIG. 2 or FIG.
3. Various motifs are known in the art, and a protein can be
evaluated for the presence of such motifs by a number of publicly
available Internet sites (see, e.g., URL addresses:
pfam.wustl.edu/; http://searchlauncher.bcm.tmc.edu/seq-search/-
struc-predict.html; psort.ims.u-tokyo.ac.jp/; www.cbs.dtu.dk/;
www.ebi.ac.uk/interpro/scan.html;
www.expasy.ch/tools/scnpsit1.html; Epimatrix.TM. and Epimer.TM.,
Brown University, www.brown.edu/Research/TB-
-HIV_Lab/epimatrix/epimatrix.html; and BIMAS,
bimas.dcrt.nih.gov/.).
[0187] Motif bearing subsequences of all 158P3D2 variant proteins
are set forth and identified in Table XVIII.
[0188] Table XX sets forth several frequently occurring motifs
based on pfam searches (see URL address pfam.wustl.edu/). The
columns of Table XX 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.
[0189] Polypeptides comprising one or more of the 158P3D2 motifs
discussed above are useful in elucidating the specific
characteristics of a malignant phenotype in view of the observation
that the 158P3D2 motifs discussed above are associated with growth
dysregulation and because 158P3D2 is overexpressed in certain
cancers (See, e.g., Table I). Casein kinase II, cAMP and
camp-dependent protein kinase, and Protein Kinase C, for example,
are enzymes known to be associated with the development of the
malignant phenotype (see e.g. Chen et al., Lab Invest., 78(2):
165-174 (1998); Gaiddon et al., Endocrinology 136(10): 4331-4338
(1995); Hall et al., Nucleic Acids Research 24(6): 1119-1126
(1996); Peterziel et al., Oncogene 18(46): 6322-6329 (1999) and
O'Brian, Oncol. Rep. 5(2): 305-309 (1998)). Moreover, both
glycosylation and myristoylation are protein modifications also
associated with cancer and cancer progression (see e.g. Dennis et
al., Biochem. Biophys. Acta 1473(1):21-34 (1999); Raju et al., Exp.
Cell Res. 235(1): 145-154 (1997)). Amidation is another protein
modification also associated with cancer and cancer progression
(see e.g., Treston et al., J. Natl. Cancer Inst. Monogr. (13):
169-175 (1992)).
[0190] In another embodiment, proteins of the invention comprise
one or more of the immunoreactive epitopes identified in accordance
with art-accepted methods, such as the peptides set forth in Tables
V-XIX. CTL epitopes can be determined using specific algorithms to
identify peptides within an 158P3D2 protein that are capable of
optimally binding to specified HLA alleles (e.g., Table IV;
Epimatrix.TM. and Epimer.TM., Brown University, URL
www.brown.edu/Research/TB-HIV_Lab/epimatrix/epimatr- ix.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.
[0191] Also known in the art are principles for creating analogs of
such epitopes in order to modulate immunogenicity. For example, one
begins with an epitope that bears a CTL or HTL motif (see, e.g.,
the HLA Class I and HLA Class II motifs/supermotifs of Table IV).
The epitope is analoged by substituting out an amino acid at one of
the specified positions, and replacing it with another amino acid
specified for that position. For example, one can substitute out a
deleterious residue in favor of any other residue, such as a
preferred residue as defined in Table IV; substitute a
less-preferred residue with a preferred residue as defined in Table
IV; or substitute an originally-occurring preferred residue with
another preferred residue as defined in Table IV. Substitutions can
occur at primary anchor positions or at other positions in a
peptide; see, e.g., Table IV.
[0192] 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 9733602 to Chesnut
et al.; Sette, Immunogenetics 1999 50(3-4): 201-212; Sette et al.,
J. Immunol. 2001 166(2): 1389-1397; Sidney et al., Hum. Immunol.
1997 58(1): 12-20; Kondo et al., Immunogenetics 1997 45(4):
249-258; Sidney et al., J. Immunol. 1996 157(8): 3480-90; and Falk
et al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3
(1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et
al., J. Immunol. 152:163-75 (1994)); Kast et al., 1994 152(8):
3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3): 266-278;
Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633;
Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivanet 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.
[0193] Related embodiments of the inventions include polypeptides
comprising combinations of the different motifs set forth in Table
XXI, and/or, one or more of the predicted CTL epitopes of Table V
through Table XIX, 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 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.
[0194] 158P3D2-related proteins are embodied in many forms,
preferably in isolated form. A purified 158P3D2 protein molecule
will be substantially free of other proteins or molecules that
impair the binding of 158P3D2 to antibody, T cell or other ligand.
The nature and degree of isolation and purification will depend on
the intended use. Embodiments of a 158P3D2-related proteins include
purified 158P3D2-related proteins and functional, soluble
158P3D2-related proteins. In one embodiment, a functional, soluble
158P3D2 protein or fragment thereof retains the ability to be bound
by antibody, T cell or other ligand.
[0195] The invention also provides 158P3D2 proteins comprising
biologically active fragments of a 158P3D2 amino acid sequence
shown in FIG. 2 or FIG. 3. Such proteins exhibit properties of the
starting 158P3D2 protein, such as the ability to elicit the
generation of antibodies that specifically bind an epitope
associated with the starting 158P3D2 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.
[0196] 158P3D2-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 on the
basis of immunogenicity. Fragments that contain such structures are
particularly useful in generating subunit-specific anti-158P3D2
antibodies, or T cells or in identifying cellular factors that bind
to 158P3D2. For example, hydrophilicity profiles can be generated,
and immunogenic peptide fragments identified, using the method of
Hopp, T. P. and Woods, K. R., 1981, Proc. Natl. Acad. Sci. U.S.A.
78:3824-3828. Hydropathicity profiles can be generated, and
immunogenic peptide fragments identified, using the method of Kyte,
J. and Doolittle, R. F., 1982, J. Mol. Biol. 157:105-132. Percent
(%) Accessible Residues profiles can be generated, and immunogenic
peptide fragments identified, using the method of Janin J., 1979,
Nature 277:491-492. Average Flexibility profiles can be generated,
and immunogenic peptide fragments identified, using the method of
Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res.
32:242-255. Beta-turn profiles can be generated, and immunogenic
peptide fragments identified, using the method of Deleage, G., Roux
B., 1987, Protein Engineering 1:289-294.
[0197] CTL epitopes can be determined using specific algorithms to
identify peptides within an 158P3D2 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 (www.brown.edu/Research/TB-HIV
Lab/epimatrix/epimatrix.html); and BIMAS, URL bimas.dcrt.nih.gov/).
Illustrating this, peptide epitopes from 158P3D2 that are presented
in the context of human MHC class I molecules HLA-A1, A2, A3, A11,
A24, B7 and B35 were predicted (Tables V-XIX). Specifically, the
complete amino acid sequence of the 158P3D2 protein and relevant
portions of other variants, i.e., for HLA Class I predictions 9
flanking residues on either side of a point mutation, and for HLA
Class II predictions 14 flanking residues on either side of a point
mutation, were entered into the HLA Peptide Motif Search algorithm
found in the Bioinformatics and Molecular Analysis Section (BIMAS)
web site listed above; for HLA Class II the site SYFPEITHI at URL
syfpeithi.bmi-heidelber- g.com/ was used.
[0198] 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 158P3D2 predicted binding peptides are
shown in Tables V-XIX herein. In Tables V-XIX, the top ranking
candidates, 9-mers, 10-mers and 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.
[0199] 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.
[0200] It is to be appreciated that every epitope predicted by the
BIMAS site, Epimer.TM. and Epimatrix.TM. sites, or specified by the
HLA class I or class II motifs available in the art or which become
part of the art such as set forth in Table IV (or determined using
World Wide Web site URL syfpeithi.bmi-heidelberg.com/, or BIMAS,
bimas.dcrt.nih.gov/) are to be "applied" to a 158P3D2 protein in
accordance with the invention. As used in this context "applied"
means that a 158P3D2 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 158P3D2 protein
of 8, 9, 10, or II 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.
[0201] III.B.) Expression of 158P3D2-Related Proteins
[0202] In an embodiment described in the examples that follow,
158P3D2 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 158P3D2 with a C-terminal
6.times.His and MYC tag (pcDNA3.1/mycHIS, Invitrogen or Tag5,
GenHunter Corporation, Nashville Tenn.). The Tag5 vector provides
an IgGK secretion signal that can be used to facilitate the
production of a secreted 158P3D2 protein in transfected cells. The
secreted HIS-tagged 158P3D2 in the culture media can be purified,
e.g., using a nickel column using standard techniques.
[0203] III.C.) Modifications of 158P3D2-Related Proteins
[0204] Modifications of 158P3D2-related proteins such as covalent
modifications are included within the scope of this invention. One
type of covalent modification includes reacting targeted amino acid
residues of a 158P3D2 polypeptide with an organic derivatizing
agent that is capable of reacting with selected side chains or the
N- or C-terminal residues of a 158P3D2 protein. Another type of
covalent modification of a 158P3D2 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 158P3D2 comprises linking a 158P3D2 polypeptide to
one of a variety of nonproteinaceous polymers, e.g., polyethylene
glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the
manner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144;
4,670,417; 4,791,192 or 4,179,337.
[0205] The 158P3D2-related proteins of the present invention can
also be modified to form a chimeric molecule comprising 158P3D2
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 158P3D2 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 158P3D2. A chimeric
molecule can comprise a fusion of a 158P3D2-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 158P3D2 protein. In an alternative
embodiment, the chimeric molecule can comprise a fusion of a
158P3D2-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 158P3D2 polypeptide in
place of at least one variable region within an Ig molecule. In a
preferred embodiment, the immunoglobulin fusion includes the hinge,
CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgG1
molecule. For the production of immunoglobulin fusions see, e.g.,
U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
[0206] III.D.) Uses of 158P3D2-Related Proteins
[0207] The proteins of the invention have a number of different
specific uses. As 158P3D2 is highly expressed in prostate and other
cancers, 158P3D2-related proteins are used in methods that assess
the status of 158P3D2 gene products in normal versus cancerous
tissues, thereby elucidating the malignant phenotype. Typically,
polypeptides from specific regions of a 158P3D2 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 158P3D2-related proteins comprising the amino
acid residues of one or more of the biological motifs contained
within a 1 58P3D2 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,
158P3D2-related proteins that contain the amino acid residues of
one or more of the biological motifs in a 158P3D2 protein are used
to screen for factors that interact with that region of
158P3D2.
[0208] 158P3D2 protein fragments/subsequences are particularly
useful in generating and characterizing domain-specific antibodies
(e.g., antibodies recognizing an extracellular or intracellular
epitope of an 158P3D2 protein), for identifying agents or cellular
factors that bind to 158P3D2 or a particular structural domain
thereof, and in various therapeutic and diagnostic contexts,
including but not limited to diagnostic assays, cancer vaccines and
methods of preparing such vaccines.
[0209] Proteins encoded by the 158P3D2 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 an 158P3D2 gene product Antibodies raised against an
158P3D2 protein or fragment thereof are useful in diagnostic and
prognostic assays, and imaging methodologies in the management of
human cancers characterized by expression of 158P3D2 protein, such
as those listed in Table I. Such antibodies can be expressed
intracellularly and used in methods of treating patients with such
cancers. 158P3D2-related nucleic acids or proteins are also used in
generating HTL or CTL responses.
[0210] Various immunological assays useful for the detection of
158P3D2 proteins are used, including but not limited to various
types of radioimmunoassays, enzyme-linked immunosorbent assays
(ELISA), enzyme-linked immunofluorescent assays (ELIFA),
immunocytochemical methods, and the like. Antibodies can be labeled
and used as immunological imaging reagents capable of detecting
158P3D2-expressing cells (e.g., in radioscintigraphic imaging
methods). 158P3D2 proteins are also particularly useful in
generating cancer vaccines, as further described herein.
[0211] IV.) 158P3D2 Antibodies
[0212] Another aspect of the invention provides antibodies that
bind to 158P3D2-related proteins. Preferred antibodies specifically
bind to a 158P3D2-related protein and do not bind (or bind weakly)
to peptides or proteins that are not 158P3D2-related proteins. For
example, antibodies that bind 158P3D2 can bind 158P3D2-related
proteins such as the homologs or analogs thereof.
[0213] 158P3D2 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 158P3D2 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 158P3D2 is involved, such as
advanced or metastatic prostate cancers.
[0214] The invention also provides various immunological assays
useful for the detection and quantification of 158P3D2 and mutant
158P3D2-related proteins. Such assays can comprise one or more
158P3D2 antibodies capable of recognizing and binding a
158P3D2-related protein, as appropriate. These assays are performed
within various immunological assay formats well known in the art,
including but not limited to various types of radioimmunoassays,
enzyme-linked immunosorbent assays (ELISA), enzyme-linked
immunofluorescent assays (ELIFA), and the like.
[0215] Immunological non-antibody assays of the invention also
comprise T cell immunogenicity assays (inhibitory or stimulatory)
as well as major histocompatibility complex (MHC) binding
assays.
[0216] In addition, immunological imaging methods capable of
detecting prostate cancer and other cancers expressing 158P3D2 are
also provided by the invention, including but not limited to
radioscintigraphic imaging methods using labeled 158P3D2
antibodies. Such assays are clinically useful in the detection,
monitoring, and prognosis of 158P3D2 expressing cancers such as
prostate cancer.
[0217] 158P3D2 antibodies are also used in methods for purifying a
158P3D2-related protein and for isolating 158P3D2 homologues and
related molecules. For example, a method of purifying a
158P3D2-related protein comprises incubating an 158P3D2 antibody,
which has been coupled to a solid matrix, with a lysate or other
solution containing a 158P3D2-related protein under conditions that
permit the 158P3D2 antibody to bind to the 158P3D2-related protein;
washing the solid matrix to eliminate impurities; and eluting the
158P3D2-related protein from the coupled antibody. Other uses of
158P3D2 antibodies in accordance with the invention include
generating anti-idiotypic antibodies that mimic a 158P3D2
protein.
[0218] 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 158P3D2-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 158P3D2 can also be used, such as a
158P3D2 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 158P3D2-related
protein is synthesized and used as an immunogen.
[0219] In addition, naked DNA immunization techniques known in the
art are used (with or without purified 158P3D2-related protein or
158P3D2 expressing cells) to generate an immune response to the
encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev.
Immunol. 15: 617-648).
[0220] The amino acid sequence of a 158P3D2 protein as shown in
FIG. 2 or FIG. 3 can be analyzed to select specific regions of the
158P3D2 protein for generating antibodies. For example,
hydrophobicity and hydrophilicity analyses of a 158P3D2 amino acid
sequence are used to identify hydrophilic regions in the 158P3D2
structure. Regions of a 158P3D2 protein that show immunogenic
structure, as well as other regions and domains, can readily be
identified using various other methods known in the art, such as
Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg,
Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles
can be generated using the method of Hopp, T. P. and Woods, K. R.,
1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity
profiles can be generated using the method of Kyte, J. and
Doolittle, R. F., 1982, J. Mol. Biol. 157:105-132. Percent (%)
Accessible Residues profiles can be generated using the method of
Janin J., 1979, Nature 277:491-492. Average Flexibility profiles
can be generated using the method of Bhaskaran R., Ponnuswamy P.
K., 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-turn profiles
can be generated using the method of Deleage, G., Roux B., 1987,
Protein Engineering 1:289-294. Thus, each region identified by any
of these programs or methods is within the scope of the present
invention. Methods for the generation of 158P3D2 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
158P3D2 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.
[0221] 158P3D2 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
158P3D2-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.
[0222] The antibodies or fragments of the invention can also be
produced, by recombinant means. Regions that bind specifically to
the desired regions of a 158P3D2 protein can also be produced in
the context of chimeric or complementarity determining region (CDR)
grafted antibodies of multiple species origin. Humanized or human
158P3D2 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 a., 1988, Nature 332: 323-327; Verhoeyen et at, 1988,
Science 239: 1534-1536). See also, Carter et at, 1993, Proc. Natl.
Acad. Sci. USA 89: 4285 and Sims et at, 1993, J. Immunol. 151:
2296.
[0223] Methods for producing fully human monoclonal antibodies
include phage display and transgenic methods (for review, see
Vaughan et a., 1998, Nature Biotechnology 16: 535-539). Fully human
158P3D2 monoclonal antibodies can be generated using cloning
technologies employing large human Ig gene combinatorial libraries
(i.e., phage display) (Griffiths and Hoogenboom, Building an in
vitro immune system: human antibodies from phage display libraries.
In: Protein Engineering of Antibody Molecules for Prophylactic and
Therapeutic Applications in Man, Clark, M. (Ed.), Nottingham
Academic, pp 45-64 (1993); Burton and Barbas, Human Antibodies from
combinatorial libraries. Id., pp 65-82). Fully human 158P3D2
monoclonal antibodies can also be produced using transgenic mice
engineered to contain human immunoglobulin gene loci as described
in PCT Patent Application WO 98/24893, Kucherlapati and Jakobovits
et al., published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp.
Opin. Invest. Drugs 7(4): 607-614; U.S. Pat. No. 6,162,963 issued
Dec. 19, 2000; U.S. Pat. No. 6,150,584 issued Nov. 12, 2000; and,
U.S. Pat. No. 6,114,598 issued Sep. 5, 2000). This method avoids
the in vitro manipulation required with phage display technology
and efficiently produces high affinity authentic human
antibodies.
[0224] Reactivity of 158P3D2 antibodies with an 158P3D2-related
protein can be established by a number of well known means,
including Western blot, immunoprecipitation, ELISA, and FACS
analyses using, as appropriate, 158P3D2-related proteins,
158P3D2-expressing cells or extracts thereof. A 158P3D2 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 158P3D2 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).
[0225] V.) 158P3D2 Cellular Immune Responses
[0226] 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.
[0227] 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 a., J. Immunol.
160:3363, 1998; Rammensee, et a., Immunogenetics 41:178, 1995;
Rammensee et al., SYFPEITHI, access via World Wide Web at URL
syfpeithi.bmi-heidelberg.com/; Sette, A. and Sidney, J. Curr. Opin.
Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13,
1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992;
Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et
al., Cell 74:929-937, 1993; Kondo et al., J. Immunol.
155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490,
1996; Sidney et al., Human Immunol. 45:79-93, 1996; Sette, A. and
Sidney, J. Immunogenetics November 1999; 50(3-4):201-12,
Review).
[0228] 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; Stem 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.)
[0229] 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).
[0230] 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.
[0231] Various strategies can be utilized to evaluate cellular
immunogenicity, including:
[0232] 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.
[0233] 2) Immunization of HLA transgenic mice (see, e.g.,
Wentworth, P. A. et al., J. Immunol. 26:97, 1996; Wentworth, P. A.
et al., Int. Immunol. 8:651, 1996; Alexander, J. et al., J.
Immunol. 159:4753, 1997). For example, in such methods peptides in
incomplete Freund's adjuvant are administered subcutaneously to HLA
transgenic mice. Several weeks following immunization, splenocytes
are removed and cultured in vitro in the presence of test peptide
for approximately one week. Peptide-specific T cells are detected
using, e.g., a .sup.51Cr-release assay involving peptide sensitized
target cells and target cells expressing endogenously generated
antigen.
[0234] 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.
[0235] VI.) 158P3D2 Transgenic Animals
[0236] Nucleic acids that encode a 158P3D2-related protein can also
be used to generate either transgenic animals or "knock out"
animals that, in turn, are useful in the development and screening
of therapeutically useful reagents. In accordance with established
techniques, cDNA encoding 158P3D2 can be used to clone genomic DNA
that encodes 158P3D2. The cloned genomic sequences can then be used
to generate transgenic animals containing cells that express DNA
that encode 158P3D2. Methods for generating transgenic animals,
particularly animals such as mice or rats, have become conventional
in the art and are described, for example, in U.S. Pat. No.
4,736,866 issued Apr. 12, 1988, and U.S. Pat. No. 4,870,009 issued
Sep. 26, 1989. Typically, particular cells would be targeted for
158P3D2 transgene incorporation with tissue-specific enhancers.
[0237] Transgenic animals that include a copy of a transgene
encoding 158P3D2 can be used to examine the effect of increased
expression of DNA that encodes 158P3D2. 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.
[0238] Alternatively, non-human homologues of 158P3D2 can be used
to construct a 158P3D2 "knock out" animal that has a defective or
altered gene encoding 158P3D2 as a result of homologous
recombination between the endogenous gene encoding 158P3D2 and
altered genomic DNA encoding 158P3D2 introduced into an embryonic
cell of the animal. For example, cDNA that encodes 158P3D2 can be
used to clone genomic DNA encoding 158P3D2 in accordance with
established techniques. A portion of the genomic DNA encoding
158P3D2 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 158P3D2
polypeptide.
[0239] VII.) Methods for the Detection of 158P3D2
[0240] Another aspect of the present invention relates to methods
for detecting 158P3D2 polynucleotides and 158P3D2-related proteins,
as well as methods for identifying a cell that expresses 158P3D2.
The expression profile of 158P3D2 makes it a diagnostic marker for
metastasized disease. Accordingly, the status of 158P3D2 gene
products provides information useful for predicting a variety of
factors including susceptibility to advanced stage disease, rate of
progression, and/or tumor aggressiveness. As discussed in detail
herein, the status of 158P3D2 gene products in patient samples can
be analyzed by a variety protocols that are well known in the art
including immunohistochemical analysis, the variety of Northern
blotting techniques including in situ hybridization, RT-PCR
analysis (for example on laser capture micro-dissected samples),
Western blot analysis and tissue array analysis.
[0241] More particularly, the invention provides assays for the
detection of 158P3D2 polynucleotides in a biological sample, such
as serum, bone, prostate, and other tissues, urine, semen, cell
preparations, and the like. Detectable 158P3D2 polynucleotides
include, for example, a 158P3D2 gene or fragment thereof, 158P3D2
mRNA, alternative splice variant 158P3D2 mRNAs, and recombinant DNA
or RNA molecules that contain a 158P3D2 polynucleotide. A number of
methods for amplifying and/or detecting the presence of 158P3D2
polynucleotides are well known in the art and can be employed in
the practice of this aspect of the invention.
[0242] In one embodiment, a method for detecting an 158P3D2 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 an 158P3D2 polynucleotides as sense and
antisense primers to amplify 158P3D2 cDNAs therein; and detecting
the presence of the amplified 158P3D2 cDNA. Optionally, the
sequence of the amplified 158P3D2 cDNA can be determined.
[0243] In another embodiment, a method of detecting a 158P3D2 gene
in a biological sample comprises first isolating genomic DNA from
the sample; amplifying the isolated genomic DNA using 158P3D2
polynucleotides as sense and antisense primers; and detecting the
presence of the amplified 158P3D2 gene. Any number of appropriate
sense and antisense probe combinations can be designed from a
158P3D2 nucleotide sequence (see, e.g., FIG. 2) and used for this
purpose.
[0244] The invention also provides assays for detecting the
presence of an 158P3D2 protein in a tissue or other biological
sample such as serum, semen, bone, prostate, urine, cell
preparations, and the like. Methods for detecting a 158P3D2-related
protein are also well known and include, for example,
immunoprecipitation, immunohistochemical analysis, Western blot
analysis, molecular binding assays, ELISA, ELIFA and the like. For
example, a method of detecting the presence of a 158P3D2-related
protein in a biological sample comprises first contacting the
sample with a 158P3D2 antibody, a 158P3D2-reactive fragment
thereof, or a recombinant protein containing an antigen binding
region of a 158P3D2 antibody; and then detecting the binding of
158P3D2-related protein in the sample.
[0245] Methods for identifying a cell that expresses 158P3D2 are
also within the scope of the invention. In one embodiment, an assay
for identifying a cell that expresses a 158P3D2 gene comprises
detecting the presence of 158P3D2 mRNA in the cell. Methods for the
detection of particular mRNAs in cells are well known and include,
for example, hybridization assays using complementary DNA probes
(such as in situ hybridization using labeled 158P3D2 riboprobes,
Northern blot and related techniques) and various nucleic acid
amplification assays (such as RT-PCR using complementary primers
specific for 158P3D2, 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 158P3D2 gene comprises detecting the presence of
158P3D2-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 158P3D2-related proteins
and cells that express 158P3D2-related proteins.
[0246] 158P3D2 expression analysis is also useful as a tool for
identifying and evaluating agents that modulate 158P3D2 gene
expression. For example, 158P3D2 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 158P3D2 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 158P3D2 expression by RT-PCR, nucleic acid hybridization
or antibody binding.
[0247] VIII.) Methods for Monitoring the Status of 158P3D2-Related
Genes and Their Products
[0248] 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 158P3D2 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 158P3D2 in a biological
sample of interest can be compared, for example, to the status of
158P3D2 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 158P3D2 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 158P3D2 status in a sample.
[0249] 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 158P3D2
expressing cells) as well as the level, and biological activity of
expressed gene products (such as 158P3D2 mRNA, polynucleotides and
polypeptides). Typically, an alteration in the status of 158P3D2
comprises a change in the location of 158P3D2 and/or 158P3D2
expressing cells and/or an increase in 158P3D2 mRNA and/or protein
expression.
[0250] 158P3D2 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 158P3D2 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 158P3D2 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 158P3D2 gene), Northern analysis and/or PCR analysis of 158P3D2
mRNA (to examine, for example alterations in the polynucleotide
sequences or expression levels of 158P3D2 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
158P3D2 proteins and/or associations of 158P3D2 proteins with
polypeptide binding partners). Detectable 158P3D2 polynucleotides
include, for example, a 158P3D2 gene or fragment thereof, 158P3D2
mRNA, alternative splice variants, 158P3D2 mRNAs, and recombinant
DNA or RNA molecules containing a 158P3D2 polynucleotide.
[0251] The expression profile of 158P3D2 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 158P3D2 provides information
useful for predicting susceptibility to particular disease stages,
progression, and/or tumor aggressiveness. The invention provides
methods and assays for determining 158P3D2 status and diagnosing
cancers that express 158P3D2, such as cancers of the tissues listed
in Table I. For example, because 158P3D2 mRNA is so highly
expressed in prostate and other cancers relative to normal prostate
tissue, assays that evaluate the levels of 158P3D2 mRNA transcripts
or proteins in a biological sample can be used to diagnose a
disease associated with 158P3D2 dysregulation, and can provide
prognostic information useful in defining appropriate therapeutic
options.
[0252] The expression status of 158P3D2 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 158P3D2 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.
[0253] As described above, the status of 158P3D2 in a biological
sample can be examined by a number of well-known procedures in the
art. For example, the status of 158P3D2 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 158P3D2
expressing cells (e.g. those that express 158P3D2 mRNAs or
proteins). This examination can provide evidence of dysregulated
cellular growth, for example, when 158P3D2-expressing cells are
found in a biological sample that does not normally contain such
cells (such as a lymph node), because such alterations in the
status of 158P3D2 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
August 1995 154(2 Pt 1):474-8).
[0254] In one aspect, the invention provides methods for monitoring
158P3D2 gene products by determining the status of 158P3D2 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 158P3D2 gene products in a corresponding normal
sample. The presence of aberrant 158P3D2 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.
[0255] In another aspect, the invention provides assays useful in
determining the presence of cancer in an individual, comprising
detecting a significant increase in 158P3D2 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
158P3D2 mRNA can, for example, be evaluated in tissue samples
including but not limited to those listed in Table I. The presence
of significant 158P3D2 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 158P3D2 mRNA
or express it at lower levels.
[0256] In a related embodiment, 158P3D2 status is determined at the
protein level rather than at the nucleic acid level. For example,
such a method comprises determining the level of 158P3D2 protein
expressed by cells in a test tissue sample and comparing the level
so determined to the level of 158P3D2 expressed in a corresponding
normal sample. In one embodiment, the presence of 158P3D2 protein
is evaluated, for example, using immunohistochemical methods.
158P3D2 antibodies or binding partners capable of detecting 158P3D2
protein expression are used in a variety of assay formats well
known in the art for this purpose.
[0257] In a further embodiment, one can evaluate the status of
158P3D2 nucleotide and amino acid sequences in a biological sample
in order to identify perturbations in the structure of these
molecules. These perturbations can include insertions, deletions,
substitutions and the like. Such evaluations are useful because
perturbations in the nucleotide and amino acid sequences are
observed in a large number of proteins associated with a growth
dysregulated phenotype (see, e.g., Marrogi et al., 1999, J. Cutan.
Pathol. 26(8):369-378). For example, a mutation in the sequence of
158P3D2 may be indicative of the presence or promotion of a tumor.
Such assays therefore have diagnostic and predictive value where a
mutation in 158P3D2 indicates a potential loss of function or
increase in tumor growth.
[0258] 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 158P3D2 gene products are observed by the Northern,
Southern, Western, PCR and DNA sequencing protocols discussed
herein. In addition, other methods for observing perturbations in
nucleotide and amino acid sequences such as single strand
conformation polymorphism analysis are well known in the art (see,
e.g., U.S. Pat. No. 5,382,510 issued Sep. 7, 1999, and U.S. Pat.
No. 5,952,170 issued Jan. 17, 1995).
[0259] Additionally, one can examine the methylation status of a
158P3D2 gene in a biological sample. Aberrant demethylation and/or
hypermethylation of CpG islands in gene 5' regulatory regions
frequently occurs in immortalized and transformed cells, and can
result in altered expression of various genes. For example,
promoter hypermethylation of the pi-class glutathione S-transferase
(a protein expressed in normal prostate but not expressed in>90%
of prostate carcinomas) appears to permanently silence
transcription of this gene and is the most frequently detected
genomic alteration in prostate carcinomas (De Marzo et al., Am. J.
Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration is
present in at least 70% of cases of high-grade prostatic
intraepithelial neoplasia (PIN) (Brooks et al., Cancer Epidemiol.
Biomarkers Prev., 1998, 7:531-536). In another example, expression
of the LAGE-I tumor specific gene (which is not expressed in normal
prostate but is expressed in 25-50% of prostate cancers) is induced
by deoxy-azacytidine in lymphoblastoid cells, suggesting that
tumoral expression is due to demethylation (Lethe et al., Int. J.
Cancer 76(6): 903-908 (1998)). A variety of assays for examining
methylation status of a gene are well known in the art. For
example, one can utilize, in Southern hybridization approaches,
methylation-sensitive restriction enzymes that cannot cleave
sequences that contain methylated CpG sites to assess the
methylation status of CpG islands. In addition, MSP (methylation
specific PCR) can rapidly profile the methylation status of all the
CpG sites present in a CpG island of a given gene. This procedure
involves initial modification of DNA by sodium bisulfite (which
will convert all unmethylated cytosines to uracil) followed by
amplification using primers specific for methylated versus
unmethylated DNA. Protocols involving methylation interference can
also be found for example in Current Protocols In Molecular
Biology, Unit 12, Frederick M. Ausubel et al. eds., 1995.
[0260] Gene amplification is an additional method for assessing the
status of 158P3D2. 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.
[0261] 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 158P3D2 expression.
The presence of RT-PCR amplifiable 158P3D2 mRNA provides an
indication of the presence of cancer. RT-PCR assays are well known
in the art. RT-PCR detection assays for tumor cells in peripheral
blood are currently being evaluated for use in the diagnosis and
management of a number of human solid tumors. In the prostate
cancer field, these include RT-PCR assays for the detection of
cells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res.
25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000;
Heston et al., 1995, Clin. Chem. 41:1687-1688).
[0262] 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 158P3D2 mRNA or 158P3D2 protein in a tissue
sample, its presence indicating susceptibility to cancer, wherein
the degree of 158P3D2 mRNA expression correlates to the degree of
susceptibility. In a specific embodiment, the presence of 158P3D2
in prostate or other tissue is examined, with the presence of
158P3D2 in the sample providing an indication of prostate cancer
susceptibility (or the emergence or existence of a prostate tumor).
Similarly, one can evaluate the integrity 158P3D2 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 158P3D2 gene products in the sample is
an indication of cancer susceptibility (or the emergence or
existence of a tumor).
[0263] 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
158P3D2 mRNA or 158P3D2 protein expressed by tumor cells, comparing
the level so determined to the level of 158P3D2 mRNA or 158P3D2
protein expressed in a corresponding normal tissue taken from the
same individual or a normal tissue reference sample, wherein the
degree of 158P3D2 mRNA or 158P3D2 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 158P3D2 is
expressed in the tumor cells, with higher expression levels
indicating more aggressive tumors. Another embodiment is the
evaluation of the integrity of 158P3D2 nucleotide and amino acid
sequences in a biological sample, in order to identify
perturbations in the structure of these molecules such as
insertions, deletions, substitutions and the like. The presence of
one or more perturbations indicates more aggressive tumors.
[0264] 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 158P3D2 mRNA or 158P3D2 protein expressed by cells in a
sample of the tumor, comparing the level so determined to the level
of 158P3D2 mRNA or 158P3D2 protein expressed in an equivalent
tissue sample taken from the same individual at a different time,
wherein the degree of 158P3D2 mRNA or 158P3D2 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 158P3D2 expression in the tumor
cells over time, where increased expression over time indicates a
progression of the cancer. Also, one can evaluate the integrity
158P3D2 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.
[0265] 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 158P3D2 gene and 158P3D2 gene products (or
perturbations in 158P3D2 gene and 158P3D2 gene products) and a
factor that is associated with malignancy, as a means for
diagnosing and prognosticating the status of a tissue sample. A
wide variety of factors associated with malignancy can be utilized,
such as the expression of genes associated with malignancy (e.g.
PSA, PSCA and PSM expression for prostate cancer etc.) as well as
gross cytological observations (see, e.g., Bocking et al., 1984,
Anal. Quant. Cytol. 6(2):74-88; Epstein, 1995, Hum. Pathol.
26(2):223-9; Thorson et al., 1998, Mod. Pathol. 11(6):543-5 1;
Baisden et al., 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods
for observing a coincidence between the expression of 158P3D2 gene
and 158P3D2 gene products (or perturbations in 158P3D2 gene and
158P3D2 gene products) and another factor that is associated with
malignancy are useful, for example, because the presence of a set
of specific factors that coincide with disease provides information
crucial for diagnosing and prognosticating the status of a tissue
sample.
[0266] In one embodiment, methods for observing a coincidence
between the expression of 158P3D2 gene and 158P3D2 gene products
(or perturbations in 158P3D2 gene and 158P3D2 gene products) and
another factor associated with malignancy entails detecting the
overexpression of 158P3D2 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
158P3D2 mRNA or protein and PSA mRNA or protein overexpression (or
PSCA or PSM expression). In a specific embodiment, the expression
of 158P3D2 and PSA mRNA in prostate tissue is examined, where the
coincidence of 158P3D2 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.
[0267] Methods for detecting and quantifying the expression of
158P3D2 mRNA or protein are described herein, and standard nucleic
acid and protein detection and quantification technologies are well
known in the art. Standard methods for the detection and
quantification of 158P3D2 mRNA include in situ hybridization using
labeled 158P3D2 riboprobes, Northern blot and related techniques
using 158P3D2 polynucleotide probes, RT-PCR analysis using primers
specific for 158P3D2, 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 158P3D2 mRNA expression. Any number of primers
capable of amplifying 158P3D2 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
158P3D2 protein can be used in an immunohistochemical assay of
biopsied tissue.
[0268] IX.) Identification of Molecules that Interact with
158P3D2
[0269] The 158P3D2 protein and nucleic acid sequences disclosed
herein allow a skilled artisan to identify proteins, small
molecules and other agents that interact with 158P3D2, as well as
pathways activated by -158P3D2 via any one of a variety of art
accepted protocols. For example, one can utilize one of the
so-called interaction trap systems (also referred to as the
"two-hybrid assay"). In such systems, molecules interact and
reconstitute a transcription factor which directs expression of a
reporter gene, whereupon the expression of the reporter gene is
assayed. Other systems identify protein-protein interactions in
vivo through reconstitution of a eukaryotic transcriptional
activator, see, e.g., U.S. Pat. No. 5,955,280 issued Sep. 21, 1999,
U.S. Pat. No. 5,925,523 issued Jul. 20, 1999, U.S. Pat. No.
5,846,722 issued Dec. 8, 1998 and U.S. Pat. No. 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).
[0270] Alternatively one can screen peptide libraries to identify
molecules that interact with 158P3D2 protein sequences. In such
methods, peptides that bind to 158P3D2 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 158P3D2 protein(s).
[0271] 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 158P3D2 protein sequences are disclosed for example
in U.S. Pat. No. 5,723,286 issued Mar. 3, 1998 and U.S. Pat. No.
5,733,731 issued Mar. 31, 1998.
[0272] Alternatively, cell lines that express 158P3D2 are used to
identify protein-protein interactions mediated by 158P3D2. Such
interactions can be examined using immunoprecipitation techniques
(see, e.g., Hamilton B. J., et al. Biochem. Biophys. Res. Commun.
1999, 261:646-51). 158P3D2 protein can be immunoprecipitated from
158P3D2-expressing cell lines using anti-158P3D2 antibodies.
Alternatively, antibodies against His-tag can be used in a cell
line engineered to express fusions of 158P3D2 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.
[0273] Small molecules and ligands that interact with 158P3D2 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 158P3D2'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
158P3D2-related ion channel, protein pump, or cell communication
functions are identified and used to treat patients that have a
cancer that expresses 158P3D2 (see, e.g., Hille, B., Ionic Channels
of Excitable Membranes 2.sup.nd Ed., Sinauer Assoc., Sunderland,
Mass., 1992). Moreover, ligands that regulate 158P3D2 function can
be identified based on their ability to bind 158P3D2 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 158P3D2 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
158P3D2.
[0274] An embodiment of this invention comprises a method of
screening for a molecule that interacts with an 158P3D2 amino acid
sequence shown in FIG. 2 or FIG. 3, comprising the steps of
contacting a population of molecules with a 158P3D2 amino acid
sequence, allowing the population of molecules and the 158P3D2
amino acid sequence to interact under conditions that facilitate an
interaction, determining the presence of a molecule that interacts
with the 158P3D2 amino acid sequence, and then separating molecules
that do not interact with the 158P3D2 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 158P3D2 amino acid sequence. The identified
molecule can be used to modulate a function performed by 158P3D2.
In a preferred embodiment, the 158P3D2 amino acid sequence is
contacted with a library of peptides.
[0275] X.) Therapeutic Methods and Compositions
[0276] The identification of 158P3D2 as a protein that is normally
expressed in a restricted set of tissues, but which is also
expressed in prostate and other cancers, opens a number of
therapeutic approaches to the treatment of such cancers. As
contemplated herein, 158P3D2 functions as a transcription factor
involved in activating tumor-promoting genes or repressing genes
that block tumorigenesis.
[0277] Accordingly, therapeutic approaches that inhibit the
activity of a 158P3D2 protein are useful for patients suffering
from a cancer that expresses 158P3D2. These therapeutic approaches
generally fall into two classes. One class comprises various
methods for inhibiting the binding or association of a 158P3D2
protein with its binding partner or with other proteins. Another
class comprises a variety of methods for inhibiting the
transcription of a 158P3D2 gene or translation of 158P3D2 mRNA.
[0278] X.A.) Anti-Cancer Vaccines
[0279] The invention provides cancer vaccines comprising a
158P3D2-related protein or 158P3D2-related nucleic acid. In view of
the expression of 158P3D2, cancer vaccines prevent and/or treat
158P3D2-expressing cancers with minimal or no effects on non-target
tissues. The use of a tumor antigen in a vaccine that generates
humoral and/or cell-mediated immune responses as anti-cancer
therapy is well known in the art and has been employed in prostate
cancer using human PSMA and rodent PAP immunogens (Hodge et al.,
1995, Int. J. Cancer 63:231-237; Fong et al., 1997, J. Immunol.
159:3113-3117).
[0280] Such methods can be readily practiced by employing a
158P3D2-related protein, or an 158P3D2-encoding nucleic acid
molecule and recombinant vectors capable of expressing and
presenting the 158P3D2 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 February 1999 31(1):66-78; Maruyama et al., Cancer
Immunol Immunother June 2000 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 158P3D2 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
158P3D2 immunogen contains a biological motif, see e.g., Tables
V-XIX, or a peptide of a size range from 158P3D2 indicated in FIG.
5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.
[0281] The entire 158P3D2 protein, immunogenic regions or epitopes
thereof can be combined and delivered by various means. Such
vaccine compositions can include, for example, lipopeptides (e.g.,
Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), peptide
compositions encapsulated in poly(DL-lactide-co-glycolide) ("PLG")
microspheres (see, e.g., Eldridge, et al., Molec. Immunol.
28:287-294, 1991: Alonso et al., Vaccine 12:299-306, 1994; Jones et
al., Vaccine 13:675-681, 1995), peptide compositions contained in
immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al.,
Nature 344:873-875, 1990; Hu et al., Clin Exp Immunol. 113:235-243,
1998), multiple antigen peptide systems (MAPs) (see e.g., Tam, J.
P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J. P.,
J. Immunol. Methods 196:17-32, 1996), peptides formulated as
multivalent peptides; peptides for use in ballistic delivery
systems, typically crystallized peptides, viral delivery vectors
(Perkus, M. E. et al., In: Concepts in vaccine development,
Kaufmann, S. H. E., ed., p. 379, 1996; Chakrabarti, S. et al.,
Nature 320:535, 1986; Hu, S. L. et al., Nature 320:537, 1986;
Kieny, M. -P. et al., AIDS Bio/Technology 4:790, 1986; Top, F. H.
et al., J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al.,
Virology 175:535, 1990), particles of viral or synthetic origin
(e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996;
Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr.
et al., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel,
F. R., and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R.
K. et al., Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J.
Immunol. 148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996),
or, naked or particle absorbed cDNA (Ulmer, J. B. et al., Science
259:1745, 1993; Robinson, H. L., Hunt, L. A., and Webster, R. G.,
Vaccine 11:957, 1993; Shiver, J. W. et al., In: Concepts in vaccine
development, Kaufmann, S. H. E., ed., p. 423, 1996; Cease, K. B.,
and Berzofsky, J. A., Annu. Rev. Immunol. 12:923, 1994 and
Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993). Toxin-targeted
delivery technologies, also known as receptor mediated targeting,
such as those of Avant Immunotherapeutics, Inc. (Needham, Mass.)
may also be used.
[0282] In patients with 158P3D2-associated cancer, the vaccine
compositions of the invention can also be used in conjunction with
other treatments used for cancer, e.g., surgery, chemotherapy, drug
therapies, radiation therapies, etc. including use in combination
with immune adjuvants such as IL-2, IL-12, GM-CSF, and the
like.
[0283] Cellular Vaccines:
[0284] CTL epitopes can be determined using specific algorithms to
identify peptides within 158P3D2 protein that bind corresponding
HLA alleles (see e.g., Table IV; Epimer.TM. and Epimatrix.TM.,
Brown University (URL
www.brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.htm- l); and,
BIMAS, (URL bimas.dcrt.nih.gov/; SYFPEITHI at URL
syfpeithi.bmi-heidelberg.com/). In a preferred embodiment, a
158P3D2 immunogen contains one or more amino acid sequences
identified using techniques well known in the art, such as the
sequences shown in Tables V-XIX or a peptide of 8, 9, 10 or 11
amino acids specified by an HLA Class I motif/supermotif (e.g.,
Table IV (A), Table IV (D), or Table IV (E)) and/or a peptide of at
least 9 amino acids that comprises an HLA Class II motif/supermotif
(e.g., Table IV (B) or Table IV (C)). As is appreciated in the art,
the HLA Class I binding groove is essentially closed ended so that
peptides of only a particular size range can fit into the groove
and be bound, generally HLA Class I epitopes are 8, 9, 10, or 11
amino acids long. In contrast, the HLA Class II binding groove is
essentially open ended; therefore a peptide of about 9 or more
amino acids can be bound by an HLA Class II molecule. Due to the
binding groove differences between HLA Class I and II, HLA Class I
motifs are length specific, i.e., position two of a Class I motif
is the second amino acid in an amino to carboxyl direction of the
peptide. The amino acid positions in a Class II motif are relative
only to each other, not the overall peptide, i.e., additional amino
acids can be attached to the amino and/or carboxyl termini of a
motif-bearing sequence. HLA Class II epitopes are often 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino
acids long, or longer than 25 amino acids.
[0285] Antibody-Based Vaccines
[0286] 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 158P3D2 protein) so
that an immune response is generated. A typical embodiment consists
of a method for generating an immune response to 158P3D2 in a host,
by contacting the host with a sufficient amount of at least one
158P3D2 B cell or cytotoxic T-cell epitope or analog thereof; and
at least one periodic interval thereafter re-contacting the host
with the 158P3D2 B cell or cytotoxic T-cell epitope or analog
thereof. A specific embodiment consists of a method of generating
an immune response against a 158P3D2-related protein or a man-made
multiepitopic peptide comprising: administering 158P3D2 immunogen
(e.g. a 158P3D2 protein or a peptide fragment thereof, an 158P3D2
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 158P3D2 immunogen by:
administering in vivo to muscle or skin of the individual's body a
DNA molecule that comprises a DNA sequence that encodes an 158P3D2
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 158P3D2, in
order to generate a response to the target antigen.
[0287] Nucleic Acid Vaccines:
[0288] 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 158P3D2. Constructs comprising DNA encoding a
158P3D2-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 158P3D2 protein/immunogen.
Alternatively, a vaccine comprises a 158P3D2-related protein.
Expression of the 158P3D2-related protein immunogen results in the
generation of prophylactic or therapeutic humoral and cellular
immunity against cells that bear a 158P3D2 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 www.genweb.com). Nucleic acid-based
delivery is described, for instance, in Wolff et. al, Science
247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466;
5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720. Examples
of DNA-based delivery technologies include "naked DNA", facilitated
(bupivicaine, polymers, peptide-mediated) delivery, cationic lipid
complexes, and particle-mediated ("gene gun") or pressure-mediated
delivery (see, e.g., U.S. Pat. No. 5,922,687).
[0289] For therapeutic or prophylactic immunization purposes,
proteins of the invention can be expressed via viral or bacterial
vectors. Various viral gene delivery systems that can be used in
the practice of the invention include, but are not limited to,
vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus,
adeno-associated virus, lentivirus, and sindbis virus (see, e.g.,
Restifo, 1996, Curr. Opin. Immunol. 8:658-663; Tsang et al. J.
Natl. Cancer Inst. 87:982-990 (1995)). Non-viral delivery systems
can also be employed by introducing naked DNA encoding a
158P3D2-related protein into the patient (e.g., intramuscularly or
intradermally) to induce an anti-tumor response.
[0290] Vaccinia virus is used, for example, as a vector to express
nucleotide sequences that encode the peptides of the invention.
Upon introduction into a host, the recombinant vaccinia virus
expresses the protein immunogenic peptide, and thereby elicits a
host immune response. Vaccinia vectors and methods useful in
immunization protocols are described in, e.g., U.S. Pat. No.
4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG
vectors are described in Stover et al., Nature 351:456-460 (1991).
A wide variety of other vectors useful for therapeutic
administration or immunization of the peptides of the invention,
e.g. adeno and adeno-associated virus vectors, retroviral vectors,
Salmonella typhi vectors, detoxified anthrax toxin vectors, and the
like, will be apparent to those skilled in the art from the
description herein.
[0291] Thus, gene delivery systems are used to deliver a
158P3D2-related nucleic acid molecule. In one embodiment, the
full-length human 158P3D2 cDNA is employed. In another embodiment,
158P3D2 nucleic acid molecules encoding specific cytotoxic T
lymphocyte (CTL) and/or antibody epitopes are employed.
[0292] Ex Vivo Vaccines
[0293] Various ex vivo strategies can also be employed to generate
an immune response. One approach involves the use of antigen
presenting cells (APCS) such as dendritic cells (DC) to present
158P3D2 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 158P3D2 peptides to T cells in the context of MHC class
I or II molecules. In one embodiment, autologous dendritic cells
are pulsed with 158P3D2 peptides capable of binding to MHC class I
and/or class II molecules. In another embodiment, dendritic cells
are pulsed with the complete 158P3D2 protein. Yet another
embodiment involves engineering the overexpression of a 158P3D2
gene in dendritic cells using various implementing vectors known in
the art, such as adenovirus (Arthur et al., 1997, Cancer Gene Ther.
4:17-25), retrovirus (Henderson et al., 1996, Cancer Res.
56:3763-3770), lentivirus, adeno-associated virus, DNA transfection
(Ribas et al., 1997, Cancer Res. 57:2865-2869), or tumor-derived
RNA transfection (Ashley et al., 1997, J. Exp. Med. 186:1177-1182).
Cells that express 158P3D2 can also be engineered to express immune
modulators, such as GM-CSF, and used as immunizing agents.
[0294] X.B.) 158P3D2 as a Target for Antibody-Based Therapy
[0295] 158P3D2 is an attractive target for antibody-based
therapeutic strategies. A number of antibody strategies are known
in the art for targeting both extracellular and intracellular
molecules (see, e.g., complement and ADCC mediated killing as well
as the use of intrabodies). Because 158P3D2 is expressed by cancer
cells of various lineages relative to corresponding normal cells,
systemic administration of 158P3D2-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 158P3D2 are useful
to treat 158P3D2-expressing cancers systemically, either as
conjugates with a toxin or therapeutic agent, or as naked
antibodies capable of inhibiting cell proliferation or
function.
[0296] 158P3D2 antibodies can be introduced into a patient such
that the antibody binds to 158P3D2 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 158P3D2, inhibition of ligand binding or
signal transduction pathways, modulation of tumor cell
differentiation, alteration of tumor angiogenesis factor profiles,
and/or apoptosis.
[0297] 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 158P3D2 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. 158P3D2), the cytotoxic agent will exert its known
biological effect (i.e. cytotoxicity) on those cells.
[0298] 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-158P3D2
antibody) that binds to a marker (e.g. 158P3D2) 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 158P3D2, comprising conjugating the
cytotoxic agent to an antibody that immunospecifically binds to a
158P3D2 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.
[0299] Cancer immunotherapy using anti-158P3D2 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, 158P3D2 antibodies can be administered in conjunction with
radiation, chemotherapy or hormone ablation. Also, antibodies can
be conjugated to a toxin such as calicheamicin (e.g., Mylotarg.TM.,
Wyeth-Ayerst, Madison, N.J., a recombinant humanized IgG.sub.4
kappa antibody conjugated to antitumor antibiotic calicheamicin) or
a maytansinoid (e.g., taxane-based Tumor-Activated Prodrug, TAP,
platform, ImmunoGen, Cambridge, Mass., also see e.g., U.S. Pat. No.
5,416,064).
[0300] Although 158P3D2 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.
[0301] Although 158P3D2 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.
[0302] Cancer patients can be evaluated for the presence and level
of 158P3D2 expression, preferably using immunohistochemical
assessments of tumor tissue, quantitative 158P3D2 imaging, or other
techniques that reliably indicate the presence and degree of
158P3D2 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.
[0303] Anti-158P3D2 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-158P3D2 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-158P3D2 mAbs that exert a direct biological effect
on tumor growth are useful to treat cancers that express 158P3D2.
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-158P3D2 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.
[0304] 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 158P3D2 antigen with high
affinity but exhibit low or no antigenicity in the patient.
[0305] Therapeutic methods of the invention contemplate the
administration of single anti-158P3D2 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-158P3D2 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-158P3D2 mAbs are administered in their
"naked" or unconjugated form, or can have a therapeutic agent(s)
conjugated to them.
[0306] Anti-158P3D2 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-158P3D2 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.
[0307] Based on clinical experience with the Herceptin.TM. mAb in
the treatment of metastatic breast cancer, an initial loading dose
of approximately 4 mg/kg patient body weight IV, followed by weekly
doses of about 2 mg/kg IV of the anti-158P3D2 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 158P3D2 expression in the patient, the
extent of circulating shed 158P3D2 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.
[0308] Optionally, patients should be evaluated for the levels of
158P3D2 in a given sample (e.g. the levels of circulating 158P3D2
antigen and/or 158P3D2 expressing cells) in order to assist in the
determination of the most effective dosing regimen, etc. Such
evaluations are also used for monitoring purposes throughout
therapy, and are useful to gauge therapeutic success in combination
with the evaluation of other parameters (for example, urine
cytology and/or ImmunoCyt levels in bladder cancer therapy, or by
analogy, serum PSA levels in prostate cancer therapy).
[0309] Anti-idiotypic anti-158P3D2 antibodies can also be used in
anti-cancer therapy as a vaccine for inducing an immune response to
cells expressing a 158P3D2-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-158P3D2 antibodies that mimic an epitope on a 158P3D2-related
protein (see, for example, Wagner et al., 1997, Hybridoma 16:
33-40; Foon et al., 1995, J. Clin. Invest. 96:334-342; Herlyn et
al., 1996, Cancer Immunol. Immunother. 43:65-76). Such an
anti-idiotypic antibody can be used in cancer vaccine
strategies.
[0310] X.C.) 158P3D2 as a Target for Cellular Immune Responses
[0311] Vaccines and methods of preparing vaccines that contain an
immunogenically effective amount of one or more HLA-binding
peptides as described herein are further embodiments of the
invention. Furthermore, vaccines in accordance with the invention
encompass compositions of one or more of the claimed peptides. A
peptide can be present in a vaccine individually. Alternatively,
the peptide can exist as a homopolymer comprising multiple copies
of the same peptide, or as a heteropolymer of various peptides.
Polymers have the advantage of increased immunological reaction
and, where different peptide epitopes are used to make up the
polymer, the additional ability to induce antibodies and/or CTLs
that react with different antigenic determinants of the pathogenic
organism or tumor-related peptide targeted for an immune response.
The composition can be a naturally occurring region of an antigen
or can be prepared, e.g., recombinantly or by chemical
synthesis.
[0312] 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))
[0313] 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 158P3D2 antigen,
or derives at least some therapeutic benefit when the antigen was
tumor-associated.
[0314] 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).
[0315] 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.
[0316] 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.
[0317] 1.) Epitopes are selected which, upon administration, mimic
immune responses that have been observed to be correlated with
tumor clearance. For HLA Class I this includes 3-4 epitopes that
come from at least one tumor associated antigen (TAA). For HLA
Class II a similar rationale is employed; again 3-4 epitopes are
selected from at least one TAA (see, e.g. Rosenberg et al., Science
278:1447-1450). Epitopes from one TAA may be used in combination
with epitopes from one or more additional TAAs to produce a vaccine
that targets tumors with varying expression patterns of
frequently-expressed TAAs.
[0318] 2.) Epitopes are selected that have the requisite binding
affinity established to be correlated with immunogenicity: for HLA
Class I an IC.sub.50 of 500 nM or less, often 200 nM or less; and
for Class II an IC.sub.50 of 1000 nM or less.
[0319] 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.
[0320] 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.
[0321] 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.
[0322] 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.
[0323] 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.
[0324] X.C.1.) Minigene Vaccines
[0325] 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.
[0326] 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 158P3D2, the PADRE(D universal helper T cell
epitope (or multiple HTL epitopes from 158P3D2), and an endoplasmic
reticulum-translocating signal sequence can be engineered. A
vaccine may also comprise epitopes that are derived from other
TAAs.
[0327] 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.
[0328] 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.
[0329] 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.
[0330] 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.
[0331] 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.
[0332] 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.
[0333] 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.
[0334] 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.
[0335] 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.
[0336] Purified plasmid DNA can be prepared for injection using a
variety of formulations. The simplest of these is reconstitution of
lyophilized DNA in sterile phosphate-buffer saline (PBS). This
approach, known as "naked DNA," is currently being used for
intramuscular (IM) administration in clinical trials. To maximize
the immunotherapeutic effects of minigene DNA vaccines, an
alternative method for formulating purified plasmid DNA may be
desirable. A variety of methods have been described, and new
techniques may become available. Cationic lipids, glycolipids, and
fusogenic liposomes can also be used in the formulation (see, e.g.,
as described by WO 93/24640; Mannino & Gould-Fogerite,
BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833; WO
91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413
(1987). In addition, peptides and compounds referred to
collectively as protective, interactive, non-condensing compounds
(PINC) could also be complexed to purified plasmid DNA to influence
variables such as stability, intramuscular dispersion, or
trafficking to specific organs or cell types.
[0337] Target cell sensitization can be used as a functional assay
for expression and HLA class I presentation of minigene-encoded CTL
epitopes. For example, the plasmid DNA is introduced into a
mammalian cell line that is suitable as a target for standard CTL
chromium release assays. The transfection method used will be
dependent on the final formulation. Electroporation can be used for
"naked" DNA, whereas cationic lipids allow direct in vitro
transfection. A plasmid expressing green fluorescent protein (GFP)
can be co-transfected to allow enrichment of transfected cells
using fluorescence activated cell sorting (FACS). These cells are
then chromium-51 (.sup.51Cr) labeled and used as target cells for
epitope-specific CTL lines; cytolysis, detected by .sup.51Cr
release, indicates both production of, and HLA presentation of,
minigene-encoded CTL epitopes. Expression of HTL epitopes may be
evaluated in an analogous manner using assays to assess HTL
activity.
[0338] 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.
[0339] 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.
[0340] 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.
[0341] X.C.2.) Combinations of CTL Peptides with Helper
Peptides
[0342] 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.
[0343] For instance, the ability of a peptide to induce CTL
activity can be enhanced by linking the peptide to a sequence which
contains at least one epitope that is capable of inducing a T
helper cell response. Although a CTL peptide can be directly linked
to a T helper peptide, often CTL epitope/HTL epitope conjugates are
linked by a spacer molecule. The spacer is typically comprised of
relatively small, neutral molecules, such as amino acids or amino
acid mimetics, which are substantially uncharged under
physiological conditions. The spacers are typically selected from,
e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or
neutral polar amino acids. It will be understood that the
optionally present spacer need not be comprised of the same
residues and thus may be a hetero- or homo-oligomer. When present,
the spacer will usually be at least one or two residues, more
usually three to six residues and sometimes 10 or more residues.
The CTL peptide epitope can be linked to the T helper peptide
epitope either directly or via a spacer either at the amino or
carboxy terminus of the CTL peptide. The amino terminus of either
the immunogenic peptide or the T helper peptide may be
acylated.
[0344] 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: ______, Plasmodium falciparum
circumsporozoite (CS) protein at positions 378-398
(DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: ______), and Streptococcus 18 kD
protein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO: ______.
Other examples include peptides bearing a DR 1-4-7 supermotif, or
either of the DR3 motifs.
[0345] 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 to most preferably
bind most HLA-DR (human HLA class II) molecules. For instance, a
pan-DR-binding epitope peptide having the formula: aKXVAAWTLKAAa
(SEQ ID NO: ______, 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.
[0346] 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.
[0347] X.C.3.) Combinations of CTL Peptides with T Cell Priming
Agents
[0348] In some embodiments it may be desirable to include in the
pharmaceutical compositions of the invention at least one component
which primes B lymphocytes or T lymphocytes. Lipids have been
identified as agents capable of priming CTL in vivo. For example,
palmitic acid residues can be attached to the .epsilon.- and
.alpha.-amino groups of a lysine residue and then linked, e.g. via
one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser,
or the like, to an immunogenic peptide. The lipidated peptide can
then be administered either directly in a micelle or particle,
incorporated into a liposome, or emulsified in an adjuvant, e.g.,
incomplete Freund's adjuvant. In a preferred embodiment, a
particularly effective immunogenic composition comprises palmitic
acid attached to .epsilon.- and .alpha.-amino groups of Lys, which
is attached via linkage, e.g., Ser-Ser, to the amino terminus of
the immunogenic peptide.
[0349] As another example of lipid priming of CTL responses, E.
coli lipoproteins, such as
tripalmitoyl-S-glycerylcysteinlyseryl-serine (P.sub.3CSS) can be
used to prime virus specific CTL when covalently attached to an
appropriate peptide (see, e.g., Deres, et al., Nature 342:561,
1989). Peptides of the invention can be coupled to P.sub.3CSS, for
example, and the lipopeptide administered to an individual to
specifically prime 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.
[0350] X.C.4.) Vaccine Compositions Comprising DC Pulsed with CTL
and/or HTL Peptides
[0351] 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.
[0352] The DC can be pulsed ex vivo with a cocktail of peptides,
some of which stimulate CTL responses to 158P3D2. 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 158P3D2.
[0353] X.D.) Adoptive Immunotherapy
[0354] Antigenic 158P3D2-related peptides are used to elicit a CTL
and/or HTL response ex vivo, as well. The resulting CTL or HTL
cells, can be used to treat tumors in patients that do not respond
to other conventional forms of therapy, or will not respond to a
therapeutic vaccine peptide or nucleic acid in accordance with the
invention. Ex vivo CTL or HTL responses to a particular antigen are
induced by incubating in tissue culture the patient's, or
genetically compatible, CTL or HTL precursor cells together with a
source of antigen-presenting cells (APC), such as dendritic cells,
and the appropriate immunogenic peptide. After an appropriate
incubation time (typically about 7-28 days), in which the precursor
cells are activated and expanded into effector cells, the cells are
infused back into the patient, where they will destroy (CTL) or
facilitate destruction (HTL) of their specific target cell (e.g., a
tumor cell). Transfected dendritic cells may also be used as
antigen presenting cells.
[0355] X.E.) Administration of Vaccines for Therapeutic or
Prophylactic Purposes
[0356] Pharmaceutical and vaccine compositions of the invention are
typically used to treat and/or prevent a cancer that expresses or
overexpresses 158P3D2. 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.
[0357] 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 158P3D2. 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.
[0358] For therapeutic use, administration should generally begin
at the first diagnosis of 158P3D2-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 158P3D2, a vaccine comprising
158P3D2-specific CTL may be more efficacious in killing tumor cells
in patient with advanced disease than alternative embodiments.
[0359] It is generally important to provide an amount of the
peptide epitope delivered by a mode of administration sufficient to
effectively stimulate a cytotoxic T cell response; compositions
which stimulate helper T cell responses can also be given in
accordance with this embodiment of the invention.
[0360] 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.
[0361] 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.
[0362] 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.
[0363] 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.
[0364] 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.
[0365] The compositions may contain pharmaceutically acceptable
auxiliary substances as required to approximate physiological
conditions, such as pH-adjusting and buffering agents, tonicity
adjusting agents, wetting agents, preservatives, and the like, for
example, sodium acetate, sodium lactate, sodium chloride, potassium
chloride, calcium chloride, sorbitan monolaurate, triethanolamine
oleate, etc.
[0366] 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.
[0367] A human unit dose form of a composition is typically
included in a pharmaceutical composition that comprises a human
unit dose of an acceptable carrier, in one embodiment an aqueous
carrier, and is administered in a volume/quantity that is known by
those of skill in the art to be used for administration of such
compositions to humans (see, e.g., Remington's Pharmaceutical
Sciences, 17.sup.th Edition, A. Gennaro, Editor, Mack Publishing
Co., Easton, Pa., 1985). For example a peptide dose for initial
immunization can be from about 1 to about 50,000 .mu.g, generally
100-5,000 .mu.g, for a 70 kg patient. For example, for nucleic
acids an initial immunization may be performed using an expression
vector in the form of naked nucleic acid administered IM (or SC or
ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid
(0.1 to 1000 .mu.g) can also be administered using a gene gun.
Following an incubation period of 3-4 weeks, a booster dose is then
administered. The booster can be recombinant fowlpox virus
administered at a dose of 5-10.sup.7 to 5.times.10.sup.9 pfu.
[0368] For antibodies, a treatment generally involves repeated
administration of the anti-158P3D2 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-158P3D2
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
158P3D2 expression in the patient, the extent of circulating shed
158P3D2 antigen, the desired steady-state concentration level,
frequency of treatment, and the influence of chemotherapeutic or
other agents used in combination with the treatment method of the
invention, as well as the health status of a particular patient.
Non-limiting preferred human unit doses are, for example, 500
.mu.g-1 mg, 1 mg-50 mg, 50 mg-100 mg, 100 mg-200 mg, 200 mg-300 mg,
400 mg-500 mg, 500 mg-600 mg, 600 mg-700 mg, 700 mg-800 mg, 800
mg-900 mg, 900 mg-1 g, or 1 mg-700 mg. In certain embodiments, the
dose is in a range of 2-5 mg/kg body weight, e.g., with follow on
weekly doses of 1-3 mg/kg; 0.5 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10
mg/kg body weight followed, e.g., in two, three or four weeks by
weekly doses; 0.5-10 mg/kg body weight, e.g., followed in two,
three or four weeks by weekly doses; 225, 250, 275, 300, 325, 350,
375, 400 mg m.sup.2 of body area weekly; 1-600 mg m.sup.2 of body
area weekly; 225-400 mg m.sup.2 of body area weekly; these does can
be followed by weekly doses for 2, 3, 4, 5, 6, 7, 8, 9, 19, 11, 12
or more weeks.
[0369] 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.
[0370] 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.
[0371] 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.
[0372] 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.
[0373] 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%.
[0374] 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.
[0375] XI.) Diagnostic and Prognostic Embodiments of 158P3D2.
[0376] As disclosed herein, 158P3D2 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 Example 4).
[0377] 158P3D2 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 July 1999 4(1):99-102 and Minimoto
et al., Cancer Detect Prev 2000;24(1):1-12). Therefore, this
disclosure of 158P3D2 polynucleotides and polypeptides (as well as
158P3D2 polynucleotide probes and anti-158P3D2 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.
[0378] Typical embodiments of diagnostic methods which utilize the
158P3D2 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
158P3D2 polynucleotides described herein can be utilized in the
same way to detect 158P3D2 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 158P3D2
polypeptides described herein can be utilized to generate
antibodies for use in detecting 158P3D2 overexpression or the
metastasis of prostate cells and cells of other cancers expressing
this gene.
[0379] 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 158P3D2 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
158P3D2-expressing cells (lymph node) is found to contain
158P3D2-expressing cells such as the 158P3D2 expression seen in
LAPC4 and LAPC9, xenografts isolated from lymph node and bone
metastasis, respectively, this finding is indicative of
metastasis.
[0380] Alternatively 158P3D2 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 158P3D2 or
express 158P3D2 at a different level are found to express 158P3D2
or have an increased expression of 158P3D2 (see, e.g., the 158P3D2
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 158P3D2) such as PSA, PSCA
etc. (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237
(1996)).
[0381] Just as PSA polynucleotide fragments and polynucleotide
variants are employed by skilled artisans for use in methods of
monitoring PSA, 158P3D2 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 Example 4, where a
158P3D2 polynucleotide fragment is used as a probe to show the
expression of 158P3D2 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. November-December 1996
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
158P3D2 polynucleotide shown in FIG. 2 or variant thereof) under
conditions of high stringency.
[0382] 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. 158P3D2
polypeptide fragments and polypeptide analogs or variants can also
be used in an analogous manner. This practice of using polypeptide
fragments or polypeptide variants to generate antibodies (such as
anti-PSA antibodies or T cells) is typical in the art with a wide
variety of systems such as fusion proteins being used by
practitioners (see, e.g., Current Protocols In Molecular Biology,
Volume 2, Unit 16, Frederick M. Ausubel et al. eds., 1995). In this
context, each epitope(s) functions to provide the architecture with
which an antibody or T cell is reactive. Typically, skilled
artisans create a variety of different polypeptide fragments that
can be used in order to generate immune responses specific for
different portions of a polypeptide of interest (see, e.g., U.S.
Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it may
be preferable to utilize a polypeptide comprising one of the
158P3D2 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 158P3D2
polypeptide shown in FIG. 3).
[0383] As shown herein, the 158P3D2 polynucleotides and
polypeptides (as well as the 158P3D2 polynucleotide probes and
anti-158P3D2 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 158P3D2 gene products, in order
to evaluate the presence or onset of a disease condition described
herein, such as prostate cancer, are used to identify patients for
preventive measures or further monitoring, as has been done so
successfully with PSA. Moreover, these materials satisfy a need in
the art for molecules having similar or complementary
characteristics to PSA in situations where, for example, a definite
diagnosis of metastasis of prostatic origin cannot be made on the
basis of a test for PSA alone (see, e.g., Alanen et al., Pathol.
Res. Pract. 192(3): 233-237 (1996)), and consequently, materials
such as 158P3D2 polynucleotides and polypeptides (as well as the
158P3D2 polynucleotide probes and anti-158P3D2 antibodies used to
identify the presence of these molecules) need to be employed to
confirm a metastases of prostatic origin.
[0384] Finally, in addition to their use in diagnostic assays, the
158P3D2 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 158P3D2 gene maps (see Example 3 below). Moreover, in
addition to their use in diagnostic assays, the 158P3D2-related
proteins and polynucleotides disclosed herein have other utilities
such as their use in thc forensic analysis of tissues of unknown
origin (see, e.g., Takahama K Forensic Sci Int Jun. 28,
1996;80(1-2): 63-9).
[0385] Additionally, 158P3D2-related proteins or polynucleotides of
the invention can be used to treat a pathologic condition
characterized by the over-expression of 158P3D2. 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 158P3D2 antigen. Antibodies or other molecules that react with
158P3D2 can be used to modulate the function of this molecule, and
thereby provide a therapeutic benefit.
[0386] XII.) Inhibition of 158P3D2 Protein Function
[0387] The invention includes various methods and compositions for
inhibiting the binding of 158P3D2 to its binding partner or its
association with other protein(s) as well as methods for inhibiting
158P3D2 function.
[0388] XII.A.) Inhibition of 158P3D2 with Intracellular
Antibodies
[0389] In one approach, a recombinant vector that encodes single
chain antibodies that specifically bind to 158P3D2 are introduced
into 158P3D2 expressing cells via gene transfer technologies.
Accordingly, the encoded single chain anti-158P3D2 antibody is
expressed intracellularly, binds to 158P3D2 protein, and thereby
inhibits its function. Methods for engineering such intracellular
single chain antibodies are well known. Such intracellular
antibodies, also known as "intrabodies", are specifically targeted
to a particular compartment within the cell, providing control over
where the inhibitory activity of the treatment is focused. This
technology has been successfully applied in the art (for review,
see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodies
have been shown to virtually eliminate the expression of otherwise
abundant cell surface receptors (see, e.g., Richardson et al.,
1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al.,
1994, J. Biol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene
Ther. 1: 332-337).
[0390] 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 precisely
target 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.
[0391] In one embodiment, intrabodies are used to capture 158P3D2
in the nucleus, thereby preventing its activity within the nucleus.
Nuclear targeting signals arc engineered into such 158P3D2
intrabodies in order to achieve the desired targeting. Such 158P3D2
intrabodies are designed to bind specifically to a particular
158P3D2 domain. In another embodiment, cytosolic intrabodies that
specifically bind to a 158P3D2 protein are used to prevent 158P3D2
from gaining access to the nucleus, thereby preventing it from
exerting any biological activity within the nucleus (e.g.,
preventing 158P3D2 from forming transcription complexes with other
factors).
[0392] In order to specifically direct the expression of such
intrabodies to particular cells, the transcription of the intrabody
is placed under the regulatory control of an appropriate
tumor-specific promoter and/or enhancer. In order to target
intrabody expression specifically to prostate, for example, the PSA
promoter and/or promoter/enhancer can be utilized (See, for
example, U.S. Pat. No. 5,919,652 issued Jul. 6, 1999).
[0393] XII.B.) Inhibition of 158P3D2 with Recombinant Proteins
[0394] In another approach, recombinant molecules bind to 158P3D2
and thereby inhibit 158P3D2 function. For example, these
recombinant molecules prevent or inhibit 158P3D2 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 158P3D2 specific antibody
molecule. In a particular embodiment, the 158P3D2 binding domain of
a 158P3D2 binding partner is engineered into a dimeric fusion
protein, whereby the fusion protein comprises two 158P3D2 ligand
binding domains linked to the Fc portion of a human IgG, such as
human IgG1. Such IgG portion can contain, for example, the CH2 and
CH3 domains and the hinge region, but not the CH1 domain. Such
dimeric fusion proteins are administered in soluble form to
patients suffering from a cancer associated with the expression of
158P3D2, whereby the dimeric fusion protein specifically binds to
158P3D2 and blocks 158P3D2 interaction with a binding partner. Such
dimeric fusion proteins are further combined into multimeric
proteins using known antibody linking technologies.
[0395] XH.C.) Inhibition of 158P3D2 Transcription or
Translation
[0396] The present invention also comprises various methods and
compositions for inhibiting the transcription of the 158P3D2 gene.
Similarly, the invention also provides methods and compositions for
inhibiting the translation of 158P3D2 mRNA into protein.
[0397] In one approach, a method of inhibiting the transcription of
the 158P3D2 gene comprises contacting the 158P3D2 gene with a
158P3D2 antisense polynucleotide. In another approach, a method of
inhibiting 158P3D2 mRNA translation comprises contacting a 158P3D2
mRNA with an antisense polynucleotide. In another approach, a
158P3D2 specific ribozyme is used to cleave a 158P3D2 message,
thereby inhibiting translation. Such antisense and ribozyme based
methods can also be directed to the regulatory regions of the
158P3D2 gene, such as 158P3D2 promoter and/or enhancer elements.
Similarly, proteins capable of inhibiting a 158P3D2 gene
transcription factor are used to inhibit 158P3D2 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.
[0398] Other factors that inhibit the transcription of 158P3D2 by
interfering with 158P3D2 transcriptional activation are also useful
to treat cancers expressing 158P3D2. Similarly, factors that
interfere with 158P3D2 processing are useful to treat cancers that
express 158P3D2. Cancer treatment methods utilizing such factors
are also within the scope of the invention.
[0399] XII.D.) General Considerations for Therapeutic
Strategies
[0400] Gene transfer and gene therapy technologies can be used to
deliver therapeutic polynucleotide molecules to tumor cells
synthesizing 158P3D2 (i.e., antisense, ribozyme, polynucleotides
encoding intrabodies and other 158P3D2 inhibitory molecules). A
number of gene therapy approaches are known in the art. Recombinant
vectors encoding 158P3D2 antisense polynucleotides, ribozymes,
factors capable of interfering with 158P3D2 transcription, and so
forth, can be delivered to target tumor cells using such gene
therapy approaches.
[0401] 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.
[0402] 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 vii-o 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 158P3D2 to a binding partner, etc.
[0403] In vivo, the effect of a 158P3D2 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.
[0404] 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.
[0405] The therapeutic compositions used in the practice of the
foregoing methods can be formulated into pharmaceutical
compositions comprising a carrier suitable for the desired delivery
method. Suitable carriers include any material that when combined
with the therapeutic composition retains the anti-tumor function of
the therapeutic composition and is generally non-reactive with the
patient's immune system. Examples include, but are not limited to,
any of a number of standard pharmaceutical carriers such as sterile
phosphate buffered saline solutions, bacteriostatic water, and the
like (see, generally, Remington's Pharmaceutical Sciences 16.sup.th
Edition, A. Osal., Ed., 1980).
[0406] 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.
[0407] 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.
[0408] XIII.) Kits
[0409] 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 158P3D2-related protein or a 158P3D2 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, fluorescent, or
radioisotope label. The kit can include all or part of the amino
acid sequence of FIG. 2 or FIG. 3 or analogs thereof, or a nucleic
acid molecules that encodes such amino acid sequences.
[0410] 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, and package inserts with instructions for use.
[0411] A label can be present on the container to indicate that the
composition is used for a specific therapy or non-therapeutic
application, and can also indicate directions for either in vivo or
in vitro use, such as those described above. Directions and or
other information can also be included on an insert which is
included with the kit.
EXAMPLES
[0412] Various aspects of the invention are further described and
illustrated by way of the several examples that follow, none of
which are intended to limit the scope of the invention.
Example 1
SSH-Generated Isolation of a cDNA Fragment of the 158P3D2 Gene
[0413] To isolate genes that are over-expressed in bladder cancer,
Suppression Subtractive Hybridization (SSH) procedure using cDNA
derived from bladder cancer tissues, including invasive
transitional cell carcinoma. The 158P3D2 SSH cDNA sequence was
derived from a bladder cancer pool minus normal bladder cDNA
subtraction. Included in the driver were also cDNAs derived from 9
other normal tissues. The 158P3D2 cDNA was identified as highly
expressed in the bladder cancer tissue pool, with lower expression
seen in a restricted set of normal tissues.
[0414] The SSH DNA sequence of 312 bp (FIG. 1) shows identity to
the fer-1-like 4 (C. elegans) (FER1L4) mRNA (FIG. 4A). A 158P3D2
cDNA clone 158P3D2-BCP1 of 1994 bp was isolated from bladder cancer
cDNA, revealing an ORF of 328 amino acids (FIG. 2 and FIG. 3).
[0415] Amino acid sequence analysis of 158P3D2 reveals 100%
identity over 328 amino acid region to dJ47704. 1. 1, a novel
protein similar to otoferlin and dysferlin, isoform 1 protein
(GenBank Accession CAB89410.1.vertline., FIG. 4B).
[0416] The 158P3D2 protein has a transmembrane domain of 23
residues between amino acids 292-313 predicted by the SOSUI Signal
program
(http://sosui.proteome.bio.tuat.acjp/cgi-bin/sosui.cgi?/sosuisignal/sosui-
signal_submit.html).
[0417] Materials and Methods
[0418] Human Tissues:
[0419] 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.
[0420] RNA Isolation:
[0421] 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.
[0422] Oligonucleotides:
[0423] The following HPLC purified oligonucleotides were used.
[0424] DPNCDN (cDNA Synthesis Primer):
[0425] 5'TTTTGATCAAGCTT.sub.303' (SEQ ID NO: ______)
1 DPNCDN (cDNA synthesis primer): (SEQ ID NO:_)
5'TTTTGATCAAGTT.sub.303' Adaptor 1: (SEQ ID NO:_)
5'CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3' (SEQ ID NO:_)
3'GGCCCGTCCTAG5' Adaptor 2: (SEQ ID NO:_)
5'GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3' (SEQ ID NO:_)
3'CGGCTCCTAG5' PCR primer 1: (SEQ ID NO:_)
5'CTAATACGACTCACTATAGGGC3' Nested primer (NP)1: (SEQ ID NO:_)
5'TCGAGCGGCCGCCCGGGCAGGA- 3' Nested primer (NP)2: (SEQ ID NO:_)
5'AGCGTGGTCGCGGCCGAGGA3'
[0426] PCR Primer 1:
[0427] 5'CTAATACGACTCACTATAGGGC3' (SEQ ID NO: ______)
[0428] Nested Primer (NP)1:
[0429] 5'TCGAGCGGCCGCCCGGGCAGGA3' (SEQ ID NO: ______)
[0430] Nested Primer (NP).sub.2:
[0431] 5'AGCGTGGTCGCGGCCGAGGA3' (SEQ ID NO: ______)
[0432] Suppression Subtractive Hybridization:
[0433] Suppression Subtractive Hybridization (SSH) was used to
identify cDNAs corresponding to genes that may be differentially
expressed in bladder cancer. The SSH reaction utilized cDNA from
bladder cancer and normal tissues.
[0434] The gene 15 8P3D2 sequence was derived from a bladder cancer
pool minus normal bladder cDNA subtraction. The SSH DNA sequence
(FIG. 1) was identified.
[0435] The cDNA derived from of pool of normal bladder tissues was
used as the source of the "driver" cDNA, while the cDNA from a pool
of bladder cancer tissues was used as the source of the "tester"
cDNA. Double stranded cDNAs corresponding to tester and driver
cDNAs were synthesized from 2 .mu.g of poly(A)+ RNA isolated from
the relevant 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 at 37.degree. C. Digested cDNA was extracted
with phenol/chloroform (1:1) and ethanol precipitated.
[0436] 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.
[0437] Tester cDNA was generated by diluting 1 .mu.l of Dpn II
digested cDNA from the relevant tissue source (see above) (400 ng)
in 5 .mu.l of water. The diluted cDNA (2 .mu.l, 160 ng) was then
ligated to 2 .mu.l of Adaptor 1 and Adaptor 2 (10 .mu.M), in
separate ligation reactions, in a total volume of 10 .mu.l at
16.degree. C. overnight, using 400 u of T4 DNA ligase (CLONTECH).
Ligation was terminated with 1 .mu.l of 0.2 M EDTA and heating at
72.degree. C. for 5 min.
[0438] 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.
[0439] PCR Amplification, Cloning and Sequencing of Gene Fragments
Generated from SSH:
[0440] 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 I (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 I 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 see, 66.degree. C. for 30 see, 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.
[0441] The PCR products were inserted into pCR2.1 using the T/A
vector cloning kit (Invitrogen). Transformed E. coli were subjected
to blue/white and ampicillin selection. White colonies were picked
and arrayed into 96 well plates and were grown in liquid culture
overnight. To identify inserts, PCR amplification was performed on
1 ml of bacterial culture using the conditions of PCR1 and NP1 and
NP2 as primers. PCR products were analyzed using 2% agarose gel
electrophoresis.
[0442] 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.
[0443] RT-PCR Expression Analysis:
[0444] 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.
[0445] Normalization of the first strand cDNAs from multiple
tissues was performed by using the primers 5
'atatcgccgcgctcgtcgtcgacaa3' (SEQ ID NO: ______) and
5'agccacacgcagctcattgtagaagg 3' (SEQ ID NO: ______) to amplify
.beta.-actin. First strand cDNA (5 .mu.l) were amplified in a total
volume of 50 .mu.l containing 0.4 .mu.M primers, 0.2 .mu.M each
dNTPs, 1.times.PCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM
MgCl.sub.2, 50 mM KCl, pH 8.3) and 1.times.-Klentaq DNA polymerase
(Clontech). Five .mu.l of the PCR reaction can be removed at 18,
20, and 22 cycles and used for agarose gel electrophoresis. PCR was
performed using an MJ Research thermal cycler under the following
conditions: Initial denaturation can be at 94.degree. C. for 15
see, 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.
[0446] To determine expression levels of the 158P3D2 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 158P3D2 SSH sequence and are listed below:
[0447] 158P3D2.1
[0448] 5'CATCTATGTGAAGAGCTGGGTGAA 3' (SEQ ID NO: )
[0449] 158P3D2.2
[0450] 5' AGGTAGTCAAAGCGGAACACAAAG 3' (SEQ ID NO: )
[0451] A typical RT-PCR expression analysis is shown in FIG. 14.
RT-PCR expression analysis was performed on first strand cDNAs
generated using pools of tissues from multiple samples. The cDNAs
were shown to be normalized using beta-actin PCR. Results show
strong expression of 158P3D2 in bladder cancer pool, kidney cancer
pool and cancer metastasis pool. Expression of 158P3D2 is also
detected in colon cancer pool, lung cancer pool, ovary cancer pool,
breast cancer pool, pancreas cancer pool and prostate metastases to
lymph node, and vital pool 2, but not vital pool 1.
Example 2
Full Length Cloning of 158P3D2
[0452] The 158P3D2 SSH cDNA sequence was derived from a bladder
cancer pool minus normal bladder cDNA subtraction. The SSH cDNA
sequence (FIG. 1) was designated 158P3D2. The full-length cDNA
clone 158P3D2 v.1 clone 158P3D2-BCP1 and 158P3D2-BCP2 (FIG. 2) were
cloned from bladder cancer pool cDNA.
[0453] The SSH DNA sequence of 312 bp (FIG. 1) shows identity to
the fer-1-like 4 (C. elegans) (FER1L4) mRNA (FIG. 4A). A 158P3D2
cDNA clone 158P3D2-BCP1 of 1994 bp was isolated from bladder cancer
cDNA, revealing an ORF of 328 amino acids (FIG. 2 and FIG. 3).
[0454] Amino acid sequence analysis of 158P3D2 reveals 100%
identity over 328 amino acid region to dJ47704.1.1, a novel protein
similar to otoferlin and dysferlin, isoform 1 protein (GenBank
Accession CAB89410.1.vertline., FIG. 4B).
[0455] The 158P3D2 protein has a transmembrane domain of 23
residues between amino acids 292-313 predicted by the SOSUI Signal
program
(http://sosui.proteome.bio.tuat.acjp/cgi-bin/sosui.cgi?/sosuisignal/sosui-
signal_submit.html).
Example 3
Chromosomal Mapping of 158P3D2
[0456] 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 Al), human-rodent somatic cell
hybrid panels such as is available from the Coriell Institute
(Camden, N.J.), and genomic viewers utilizing BLAST homologies to
sequenced and mapped genomic clones (NCBI, Bethesda, Md.).
[0457] 158P3D2 maps to chromosome 8, using 158P3D2 sequence and the
NCBI BLAST tool:
(http://www.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.ht-
ml&&ORG=Hs).
Example 4
Expression Analysis of 158P3D2 in Normal Tissues and Patient
Specimens
[0458] Expression analysis by RT-PCR demonstrated that 158P3D2 is
strongly expressed in bladder 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 from 2 different patients, prostate
cancer pool, bladder cancer pool, kidney cancer pool, colon cancer
pool, lung cancer pool, ovary cancer pool, breast cancer pool,
cancer metastasis pool, and pancreas cancer pool. Normalization was
performed by PCR using primers to actin and GAPDH.
Semi-quantitative PCR, using primers to 158P3D2, was performed at
26 and 30 cycles of amplification. Results show strong expression
of 158P3D2 in bladder cancer pool, kidney cancer pool and cancer
metastasis pool. Expression of 158P3D2 is also detected in colon
cancer pool, lung cancer pool, ovary cancer pool, breast cancer
pool, pancreas cancer pool and prostate metastases to lymph node,
and vital pool 2, but not vital pool 1.
[0459] Northern blot analysis of 158P3D2 in 16 human normal tissues
is shown in FIG. 15. An approximately 8 kb transcript is detected
exclusively in placenta. Extensive analysis of expression of
158P3D2 in 76 human tissues shows restricted expression of 158P3D2
in placenta and stomach (FIG. 16).
[0460] Expression of 158P3D2 in patient cancer specimens and human
normal tissues is shown in FIG. 17. RNA was extracted from a pool
of three bladder cancers, as well as from normal prostate (NP),
normal bladder (NB), normal kidney (NK), normal colon (NC), normal
lung (NL) and normal breast (NBr). Northern blot with 10 ug of
total RNA/lane was probed with 158P3D2 sequence. The results show
expression of 158P3D2 in the bladder cancer pool but not in the
normal tissues tested. Analysis of individual patient specimens
shows strong expression of 158P3D2 in 8 different bladder cancer
tissues tested (FIG. 18). Presence of 158P3D2 transcript is also
detected in the bladder cancer cell line SCaBER. The expression
observed in normal adjacent tissue (isolated from diseased tissues)
but not in normal tissue, isolated from healthy donors, may
indicate that these tissues are not fully normal and that 158P3D2
may be expressed in early stage tumors.
[0461] The restricted expression of 158P3D2 in normal tissues and
the expression detected in bladder cancer, prostate cancer pool,
kidney cancer pool, colon cancer pool, lung cancer pool, ovary
cancer pool, breast cancer pool, pancreas cancer pool and cancer
metastases suggest that 158P3D2 is a potential therapeutic target
and a diagnostic marker for human cancers.
Example 5
Transcript Variants of 158P3D2
[0462] Transcript variants are variants of matured mRNA from the
same gene 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 may encode proteins with different functions, and may be
expressed in the same tissue at the same time, or at different
tissue, or at different times, proteins encoded by transcript
variants can have similar or different cellular or extracellular
localizations, i.e., be secreted.
[0463] Transcript variants are identified by a variety of
art-accepted methods. For example, alternative transcripts and
splice variants are identified through full-length cloning
experiments, or by use of full-length transcript and EST sequences.
First, all human ESTs were grouped into clusters which show direct
or indirect identity with each other. Second, ESTs in the same
cluster were further grouped into sub-clusters and assembled into a
consensus sequence. The original gene sequence is compared to the
consensus sequence(s) or other full-length sequences. Each
consensus sequence is a potential splice variant for that gene
(see, e.g.,
http://www.doubletwist.com/products/c11_agentsOverview.j- html).
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.
[0464] 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. April 2000;
10(4):516-22); Grail (http://compbio.oml.gov/Grail--
bin/EmptyGrailForm) and GenScan
(http://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.
[0465] 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. April 2001; 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).
[0466] 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 158P3D2 has a particular expression profile related to cancer.
Alternative transcripts and splice variants of 158P3D2 may also be
involved in cancer in the same or different tissues, thus serving
as tumor-associated markers/antigens.
[0467] The exon composition of the original transcript, designated
as 158P3D2 var1, is shown in FIG. 13 and Table XXIIIA. Using the
full-length gene and EST sequences, one alternative transcript was
identified, designated as 158P3D2 var2, which is also shown in FIG.
13 and Table XXIIIB. Transcript variant 158P3D2 var2 has two
potential open reading frames and two protein products, designated
as 158P3D2 var2a and 158P3D2 var2b. FIG. 13 shows the schematic
alignment of exons of the two transcripts. Potentially, each
different combination of exons in spatial order, e.g. exons 1, 2,
3, 4 and 7, can be a splice variant.
[0468] Tables XXIV through XXVII are set forth herein on a
variant-by-variant basis. Table XXIV shows nucleotide sequence of a
transcript variant. Table XXV shows the alignment of the transcript
variant 158P3D2 var2 with nucleic acid sequence of 158P3D2 var1.
Table XXVI lays out amino acid translation of the transcript
variant 158P3D2 var2 for the identified reading frame orientation.
Table XXVII displays alignments of the amino acid sequence encoded
by the transcript variant 158P3D2 var2 with that of 158P3D2
var1.
Example 6
Single Nucleotide Polymorphisms of 158P3D2
[0469] Single Nucleotide Polymorphism (SNP) is a single base pair
variation in nucleotide sequences. At a specific point of the
genome, there are four possible nucleotide base pairs: A/T, C/G,
G/C and T/A. Genotype refers to the base pair make-up of one or
more spots in the genome of an individual, while haplotype refers
to base pair make-up of more than one varied spots on the same DNA
molecule (chromosome in higher organism). 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 and some 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 discovery of genetic relationship
between individuals (P. Nowotny, J. M. Kwon and A. M. Goate, "SNP
analysis to dissect human traits," Curr. Opin. Neurobiol. October
2001; 11(5):637-641; M. Pirmohamed and B. K. Park, "Genetic
susceptibility to adverse drug reactions," Trends Pharmacol. Sci.
June 2001; 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," Pharmacogenonics. February 2000;
1(1):39-47; R. Judson, J. C. Stephens and A. Windemuth, "The
predictive power of haplotypes in clinical response,"
Pharmacogenomics. February 2000; 1(1):15-26).
[0470] SNPs are identified by a variety of art-accepted methods (P.
Bean, "The promising voyage of SNP target discovery," Am. Clin.
Lab. October-November 2001; 20(9): 18-20; K. M. Weiss, "In search
of human variation," Genome Res. July 1998; 8(7):691-697; M. M.
She, "Enabling large-scale pharmacogenetic studies by
high-throughput mutation detection and genotyping technologies,"
Clin. Chem. February 2001; 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. December 2000; 5(4):329-340).
[0471] SNPs are identified by directly sequencing cDNA clones of
the invention and by comparing the sequences with public and
proprietary sequences. By comparing these cDNA clones with high
quality proprietary or public sequences, seven SNPs were identified
and two of them are linked (a deletion and a substitution). The
transcripts or proteins with alternative alleles were designated as
variants 158P3D2 v.3, v.4, v.5, v.6, v.7 and v.8. FIG. 10 shows the
schematic alignment of the nucleotide 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 different transcript
variants that contain the sequence context.
Example 7
Production of Recombinant 158P3D2 in Prokaryotic Systems
[0472] To express recombinant 158P3D2 and 158P3D2 variants in
prokaryotic cells, the full or partial length 158P3D2 and 158P3D2
variant 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 158P3D2 or 158P3D2 variants are expressed in these
constructs, amino acids 1 to 328 of 158P3D2 (variant 1), amino
acids 1-236 of variant 2a, amino acids 1-181 of variant 2b, amino
acids 1-328 of variant 3, amino acids 1-328 of variant 4, amino
acids 1-178 of variant 5a, amino acids 1-181 of variant 5b; 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 158P3D2,
variants, or analogs thereof.
[0473] A. In vitro Transcription and Translation Constructs:
[0474] pCRII: To generate 158P3D2 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 158P3D2 cDNA. The pCRII vector has Sp6 and T7 promoters
flanking the insert to drive the transcription of 158P3D2 RNA for
use as probes in RNA in situ hybridization experiments. These
probes are used to analyze the cell and tissue expression of
158P3D2 at the RNA level. Transcribed 158P3D2 RNA representing the
cDNA amino acid coding region of the 158P3D2 gene is used in in
vitro translation systems such as the TnT.TM. Coupled
Reticulolysate Sytem (Promega, Corp., Madison, Wis.) to synthesize
158P3D2 protein.
[0475] B. Bacterial Constructs:
[0476] pGEX Constructs: To generate recombinant 158P3D2 proteins in
bacteria that are fused to the Glutathione S-transferase (GST)
protein, all or parts of the T-fusion vector of the pGEX family
(Amersham Pharmacia Biotech, Piscataway, N.J.). These constructs
allow controlled expression of recombinant 158P3D2 protein
sequences with GST fused at the amino-terminus and a six histidine
epitope (6.times.His) at the carboxyl-terminus. The GST and
6.times.His tags permit purification of the recombinant fusion
protein from induced bacteria with the appropriate affinity matrix
and allow recognition of the fusion protein with anti-GST and
anti-His antibodies. The 6.times.His tag is generated by adding 6
histidine codons to the cloning primer at the 3' end, e.g., of the
open reading frame (ORF). A proteolytic cleavage site, such as the
PreScission.TM. recognition site in pGEX-6P-1, may be employed such
that it permits cleavage of the GST tag from 158P3D2-related
protein. The ampicillin resistance gene and pBR322 origin permits
selection and maintenance of the pGEX plasmids in E. coli.
[0477] pMAL Constructs: To generate, in bacteria, recombinant
158P3D2 proteins that are fused to maltose-binding protein (MBP),
all or parts of the 158P3D2 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 158P3D2 protein sequences with
MBP fused at the amino-terminus and a 6.times.His epitope tag at
the carboxyl-terminus. The MBP and 6.times.His tags permit
purification of the recombinant protein from induced bacteria with
the appropriate affinity matrix and allow recognition of the fusion
protein with anti-MBP and anti-His antibodies. The 6.times.His
epitope tag is generated by adding 6 histidine codons to the 3'
cloning primer. A Factor Xa recognition site permits cleavage of
the pMAL tag from 158P3D2. 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.
[0478] pET Constructs: To express 158P3D2 in bacterial cells, all
or parts of the 158P3D2 cDNA protein coding sequence are cloned
into the pET family of vectors (Novagen, Madison, Wis.). These
vectors allow tightly controlled expression of recombinant 158P3D2
protein in bacteria with and without fusion to proteins that
enhance solubility, such as NusA and thioredoxin (Trx), and epitope
tags, such as 6.times.His and S-Tag.TM. that aid purification and
detection of the recombinant protein. For example, constructs are
made utilizing pET NusA fusion system 43.1 such that regions of the
158P3D2 protein are expressed as amino-terminal fusions to NusA. In
one embodiment, a NusA-fusion protein encompassing amino acids
412-328 of 158P3D2 with a C-terminal 6.times.His tag was expressed
in E. Coli, purified by metal chelate affinity chromatography, and
used as an immunogen for generation of antibodies.
[0479] C. Yeast Constructs:
[0480] pESC Constructs: To express 158P3D2 in the yeast species
Saccharomyces cerevisiae for generation of recombinant protein and
functional studies, all or parts of the 158P3D2 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 158P3D2. In addition, expression in
yeast yields similar post-translational modifications, such as
glycosylations and phosphorylations, that are found when expressed
in eukaryotic cells.
[0481] pESP Constructs: To express 158P3D2 in the yeast species
Saccharomyces pombe, all or parts of the 158P3D2 cDNA protein
coding sequence are cloned into the pESP family of vectors. These
vectors allow controlled high level of expression of a 158P3D2
protein sequence that is fused at either the amino terminus or at
the carboxyl terminus to GST which aids purification of the
recombinant protein. A Flag.TM. epitope tag allows detection of the
recombinant protein with anti-Flag.TM. antibody.
Example 8
Production of Recombinant 158P3D2 in Eukaryotic Systems
[0482] A. Mammalian Constructs:
[0483] To express recombinant 158P3D2 in eukaryotic cells, the full
or partial length 158P3D2 cDNA sequences were cloned into any one
of a variety of expression vectors known in the art. One or more of
the following regions of 158P3D2 were expressed in these
constructs, amino acids 1 to 328, 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 158P3D2, variants, or analogs
thereof. In certain embodiments a region of 158P3D2 was expressed
that encodes an amino acid not shared amongst at least
variants.
[0484] The constructs were 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-158P3D2 polyclonal serum,
described herein.
[0485] pcDNA4/HisMax Constructs: To express 158P3D2 in mammalian
cells, a 158P3D2 ORF, or portions thereof, of 158P3D2 are cloned
into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, Calif.).
Protein expression is driven from the cytomegalovirus (CMV)
promoter and the SP 16 translational enhancer. The recombinant
protein has Xpress.TM. and six histidine (6.times.His) epitopes
fused to the amino-terminus. The pcDNA4/HisMax vector also contains
the bovine growth hormone (BGH) polyadenylation signal and
transcription termination sequence to enhance mRNA stability along
with the SV40 origin for episomal replication and simple vector
rescue in cell lines expressing the large T antigen. The Zeocin
resistance gene allows for selection of mammalian cells expressing
the protein and the ampicillin resistance gene and ColE1 origin
permits selection and maintenance of the plasmid in E. coli.
[0486] pcDNA3.1/MycHis Constructs: To express 158P3D2 in mammalian
cells, a 158P3D2 ORF, or portions thereof, of 158P3D2 with a
consensus Kozak translation initiation site was cloned into
pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, Calif.). Protein
expression is driven from the cytomegalovirus (CMV) promoter. The
recombinant proteins have the myc epitope and 6.times.His epitope
fused to the carboxyl-terminus. The pcDNA3.1/MycHis vector also
contains the bovine growth hormone (BGH) polyadenylation signal and
transcription termination sequence to enhance mRNA stability, along
with the SV40 origin for episomal replication and simple vector
rescue in cell lines expressing the large T antigen. The Neomycin
resistance gene can be used, as it allows for selection of
mammalian cells expressing the protein and the ampicillin
resistance gene and ColE1 origin permits selection and maintenance
of the plasmid in E. coli. FIG. 19 shows expression of
158P3D2.pcDNA3.1/mychis in transiently transfected 293T cells.
[0487] pcDNA3.1/CT-GFP-TOPO Construct: To express 158P3D2 in
mammalian cells and to allow detection of the recombinant proteins
using fluorescence, a 158P3D2 ORF, or portions thereof, with a
consensus Kozak translation initiation site are cloned into
pcDNA3.1/CT-GFP-TOPO (Invitrogen, CA). Protein expression is driven
from the cytomegalovirus (CMV) promoter. The recombinant proteins
have the Green Fluorescent Protein (GFP) fused to the
carboxyl-terminus facilitating non-invasive, in vivo detection and
cell biology studies. The pcDNA3.1CT-GFP-TOPO vector also contains
the bovine growth hormone (BGH) polyadenylation signal and
transcription termination sequence to enhance mRNA stability along
with the SV40 origin for episomal replication and simple vector
rescue in cell lines expressing the large T antigen. The Neomycin
resistance gene allows for selection of mammalian cells that
express the protein, and the ampicillin resistance gene and ColE1
origin permits selection and maintenance of the plasmid in E. coli.
Additional constructs with an amino-terminal GFP fusion are made in
pcDNA3.1/NT-GFP-TOPO spanning the entire length of a 158P3D2
protein.
[0488] PAPtag: A 158P3D2 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 158P3D2 protein while fusing the IgGK signal sequence to the
amino-terminus. Constructs are also generated in which alkaline
phosphatase with an amino-terminal IgGK signal sequence is fused to
the amino-terminus of a 158P3D2 protein. The resulting recombinant
158P3D2 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 158P3D2 proteins.
Protein expression is driven from the CMV promoter and the
recombinant proteins also contain myc and 6.times.His epitopes
fused at the carboxyl-terminus that facilitates detection and
purification. The Zeocin resistance gene present in the vector
allows for selection of mammalian cells expressing the recombinant
protein and the ampicillin resistance gene permits selection of the
plasmid in E. coli. ptag5: A 158P3D2 ORF, or portions thereof, is
cloned into pTag-5. This vector is similar to pAPtag but without
the alkaline phosphatase fusion. This construct generates 158P3D2
protein with an amino-terminal IgGK signal sequence and myc and
6.times.His epitope tags at the carboxyl-terminus that facilitate
detection and affinity purification. The resulting recombinant
158P3D2 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 158P3D2 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.
[0489] PsecFc: A 158P3D2 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 158P3D2 proteins, while
fusing the IgGK signal sequence to N-terminus. 158P3D2 fusions
utilizing the murine IgG1 Fc region are also used. The resulting
recombinant 158P3D2 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 158P3D2 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.
[0490] pSR.alpha. Constructs: To generate mammalian cell lines that
express 158P3D2 constitutively, 158P3D2 ORF, or portions thereof,
of 158P3D2 are cloned into pSR.alpha. constructs. Amphotropic and
ecotropic retroviruses are generated by transfection of pSR.alpha.
constructs into the 293T-10A1 packaging line or co-transfection of
pSR.alpha. and a helper plasmid (containing deleted packaging
sequences) into the 293 cells, respectively. The retrovirus is used
to infect a variety of mammalian cell lines, resulting in the
integration of the cloned gene, 158P3D2, 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.
[0491] Additional pSR.alpha. constructs are made that fuse an
epitope tag such as the FLAG.TM. tag to the carboxyl-terminus of
158P3D2 sequences to allow detection using anti-Flag antibodies.
For example, the FLAG.TM. sequence 5' gat tac aag gat gac gac gat
aag 3' is added to cloning primer at the 3' end of the ORF.
Additional pSR.alpha. constructs are made to produce both
amino-terminal and carboxyl-terminal GFP and myc/6.times.His fusion
proteins of the full-length 158P3D2 proteins.
[0492] Additional Viral Vectors: Additional constructs are made for
viral-mediated delivery and expression of 158P3D2. High virus titer
leading to high level expression of 158P3D2 is achieved in viral
delivery systems such as adenoviral vectors and herpes amplicon
vectors. A 158P3D2 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, 158P3D2 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-I
cells.
[0493] Regulated Expression Systems: To control expression of
158P3D2 in mammalian cells, coding sequences of 158P3D2, or
portions thereof, are cloned into regulated mammalian expression
systems such as the T-Rex System (Invitrogen), the GeneSwitch
System (Invitrogen) and the tightly-regulated Ecdysone System
(Sratagene). These systems allow the study of the temporal and
concentration dependent effects of recombinant 158P3D2. These
vectors are thereafter used to control expression of 158P3D2 in
various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
[0494] B. Baculovirus Expression Systems
[0495] To generate recombinant 158P3D2 proteins in a baculovirus
expression system, 158P3D2 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-158P3D2 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.
[0496] Recombinant 158P3D2 protein is then generated by infection
of HighFive insect cells (Invitrogen) with purified baculovirus.
Recombinant 158P3D2 protein can be detected using anti-158P3D2 or
anti-His-tag antibody. 158P3D2 protein can be purified and used in
various cell-based assays or as immunogen to generate polyclonal
and monoclonal antibodies specific for 158P3D2.
Example 9
Antigenicity Profiles and Secondary Structure
[0497] FIG. 5A, FIG. 6A, FIG. 7A, FIG. 8A, and FIG. 9A depict
graphically five amino acid profiles of the 158P3D2 variant 1 amino
acid sequence; FIG. 5B, FIG. 6B, FIG. 7B, FIG. 8B, and FIG. 9B
depict graphically five amino acid profiles of the 158P3D2 variant
2A amino acid sequence, and FIG. 5C, FIG. 6C, FIG. 7C, FIG. 8C, and
FIG. 9C depict graphically five amino acid profiles of the 158P3D2
variant 5A amino acid sequence, each assessment available by
accessing the ProtScale website (URL
www.expasy.ch/cgi-bin/protscale.pl) on the ExPasy molecular biology
server.
[0498] 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 158P3D2 protein. Each of the above amino acid
profiles of 158P3D2 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.
[0499] 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.
[0500] 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.
[0501] Antigenic sequences of the 158P3D2 protein and of the
variant proteins indicated, e.g., by the profiles set forth in
FIGS. 5A-C, FIGS. 6A-C, FIGS. 7A-C, FIGS. 8A-C, and/or FIGS. 9A-C
are used to prepare immunogens, either peptides or nucleic acids
that encode them, to generate therapeutic and diagnostic
anti-158P3D2antibodies. 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 158P3D2
protein or of 158P3D2 variants. In particular, peptide immunogens
of the invention can comprise, a peptide region of at least 5 amino
acids of FIG. 2 in any whole number increment up to 328 that
includes an amino acid position having a value greater than 0.5 in
the Hydrophilicity profile of FIG. 5; a peptide region of at least
5 amino acids of FIG. 2 in any whole number increment up to 328
that includes an amino acid position having a value less than 0.5
in the Hydropathicity profile of FIG. 6; a peptide region of at
least 5 amino acids of FIG. 2 in any whole number increment up to
328 that includes an amino acid position having a value greater
than 0.5 in the Percent Accessible Residues profile of FIG. 7; a
peptide region of at least 5 amino acids of FIG. 2 in any whole
number increment up to 328 that includes an amino acid position
having a value greater than 0.5 in the Average Flexibility profile
on FIG. 8; and, a peptide region of at least 5 amino acids of FIG.
2 in any whole number increment up to 328 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.
[0502] 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.
[0503] The secondary structure of 158P3D2 variant 1 and variants 2a
and 5a, 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
(http://www.expasy.ch/t- ools/). The analysis indicates that
158P3D2 variant 1 is composed 32.93% alpha helix, 18.29% extended
strand, and 48.78% random coil (FIG. 12A), variant 2a is composed
of 25.85% alpha helix, 18.22% extended strand, and 55.93% random
coil (FIG. 12B), and variant 5a is composed of 9.55% alpha helix,
26.40% extended strand, and 64.04% random coil (FIG. 12C).
[0504] Analysis for the potential presence of transmembrane domains
in 158P3D2 variant 1 was carried out using a variety of
transmembrane prediction algorithms accessed from the ExPasy
molecular biology server (http://www.expasy.ch/tools/). The
programs predict the presence of a single transmembrane domain in
15.8P3D2 variant 1. Shown graphically in FIGS. 12D and 12E are the
results of analysis using the TMpred (FIG. 12D) and TMHMM (FIG.
12E) prediction programs depicting the location of the
transmembrane domain. The results of each program, namely the amino
acids encoding the transmembrane domain are summarized in Table
XXII. Variants 2b, 3, 4, and 5b, also contain the amino acids
predicted to encode the transmembrane domain. No transmembrane
domains are predicted in variants 2a and 5a.
Example 10
Generation of 158P3D2 Polyclonal Antibodies
[0505] Polyclonal antibodies can be raised in a mammal, for
example, by one or more injections of an immunizing agent and, if
desired, an adjuvant. Typically, the immunizing agent and/or
adjuvant will be injected in the mammal by multiple subcutaneous or
intraperitoneal injections. In addition to immunizing with the full
length 158P3D2 protein, computer algorithms are employed in design
of immunogens that, based on amino acid sequence analysis contain
characteristics of being antigenic and available for recognition by
the immune system of the immunized host (see Example 9): 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. 5A-C, FIGS. 6A-C, FIGS. 7A-C, FIGS. 8A-C,
or FIGS. 9A-C for amino acid profiles that indicate such regions of
158P3D2 and variants).
[0506] For example, 158P3D2 recombinant bacterial fusion proteins
or peptides containing hydrophilic, flexible, beta-turn regions of
the 158P3D2, such as regions amino terminal to the predicted
transmembrane domain of variant 1 (predicted to be extracellular),
are used as antigens to generate polyclonal antibodies in New
Zealand White rabbits. For example, such regions include, but are
not limited to, amino acids 1-25, amino acids 37-54, amino acids
60-73, and amino acids 187-225. 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 200-225 of 158P3D2 is
conjugated to KLH and used to immunize the rabbit. Alternatively
the immunizing agent may include all or portions of the 158P3D2
protein, analogs or fusion proteins thereof. For example, the
158P3D2 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.
[0507] In one embodiment, a GST-fusion protein encoding the
predicted extracellular domain, amino acids 1-291, 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
Example 7 and Current Protocols In Molecular Biology, Volume 2,
Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, P.S.,
Brady, W., Urnes, M., Grosmaire, L., Damle, N., and Ledbetter,
L.(1991) J.Exp. Med. 174, 561-566).
[0508] In addition to bacterial derived fusion proteins, mammalian
expressed protein antigens are also used. These antigens are
expressed from mammalian expression vectors such as the Tag5 and
Fc-fusion vectors (see Example 8), and retain post-translational
modifications such as glycosylations found in native protein. 10.
In one embodiment, amino acids 185-225 is cloned into the TagS
mammalian secretion vector. In another embodiment, the predicted
extracellular domain, amino acids 1-291 is cloned into the TagS
expression vector. The recombinant proteins are purified by metal
chelate chromatography from tissue culture supernatants of 293T
cells stably expressing the recombinant vector. The purified Tag5
158P3D2 proteins are then individually used as immunogens.
[0509] 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).
[0510] 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.
[0511] To test reactivity and specificity of immune serum, such as
the rabbit serum derived from immunization with TagS 158P3D2
encoding amino acids 1-291, the full-length 158P3D2 cDNA is cloned
into pcDNA 3.1 myc-his expression vector (Invitrogen, see Example
7). After transfection of the constructs into 293T cells, cell
lysates are probed with the anti-158P3D2 serum and with anti-His
antibody (Santa Cruz Biotechnologies, Santa Cruz, Calif.) to
determine specific reactivity to denatured 158P3D2 protein using
the Western blot technique. Shown in FIG. 19 is expression of Myc
His tagged 158P3D2 protein in 293T cells as detected by Western
blot with anti-His antibody. The immune serum is then tested by the
Western blot technique against 293T-158P3D2 cells. In addition, the
immune serum is tested by fluorescence microscopy, flow cytometry
and immunoprecipitation against 293T and other recombinant
158P3D2-expressing cells to determine specific recognition of
native protein. Western blot, immunoprecipitation, fluorescent
microscopy, and flow cytometric techniques using cells that
endogenously express 158P3D2 are also carried out to test
reactivity and specificity.
[0512] Anti-serum from rabbits immunized with 158P3D2 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-158P3D2 fusion protein
encoding amino acids 1-291 is first purified by passage over a
column of GST protein covalently coupled to AffiGel matrix (BioRad,
Hercules, Calif.). The antiserum is then affinity purified by
passage over a column composed of a MBP-fusion protein also
encoding amino acids 1-291 covalently coupled to Affigel matrix.
The serum is then further purified by protein G affinity
chromatography to isolate the IgG fraction. Sera from other
His-tagged antigens and peptide immunized rabbits as well as fusion
partner depleted sera are affinity purified by passage over a
column matrix composed of the original protein immunogen or free
peptide.
Example 11
Generation of 158P3D2 Monoclonal Antibodies (mAbs)
[0513] In one embodiment, therapeutic mAbs to 158P3D2 comprise
those that react with epitopes of the protein that would disrupt or
modulate the biological function of 158P3D2, for example those that
would disrupt its interaction with ligands and binding partners.
Therapeutic mAbs also comprise those that specifically bind
epitopes of 158P3D2 exposed on the cell surface and thus are useful
in targeting mAb-toxin conjugates. Immunogens for generation of
such mAbs include those designed to encode or contain the entire
158P3D2 protein, regions of the 158P3D2 protein predicted to be
antigenic from computer analysis of the amino acid sequence (see,
e.g., FIGS. 5A-C, FIGS. 6A-C, FIGS. 7A-C, FIGS. 8A-C, or FIGS.
9A-C, and Example 9) such as regions in the extracellular domain of
variant 1. Immunogens include peptides, recombinant bacterial
proteins, and mammalian expressed Tag 5 proteins and human and
murine IgG FC fusion proteins. In addition, cells expressing high
levels of 158P3D2, such as 293T-158P3D2 or 300.19-158P3D2 murine
Pre-B cells, are used to immunize mice.
[0514] To generate mAbs to 158P3D2, mice are first immunized
intraperitoneally (IP) with, typically, 10-50 .mu.g of protein
immunogen or 10.sup.7 158P3D2-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 158P3D2 sequence is used to
immunize mice by direct injection of the plasmid DNA. For example,
amino acids 1-291 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 158P3D2 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
158P3D2.
[0515] 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).
[0516] In one embodiment for generating 158P3D2 monoclonal
antibodies, a Tag5-158P3D2 antigen encoding amino acids 1-291, the
predicted extracellular domain, is expressed and purified from
stably transfected 293T cells. Balb C mice are initially immunized
intraperitoneally with 25 .mu.g of the Tag5-158P3D2 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 158P3D2 protein
is monitored by Western blotting, immunoprecipitation and flow
cytometry using 293T cells transfected with an expression vector
encoding the 158P3D2 cDNA (see e.g., Example 8). Other recombinant
158P3D2-expressing cells or cells endogenously expressing 158P3D2
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 158P3D2 specific
antibody-producing clones.
[0517] Monoclonal antibodies are also derived that react only with
specific 158P3D2 variants, such as variants 2a and 5a. To this end,
immunogens are designed to encode amino acid regions specific to
the respective variant. For example, a Tag5 immunogen is encoding
amino acids 1-236 of variant 2a is produced, purified, and used to
immunize mice to generate hybridomas. In another example, a Tag5
immunogen encoding amino acids 130-178 of variant 5a is produced,
purified, and used as immunogen. Monoclonal antibodies raised to
these immunogens are then screened for reactivity to cells
expressing the respective variants but not to other 158P3D2
variants. These strategies for raising 158P3D2 variant specific
monoclonal antibodies are also applied to polyclonal reagents
described in Example 10.
[0518] The binding affinity of a 158P3D2 monoclonal antibody is
determined using standard technologies. Affinity measurements
quantify the strength of antibody to epitope binding and are used
to help define which 158P3D2 monoclonal antibodies preferred for
diagnostic or therapeutic use, as appreciated by one of skill in
the art. The BIAcore system (Uppsala, Sweden) is a preferred method
for determining binding affinity. The BIAcore system uses surface
plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23: 1;
Morton and Myszka, 1998, Methods in Enzymology 295: 268) to monitor
biomolecular interactions in real time. BIAcore analysis
conveniently generates association rate constants, dissociation
rate constants, equilibrium dissociation constants, and affinity
constants.
Example 12
HLA Class I and Class II Binding Assays
[0519] HLA class I and class II binding assays using purified HLA
molecules are performed in accordance with disclosed protocols
(e.g., PCT publications WO 94/20127 and WO 94/03205; Sidney et al.,
Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J.
Immunol. 154:247 (1995); Sette, et al., Mol. Immunol. 31:813
(1994)). Briefly, purified MHC molecules (5 to 500 nM) are
incubated with various unlabeled peptide inhibitors and 1-10 nM
.sup.125I-radiolabeled probe peptides as described. Following
incubation, MHC-peptide complexes are separated from free peptide
by gel filtration and the fraction of peptide bound is determined.
Typically, in preliminary experiments, each MHC preparation is
titered in the presence of fixed amounts of radiolabeled peptides
to determine the concentration of HLA molecules necessary to bind
10-20% of the total radioactivity. All subsequent inhibition and
direct binding assays are performed using these HLA
concentrations.
[0520] 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.
[0521] Binding assays as outlined above may be used to analyze HLA
supermotif and/or HLA motif-bearing peptides.
Example 13
Identification of HLA Supermotif- and Motif-Bearing CTL Candidate
Epitopes
[0522] HLA vaccine compositions of the invention can include
multiple epitopes. The multiple epitopes can comprise multiple HLA
supermotifs or motifs to achieve broad population coverage. This
example illustrates the identification and confirmation of
supermotif- and motif-bearing epitopes for the inclusion in such a
vaccine composition. Calculation of population coverage is
performed using the strategy described below.
[0523] Computer Searches and Algorithms for Identification of
Supermotif and/or Motif-Bearing Epitopes
[0524] The searches performed to identify the motif-bearing peptide
sequences in Example 9 and Tables V-XIX employ the protein sequence
data from the gene product of 158P3D2 set forth in FIGS. 2 and
3.
[0525] Computer searches for epitopes bearing HLA Class I or Class
II supermotifs or motifs are performed as follows. All translated
158P3D2 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.
[0526] 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
[0527] 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.
[0528] 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.
[0529] Selection of HLA-A2 Supertype Cross-Reactive Peptides
[0530] Protein sequences from 158P3D2 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).
[0531] 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.
[0532] Selection of HLA-A3 Supermotif-Bearing Epitopes
[0533] The 158P3D2 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 <500 nM, often <200 nM, are then tested for
binding cross-reactivity to the other common A3-supertype alleles
(e.g., A*3101, A*3301, and A*6801) to identify those that can bind
at least three of the five HLA-A3-supertype molecules tested.
[0534] Selection of HLA-B7 Supermotif Bearing Epitopes
[0535] The 158P3D2 protein(s) scanned above is also analyzed for
the presence of 8-, 9-10-, or 1 1-mer peptides with the
HLA-B7-supermotif. Corresponding peptides are synthesized and
tested for binding to HLA-B*0702, the molecule encoded by the most
common B7-supertype allele (i.e., the prototype B7 supertype
allele). Peptides binding B*0702 with IC.sub.50 of <500 nM are
identified using standard methods. These peptides are then tested
for binding to other common B7-supertype molecules (e.g., B*3501,
B*5101, B*5301, and B*5401). Peptides capable of binding to three
or more of the five B7-supertype alleles tested are thereby
identified.
[0536] Selection of A1 and A24 Motif-Bearing Epitopes
[0537] To further increase population coverage, HLA-A1 and -A24
epitopes can also be incorporated into vaccine compositions. An
analysis of the 158P3D2 protein can also be performed to identify
HLA-A1- and A24-motif-containing sequences.
[0538] High affinity and/or cross-reactive binding epitopes that
bear other motif and/or supermotifs arc identified using analogous
methodology.
Example 14
Confirmation of Immunogenicity
[0539] 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:
[0540] Target Cell Lines for Cellular Screening:
[0541] The 0.221A2.1 cell line, produced by transferring the
HLA-A2.1 gene into the HLA-A, -B, -C null mutant human
B-lymphoblastoid cell line 721.221, is used as the peptide-loaded
target to measure activity of HLA-A2.1-restricted CTL. This cell
line is grown in RPMI-1640 medium supplemented with antibiotics,
sodium pyruvate, nonessential amino acids and 10% (v/v) heat
inactivated FCS. Cells that express an antigen of interest, or
transfectants comprising the gene encoding the antigen of interest,
can be used as target cells to confirm the ability of
peptide-specific CTLs to recognize endogenous antigen.
[0542] Primary CTL Induction Cultures:
[0543] Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI
with 30 .mu.g/ml DNAse, washed twice and resuspended in complete
medium (RPMI-1640 plus 5% AB human serum, non-essential amino
acids, sodium pyruvate, L-glutamine and penicillin/streptomycin).
The monocytes are purified by plating 10.times.10.sup.6 PBMC/well
in a 6-well plate. After 2 hours at 37.degree. C., the non-adherent
cells are removed by gently shaking the plates and aspirating the
supernatants. The wells are washed a total of three times with 3 ml
RPMI to remove most of the non-adherent and loosely adherent cells.
Three ml of complete medium containing 50 ng/ml of GM-CSF and 1,000
U/ml of IL-4 are then added to each well. TNF.alpha. is added to
the DCs on day 6 at 75 ng/ml and the cells are used for CTL
induction cultures on day 7.
[0544] Induction of CTL with DC and Peptide: CD8+T-cells are
isolated by positive selection with Dynal immunomagnetic beads
(Dynabeads.RTM. M-450) and the detacha-bead.RTM. reagent. Typically
about 200-250.times.10.sup.6 PBMC are processed to obtain
24.times.10.sup.6 CD8+ T-cells (enough for a 48-well plate
culture). Briefly, the PBMCs are thawed in RPMI with 30 .mu.g/ml
DNAse, washed once with PBS containing 1% human AB serum and
resuspended in PBS/1% AB serum at a concentration of
20.times.10.sup.6cells/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.
[0545] 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.
[0546] 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/mil 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 0.25mil RPMI/5%AB per
well for 2 hours at 37.degree. C. Peptide solution from each well
is aspirated and the wells are washed once with RPMI. Most of the
media is aspirated from the induction cultures (CD8+cells) and
brought to 0.5 ml with fresh media. The cells are then transferred
to the wells containing the peptide-pulsed adherent cells. Twenty
four hours later recombinant human IL-10 is added at a final
concentration of 10 ng/ml and recombinant human IL2 is added the
next day and again 2-3 days later at 50 IU/ml (Tsai et al.,
Critical Reviews in Immunology 18(1-2):65-75, 1998). Seven days
later, the cultures are assayed for CTL activity in a .sup.51Cr
release assay. In some experiments the cultures are assayed for
peptide-specific recognition in the in situ IFN.gamma. ELISA at the
time of the second restimulation followed by assay of endogenous
recognition 7 days later. After expansion, activity is measured in
both assays for a side-by-side comparison.
[0547] Measurement of CTL Lytic Activity by .sup.51Cr Release.
[0548] Seven days after the second restimulation, cytotoxicity is
determined in a standard (5 hr) .sup.51Cr release assay by assaying
individual wells at a single E:T. Peptide-pulsed targets are
prepared by incubating the cells with 10 .mu.g/ml peptide overnight
at 37.degree. C.
[0549] 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 106 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.
[0550] 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.
[0551] In situ Measurement of Human IFN.gamma. Production as an
Indicator of Peptide-Specific and Endogenous Recognition
[0552] Immulon 2 plates are coated with mouse anti-human IFN.gamma.
monoclonal antibody (4 .mu.g/ml 0.1M NaHCO.sub.3, pH 8.2) 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.
[0553] Recombinant human IFN-gamma is added to the standard wells
starting at 400 .mu.g or 1200 .mu.g/100 microliter/well and the
plate incubated for two hours at 37.degree. C. The plates are
washed and 100 .mu.l of biotinylated mouse anti-human IFN-gamma
monoclonal antibody (2 microgram/ml in PBS/3%FCS/0.05% Tween 20)
are added and incubated for 2 hours at room temperature. After
washing again, 100 microliter HRP-streptavidin (1:4000) are added
and the plates incubated for one hour at room temperature. The
plates are then washed 6.times.with wash buffer, 100
microliter/well developing solution (TMB 1:1) are added, and the
plates allowed to develop for 5-15 minutes. The reaction is stopped
with 50 microliter/well 1M H.sub.3PO.sub.4 and read at OD450. A
culture is considered positive if it measured at least 50 pg of
IFN-gamma/well above background and is twice the background level
of expression.
[0554] CTL Expansion.
[0555] Those cultures that demonstrate specific lytic activity
against peptide-pulsed targets and/or tumor targets are expanded
over a two week period with anti-CD3. Briefly, 5.times.10.sup.4
CD8+ cells are added to a T25 flask containing the following:
1.times.10.sup.6 irradiated (4,200 rad) PBMC (autologous or
allogeneic) per ml, 2.times.10.sup.5 irradiated (8,000 rad)
EBV-transformed cells per ml, and OKT3 (anti-CD3) at 30 ng per ml
in RPMI-1640 containing 10% (v/v) human AB serum, non-essential
amino acids, sodium pyruvate, 25 .mu.M 2-mercaptoethanol,
L-glutamine and penicillin/streptomycin. Recombinant human IL2 is
added 24 hours later at a final concentration of 200 IU/ml and
every three days thereafter with fresh media at 50 IU/ml. The cells
are split if the cell concentration exceeds 1.times.10.sup.6/ml and
the cultures are assayed between days 13 and 15 at E:T ratios of
30, 10, 3 and 1:1 in the .sup.51Cr release assay or at
1.times.10.sup.6/ml in the in situ IFN.gamma. assay using the same
targets as before the expansion.
[0556] Cultures are expanded in the absence of anti-CD3.sup.+ as
follows. Those cultures that demonstrate specific lytic activity
against peptide and endogenous targets are selected and
5.times.10.sup.4 CD8.sup.+ cells are added to a T25 flask
containing the following: 1.times.10.sup.6 autologous PBMC per ml
which have been peptide-pulsed with 10 .mu.g/ml peptide for two
hours at 37.degree. C. and irradiated (4,200 rad); 2.times.10.sup.5
irradiated (8,000 rad) EBV-transformed cells per ml RPMI-1640
containing 10%(v/v) human AB serum, non-essential AA, sodium
pyruvate, 25 mM 2-ME, L-glutamine and gentamicin.
[0557] Immunogenicity of A2 Supermotif-Bearing Peptides
[0558] 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.
[0559] Immunogenicity can also be confirmed using PBMCs isolated
from patients bearing a tumor that expresses 158P3D2. 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.
[0560] Evaluation of A*03/A11 Immunogenicity
[0561] 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.
[0562] Evaluation of B7 Immunogenicity
[0563] 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.
[0564] Peptides bearing other supermotifs/motifs, e.g., HLA-A1,
HLA-A24 etc. are also confirmed using similar methodology
Example 15
Implementation of the Extended Supermotif to Improve the Binding
Capacity of Native Epitopes by Creating Analogs
[0565] 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.
[0566] Analoging at Primary Anchor Residues
[0567] 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.
[0568] 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.
[0569] 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.
[0570] 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).
[0571] 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.
[0572] Analoging of HLA-A3 and B7-Supermotif-Bearing Peptides
[0573] 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.
[0574] 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.
[0575] 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).
[0576] Analoging at primary anchor residues of other motif and/or
supermotif-bearing epitopes is performed in a like manner.
[0577] 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.
[0578] Analoging at Secondary Anchor Residues
[0579] 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.
[0580] 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 158P3D2-expressing tumors.
[0581] Other Analoging Strategies
[0582] 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).
[0583] Thus, by the use of single amino acid substitutions, the
binding properties and/or cross-reactivity of peptide ligands for
HLA supertype molecules can be modulated.
Example 16
Identification and Confirmation of 158P3D2-Derived Sequences with
HLA-DR Binding Motifs
[0584] 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.
[0585] Selection of HLA-DR-Supermotif-Bearing Epitopes.
[0586] To identify 158P3D2-derived, HLA class II HTL epitopes, a
158P3D2 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).
[0587] 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.
[0588] The 158P3D2-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. 158P3D2-derived peptides found to bind
common HLA-DR alleles are of particular interest.
[0589] Selection of DR3 Motif Peptides
[0590] 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.
[0591] To efficiently identify peptides that bind DR3, target
158P3D2 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.
[0592] DR3 binding epitopes identified in this manner are included
in vaccine compositions with DR supermotif-bearing peptide
epitopes.
[0593] Similarly to the case of HLA class I motif-bearing peptides,
the class II motif-bearing peptides are analoged to improve
affinity or cross-reactivity. For example, aspartic acid at
position 4 of the 9-mer core sequence is an optimal residue for DR3
binding, and substitution for that residue often improves DR 3
binding.
Example 17
Immunogenicity of 158P3D2-Derived HTL Epitopes
[0594] This example determines immunogenic DR supermotif- and DR3
motif-bearing epitopes among those identified using the methodology
set forth herein.
[0595] 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 158P3D2-expressing
tumors.
Example 18
Calculation of Phenotypic Frequencies of HLA-Supertypes in Various
Ethnic Backgrounds to Determine Breadth of Population Coverage
[0596] 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.
[0597] 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].
[0598] 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).
[0599] Population coverage achieved by combining the A2-, A3- and
B7-supertypes is approximately 86% in five major ethnic groups.
Coverage may be extended by including peptides bearing the A1 and
A24 motifs. On average, A1 is present in 12% and A24 in 29% of the
population across five different major ethnic groups (Caucasian,
North American Black, Chinese, Japanese, and Hispanic). Together,
these alleles are represented with an average frequency of 39% in
these same ethnic populations. The total coverage across the major
ethnicities when A1 and A24 are combined with the coverage of the
A2-, A3- and B7-supertype alleles is >95%. An analogous approach
can be used to estimate population coverage achieved with
combinations of class II motif-bearing epitopes.
[0600] 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.
[0601] With a sufficient number of epitopes (as disclosed herein
and from the art), an average population coverage is predicted to
be greater than 95% in each of five major ethnic populations. The
game theory Monte Carlo simulation analysis, which is known in the
art (see e.g., Osborne, M. J. and Rubinstein, A. "A course in game
theory" MIT Press, 1994), can be used to estimate what percentage
of the individuals in a population comprised of the Caucasian,
North American Black, Japanese, Chinese, and Hispanic ethnic groups
would recognize the vaccine epitopes described herein. A preferred
percentage is 90%. A more preferred percentage is 95%.
Example 19
CTL Recognition of Endogenously Processed Antigens after
Priming
[0602] 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.
[0603] 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 158P3D2
expression vectors.
[0604] The results demonstrate that CTL lines obtained from animals
primed with peptide epitope recognize endogenously synthesized
158P3D2 antigen. The choice of transgenic mouse model to be used
for such an analysis depends upon the epitope(s) that are being
evaluated. In addition to HLA-A*0201/K.sup.b transgenic mice,
several other transgenic mouse models including mice with human
A11, which may also be used to evaluate A3 epitopes, and B7 alleles
have been characterized and others (e.g., transgenic mice for
HLA-A1 and A24) are being developed. HLA-DR1 and HLA-DR3 mouse
models have also been developed, which may be used to evaluate HTL
epitopes.
Example 20
Activity of CTL-HTL Conjugated Epitopes in Transgenic Mice
[0605] This example illustrates the induction of CTLs and HTLs in
transgenic mice, by use of a 158P3D2-derived CTL and HTL peptide
vaccine compositions. The vaccine composition used herein comprise
peptides to be administered to a patient with a 158P3D2-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.
[0606] Immunization procedures: Immunization of transgenic mice is
performed as described (Alexander et al., J. Immunol. 159:47534761,
1997). For example, A2/1 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.
[0607] 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)
[0608] 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.
[0609] 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.
[0610] The results are analyzed to assess the magnitude of the CTL
responses of animals injected with the immunogenic CTL/HTL
conjugate vaccine preparation and are compared to the magnitude of
the CTL response achieved using, for example, CTL epitopes as
outlined above in Example 14. Analyses similar to this may be
performed to confirm the immunogenicity of peptide conjugates
containing multiple CTL epitopes and/or multiple HTL epitopes. In
accordance with these procedures, it is found that a CTL response
is induced, and concomitantly that an HTL response is induced upon
administration of such compositions.
Example 21
Selection of CTL and HTL Epitopes for Inclusion in an
158P3D2-Specific Vaccine
[0611] 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.
[0612] 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.
[0613] Epitopes are selected which, upon administration, mimic
immune responses that are correlated with 158P3D2 clearance. The
number of epitopes used depends on observations of patients who
spontaneously clear 158P3D2. For example, if it has been observed
that patients who spontaneously clear 158P3D2 generate an immune
response to at least three (3) from 158P3D2 antigen, then three or
four (3-4) epitopes should be included for HLA class I. A similar
rationale is used to determine HLA class II epitopes.
[0614] 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/.
[0615] 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.
[0616] 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 158P3D2, 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.
[0617] 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 158P3D2.
Example 22
Construction of "Minigene" Multi-Epitope DNA Plasmids
[0618] 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.
[0619] 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 158P3D2, are
selected such that multiple supermotifs/motifs are represented to
ensure broad population coverage. Similarly, HLA class II epitopes
are selected from 158P3D2 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.
[0620] Such a construct may additionally include sequences that
direct the HTL epitopes to the endoplasmic reticulum. For example,
the Ii protein may be fused to one or more HTL epitopes as
described in the art, wherein the CLIP sequence of the Ii protein
is removed and replaced with an HLA class II epitope sequence so
that HLA class II epitope is directed to the endoplasmic reticulum,
where the epitope binds to an HLA class II molecules.
[0621] 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.
[0622] The minigene DNA plasmid of this example contains a
consensus Kozak sequence and a consensus murine kappa Ig-light
chain signal sequence followed by CTL and/or HTL epitopes selected
in accordance with principles disclosed herein. The sequence
encodes an open reading frame fused to the Myc and His antibody
epitope tag coded for by the pcDNA 3.1 Myc-His vector.
[0623] 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.
[0624] For example, a minigene is prepared as follows. For a first
PCR reaction, 5 .mu.g of each of two oligonucleotides are annealed
and extended: In an example using eight oligonucleotides, i.e.,
four pairs of primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are
combined in 100 .mu.l reactions containing Pfu polymerase buffer
(1.times.=10 mM KCL, 10 mM (NH4).sub.2SO.sub.4, 20 mM
Tris-chloride, pH 8.75, 2 mM MgSO.sub.4, 0.1% Triton X-100, 100
.mu.g/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. The
full-length dimer products are gel-purified, and two reactions
containing the product of 1+2 and 3+4, and the product of 5+6 and
7+8 are mixed, annealed, and extended for 10 cycles. Half of the
two reactions are then mixed, and 5 cycles of annealing and
extension carried out before flanking primers are added to amplify
the full length product. The full-length product is gel-purified
and cloned into pCR-blunt (Invitrogen) and individual clones are
screened by sequencing.
Example 23
The Plasmid Construct and the Degree to Which it Induces
Immunogenicity
[0625] 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).
[0626] 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.
[0627] 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.
[0628] 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.
[0629] 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.
[0630] To confirm the capacity of a class II epitope-encoding
minigene to induce HTLs in vivo, DR transgenic mice, or for those
epitopes that cross react with the appropriate mouse MHC molecule,
I-Ab.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.sup.+ 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.
[0631] 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).
[0632] For example, the efficacy of the DNA minigene used in a
prime boost protocol is initially evaluated in transgenic mice. In
this example, A2.1/K.sup.b transgenic mice are immunized IM with
100 .mu.g of a DNA minigene encoding the immunogenic peptides
including at least one HLA-A2 supermotif-bearing peptide. After an
incubation period (ranging from 3-9 weeks), the mice are boosted IP
with 10.sup.7 pfu/mouse of a recombinant vaccinia virus expressing
the same sequence encoded by the DNA minigene. Control mice are
immunized with 100 .mu.g of DNA or recombinant vaccinia without the
minigene sequence, or with DNA encoding the minigene, but without
the vaccinia boost. After an additional incubation period of two
weeks, splenocytes from the mice are immediately assayed for
peptide-specific activity in an ELISPOT assay. Additionally,
splenocytes are stimulated in vitro with the A2-restricted peptide
epitopes encoded in the minigene and recombinant vaccinia, then
assayed for peptide-specific activity in an alpha, beta and/or
gamma IFN ELISA.
[0633] 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 Example 31.
Example 24
Peptide Compositions for Prophylactic Uses
[0634] Vaccine compositions of the present invention can be used to
prevent 158P3D2 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
158P3D2-associated tumor.
[0635] 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 158P3D2-associated disease.
[0636] Alternatively, a composition typically comprising
transfecting agents is used for the administration of a nucleic
acid-based vaccine in accordance with methodologies known in the
art and disclosed herein.
Example 25
Polyepitopic Vaccine Compositions Derived from Native 158P3D2
Sequences
[0637] A native 158P3D2 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 is selected; it
can be used to generate a minigene construct. The construct is
engineered to express the peptide, which corresponds to the native
protein sequence. The "relatively short" peptide is generally less
than 250 amino acids in length, often less than 100 amino acids in
length, preferably less than 75 amino acids in length, and more
preferably less than 50 amino acids in length. The protein sequence
of the vaccine composition is selected because it has maximal
number of epitopes contained within the sequence, i.e., it has a
high concentration of epitopes. As noted herein, epitope motifs may
be nested or overlapping (i.e., frame shifted relative to one
another). For example, with overlapping epitopes, two 9-mer
epitopes and one 10-mer epitope can be present in a 10 amino acid
peptide. Such a vaccine composition is administered for therapeutic
or prophylactic purposes.
[0638] The vaccine composition will include, for example, multiple
CTL epitopes from 158P3D2 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.
[0639] 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 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 158P3D2, 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.
[0640] Related to this embodiment, computer programs are available
in the art which can be used to identify in a target sequence, the
greatest number of epitopes per sequence length.
Example 26
Polyepitopic Vaccine Compositions from Multiple Antigens
[0641] The 158P3D2 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
158P3D2 and such other antigens. For example, a vaccine composition
can be provided as a single polypeptide that incorporates multiple
epitopes from 158P3D2 as well as tumor-associated antigens that are
often expressed with a target cancer associated with 158P3D2
expression, or can be administered as a composition comprising a
cocktail of one or more discrete epitopes. Alternatively, the
vaccine can be administered as a minigene construct or as dendritic
cells which have been loaded with the peptide epitopes in
vitro.
Example 27
Use of Peptides to Evaluate an Immune Response
[0642] Peptides of the invention may be used to analyze an immune
response for the presence of specific antibodies, CTL or HTL
directed to 158P3D2. 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.
[0643] In this example highly sensitive human leukocyte antigen
tetrameric complexes ("tetramers") are used for a cross-sectional
analysis of, for example, 158P3D2 HLA-A*0201-specific CTL
frequencies from HLA A*0201-positive individuals at different
stages of disease or following immunization comprising an 158P3D2
peptide containing an A*0201 motif. Tetrameric complexes are
synthesized as described (Musey et al., N. Engl. J. Med. 337:1267,
1997). Briefly, purified HLA heavy chain (A*0201 in this example)
and .beta.2-microglobulin are synthesized by means of a prokaryotic
expression system. The heavy chain is modified by deletion of the
transmembrane-cytosolic tail and COOH-terminal addition of a
sequence containing a BirA enzymatic biotinylation site. The heavy
chain, .beta.2-microglobulin, and peptide are refolded by dilution.
The 45-kD refolded product is isolated by fast protein liquid
chromatography and then biotinylated by BirA in the presence of
biotin (Sigma, St. Louis, Mo.), adenosine 5' triphosphate and
magnesium. Streptavidin-phycoerythrin conjugate is added in a 1:4
molar ratio, and the tetrameric product is concentrated to 1 mg/ml.
The resulting product is referred to as tetramer-phycoerythrin.
[0644] 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 158P3D2 epitope, and thus the
status of exposure to 158P3D2, or exposure to a vaccine that
elicits a protective or therapeutic response.
Example 28
Use of Peptide Epitopes to Evaluate Recall Responses
[0645] 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 158P3D2-associated disease or who have been
vaccinated with an 158P3D2 vaccine.
[0646] For example, the class I restricted CTL response of persons
who have been vaccinated may be analyzed. The vaccine may be any
158P3D2 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.
[0647] 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 (50U/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.
[0648] 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 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).
[0649] 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).
[0650] 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.
[0651] Cytolytic activity is determined in a standard 4-h, split
well .sup.51Cr release assay using U-bottomed 96 well plates
containing 3,000 targets/well. Stimulated PBMC are tested at
effector/target (E/T) ratios of 20-50:1 on day 14. Percent
cytotoxicity is determined from the formula:
100.times.[(experimental release-spontaneous release)/maximum
release-spontaneous release)]. Maximum release is determined by
lysis of targets by detergent (2% Triton X-100; Sigma Chemical Co.,
St. Louis, Mo.). Spontaneous release is <25% of maximum release
for all experiments.
[0652] The results of such an analysis indicate the extent to which
HLA-restricted CTL populations have been stimulated by previous
exposure to 158P3D2 or an 158P3D2 vaccine.
[0653] 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/mil synthetic peptide of the invention, whole 158P3D2
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
Induction of Specific CTL Response in Humans
[0654] 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:
[0655] A total of about 27 individuals are enrolled and divided
into 3 groups:
[0656] Group I: 3 subjects are injected with placebo and 6 subjects
are injected with 5 .mu.g of peptide composition;
[0657] Group II: 3 subjects are injected with placebo and 6
subjects are injected with 50 .mu.g peptide composition;
[0658] Group III: 3 subjects are injected with placebo and 6
subjects are injected with 500 .mu.g of peptide composition.
[0659] After 4 weeks following the first injection, all subjects
receive a booster inoculation at the same dosage.
[0660] 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.
[0661] Safety: The incidence of adverse events is monitored in the
placebo and drug treatment group and assessed in terms of degree
and reversibility.
[0662] 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.
[0663] The vaccine is found to be both safe and efficacious.
Example 30
Phase II Trials in Patients Expressing 158P3D2
[0664] Phase II trials are performed to study the effect of
administering the CTL-HTL peptide compositions to patients having
cancer that expresses 158P3D2. The main objectives of the trial are
to determine an effective dose and regimen for inducing CTLs in
cancer patients that express 158P3D2, 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:
[0665] 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.
[0666] 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 158P3D2.
[0667] 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 158P3D2-associated disease.
Example 31
Induction of CTL Responses Using a Prime Boost Protocol
[0668] 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 Example 23, 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.
[0669] For example, the initial immunization may be performed using
an expression vector, such as that constructed in Example 22 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.
[0670] Analysis of the results indicates that a magnitude of
response sufficient to achieve a therapeutic or protective immunity
against 158P3D2 is generated.
Example 32
Administration of Vaccine Compositions Using Dendritic Cells
(DC)
[0671] 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
158P3D2 protein from which the epitopes in the vaccine are
derived.
[0672] 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.
[0673] 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.
[0674] 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.
[0675] Ex vivo Activation of CTL/HTL Responses
[0676] Alternatively, ex vivo CTL or HTL responses to 158P3D2
antigens can be induced by incubating, in tissue culture, the
patient's, or genetically compatible, CTL or HTL precursor cells
together with a source of APC, such as DC, and immunogenic
peptides. After an appropriate incubation time (typically about
7-28 days), in which the precursor cells are activated and expanded
into effector cells, the cells are infused into the patient, where
they will destroy (CTL) or facilitate destruction (HTL) of their
specific target cells, i.e., tumor cells.
Example 33
An Alternative Method of Identifying and Confirming Motif-Bearing
Peptides
[0677] 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. 158P3D2.
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.
[0678] 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
158P3D2 to isolate peptides corresponding to 158P3D2 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.
[0679] As appreciated by one in the art, one can perform a similar
analysis on a cell bearing more than one HLA allele and
subsequently determine peptides specific for each HLA allele
expressed. Moreover, one of skill would also recognize that means
other than transfection, such as loading with a protein antigen,
can be used to provide a source of antigen to the cell.
Example 34
Complementary Polynucleotides
[0680] Sequences complementary to the 158P3D2-encoding sequences,
or any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring 158P3D2. 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 158P3D2. 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 158P3D2-encoding
transcript.
Example 35
Purification of Naturally-Occurring or Recombinant 158P3D2 Using
158P3D2 Specific Antibodies
[0681] Naturally occurring or recombinant 158P3D2 is substantially
purified by immunoaffinity chromatography using antibodies specific
for 158P3D2. An immunoaffinity column is constructed by covalently
coupling anti-158P3D2 antibody to an activated chromatographic
resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia
Biotech). After the coupling, the resin is blocked and washed
according to the manufacturer's instructions.
[0682] Media containing 158P3D2 are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of 158P3D2 (e.g., high ionic strength
buffers in the presence of detergent). The column is eluted under
conditions that disrupt antibody/158P3D2 binding (e.g., a buffer of
pH 2 to pH 3, or a high concentration of a chaotrope, such as urea
or thiocyanate ion), and GCR.P is collected.
Example 36
Identification of Molecules Which Interact with 158P3D2
[0683] 158P3D2, 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
158P3D2, washed, and any wells with labeled 158P3D2 complex are
assayed. Data obtained using different concentrations of 158P3D2
are used to calculate values for the number, affinity, and
association of 158P3D2 with the candidate molecules.
Example 37
In vivo Assay for 158P3D2 Tumor Growth Promotion
[0684] The effect of the 158P3D2 protein on tumor cell growth is
evaluated in vivo by gene overexpression in tumor-bearing mice. For
example, SCID mice are injected subcutaneously on each flank with
1.times.10.sup.6 of either NIH-3T3 cells, bladder cancer lines
(UM-UC3, J82 or SCABER) and kidney cancer cells (CaKil, 769-P)
containing tkNeo empty vector or 158P3D2. At least two strategies
may be used: (1) Constitutive 158P3D2 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), adenovirus (such as Adenovirus
2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a
retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or from
heterologous mammalian promoters, e.g., the actin promoter or an
immunoglobulin promoter, provided such promoters are compatible
with the host cell systems, and (2) Regulated expression under
control of an inducible vector system, such as ecdysone, tet, etc.,
provided such promoters are compatible with the host cell systems.
Tumor volume is then monitored at the appearance of palpable tumors
and followed over time to determine if 158P3D2-expressing cells
grow at a faster rate and whether tumors produced by
158P3D2-expressing cells demonstrate characteristics of altered
aggressiveness (e.g. enhanced metastasis, vascularization, reduced
responsiveness to chemotherapeutic drugs).
[0685] Additionally, mice can be implanted with 1.times.10.sup.5 of
the same cells orthotopically to determine if 158P3D2 has an effect
on local growth in the prostate or on the ability of the cells to
metastasize, specifically to lungs, lymph nodes, and bone
marrow.
[0686] The assay is also useful to determine the 158P3D2 inhibitory
effect of candidate therapeutic compositions, such as for example,
158P3D2 intrabodies, 158P3D2 antisense molecules and ribozymes.
Example 38
158P3D2 Monoclonal Antibody-Mediated Inhibition of Bladder Tumors
in vivo
[0687] The significant expression of 158P3D2 in cancer tissues, its
restrictive expression in normal tissues together with its expected
cell surface expression makes 158P3D2 an excellent target for
antibody therapy. Similarly, 158P3D2 is a target for T cell-based
immunotherapy. Thus, the therapeutic efficacy of anti-158P3D2 mAbs
in human bladder cancer xenograft mouse models is evaluated by
using recombinant cell lines UM-UC3-158P3D2 and J28-158P3D2.
Similarly, anti-158P3D2 mAbs are evaluated in human kidney cancer
xenograft models such as AGS-K3 and AGS-K6 and in recombinant
kidney cell lines such as Caki-158P3D2.
[0688] Antibody efficacy on tumor growth and metastasis formation
is studied, e.g., in a mouse orthotopic bladder cancer xenograft
models and mouse kidney xenograft models. The antibodies can be
unconjugated, as discussed in this Example, or can be conjugated to
a therapeutic modality, as appreciated in the art. Anti-158P3D2
mAbs inhibit formation of both Caki-158P3D2 and UMUC3-158P3D2 tumor
xenografts. Anti-158P3D2 mAbs also retard the growth of established
orthotopic tumors and prolonged survival of tumor-bearing mice.
These results indicate the utility of anti-158P3D2 mAbs in the
treatment of local and advanced stages of kidney and bladder
cancer. (See, e.g., (Saffran, D., et al., PNAS 10: 1073-1078 or
www.pnas.org/cgi/doi/10.1073/pnas.051624698). These results
indicate the use of anti-158P3D2 mAbs in the treatment of bladder
and kidney cancer.
[0689] Administration of the anti-158P3D2 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 158P3D2
as an attractive target for immunotherapy and demonstrate the
therapeutic potential of anti-158P3D2 mAbs for the treatment of
local and metastatic prostate cancer. This example demonstrates
that unconjugated 158P3D2 monoclonal antibodies are effective to
inhibit the growth of human bladder tumor xenografts and human
kidney xenografts grown in SCID mice; accordingly a combination of
such efficacious monoclonal antibodies is also effective.
[0690] Tumor Inhibition Using Multiple Unconjugated 158P3D2 mAbs
Materials and Methods
[0691] 158P3D2 Monoclonal Antibodies:
[0692] Monoclonal antibodies are raised against 158P3D2 as
described in Example 11. The antibodies are characterized by ELISA,
Western blot, FACS, and immunoprecipitation for their capacity to
bind 158P3D2. Epitope mapping data for the anti-158P3D2 mAbs, as
determined by ELISA and Western analysis, recognize epitopes on the
158P3D2 protein. Immunohistochemical analysis of prostate cancer
tissues and cells with these antibodies is performed.
[0693] 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 LAPC-9 prostate tumor xenografts.
[0694] Cancer Xenografts and Cell Lines
[0695] Human cancer xenograft models, such as bladder and kidney
cancer models, as well as ICR-severe combined immunodeficient
(SCID) mice injected with human cell lines expressing or lacking
158P3D2 are used to confirm the role of 158P3D2 in tumor growth and
progression. The bladder xenograft is passaged in 6- to 8-week-old
male ICR-severe combined immunodeficient (SCID) mice (Taconic
Farms) by s.c. trocar implant (Craft, N., et al., supra). The
AGS-K3 and AGS-K6 kidney xenografts are also passaged by
subcutaneous implants in 6- to 8-week old SCID mice. Single-cell
suspensions of tumor cells are prepared as described in Craft, et
al. The bladder and kidney carcinoma cell lines UM-UC3, SCABER,
J82, 769-P and CaKi (American Type Culture Collection) are
maintained in DMEM supplemented with L-glutamine and 10% FBS.
[0696] A UMUC3-158P3D2, J82-158P3D2, 769-P-158P3D2 and CaKi-158P3D2
cell populations are generated by retroviral gene transfer as
described in Hubert, R. S., et al., STEAP: a prostate-specific
cell-surface antigen highly expressed in human prostate tumors.
Proc Natl Acad Sci USA, 1999. 96(25): p. 14523-8. Anti-158P3D2
staining is detected by using an FITC-conjugated goat anti-mouse
antibody (Southern Biotechnology Associates) followed by analysis
on a Coulter Epics-XL f low cytometer.
[0697] Xenograft Mouse Models.
[0698] Subcutaneous (s.c.) tumors are generated by injection of
1.times.10 .sup.6 AGS-K3, AGS-K6, A UMUC3-158P3D2, SCABER-158P3D2,
769-P-158P3D2 and CaKi-158P3D2 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 vernier caliper measurements, and the tumor volume is
calculated as length.times.width.times.height. Mice with s.c.
tumors greater than 1.5 cm in diameter are sacrificed. PSA levels
are determined by using a PSA ELISA kit (Anogen, Mississauga,
Ontario). Circulating levels of anti-158P3D2 mAbs are determined by
a capture ELISA kit (Bethyl Laboratories, Montgomery, Tex.). (See,
e.g., (Saffran, D., et al., PNAS 10:1073-1078 or
www.pnas.org/cgi/doi/10.1073/pnas.051624698)
[0699] Orthotopic injections are performed under anesthesia by
using ketamine/xylazine. For bladder orthotopic studies, an
incision is made through the abdominal muscles to expose the
bladder. Cells (5.times.10.sup.5) mixed with Matrigel are injected
into the bladder in a 10-.mu.l volume. For kidney orthopotic
models, an incision is made through the abdominal muscles to expose
the kidney. AGS-K3 or AGS-K6 cells mixed with Matrigel are injected
under the kidney capsule. The mice are segregated into groups for
the appropriate treatments, with anti-158P3D2 or control mAbs being
injected i.p.
[0700] Anti-158P3D2 mAbs Inhibit Growth of 158P3D2-Expressing
Xenograft-Cancer Tumors
[0701] The effect of anti-158P3D2 mAbs on tumor formation is tested
by using bladder and kidney orthotopic models. As compared with the
s.c. tumor model, the orthotopic model, which requires injection of
tumor cells directly in the mouse bladder or kidney, respectively,
results in a local tumor growth, development of metastasis in
distal sites, deterioration of mouse health, and subsequent death
(Saffran, D., et al., PNAS supra; Fu, X., et al., Int J Cancer,
1992. 52(6): p. 987-90; Kubota, T., J Cell Biochem, 1994. 56(1): p.
4-8). The features make the orthotopic model more representative of
human disease progression and allowed us to follow the therapeutic
effect of mAbs on clinically relevant end points.
[0702] Accordingly, tumor cells are injected into the mouse bladder
or kidney, and 2 days later, the mice are segregated into two
groups and treated with either: a) 200-500 .mu.g, of anti-158P3D2
Ab, or b) PBS three times per week for two to five weeks.
[0703] A major advantage of the orthotopic cancer model is the
ability to study the development of metastases. Formation of
metastasis in mice bearing established orthotopic tumors is studies
by IHC analysis on lung sections using an antibody against BTA, a
bladder specific antigen (Hubert, R. S., et al., Proc Natl Acad Sci
USA, 1999. 96(25): p. 14523-8) or anti-G250 antibody for kidney
cancer models.
[0704] Mice bearing established orthotopic tumors are administered
1000 .mu.g injections of either anti-158P3D2 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 bladder/kidney and
lungs are analyzed for the presence of tumor cells by IHC
analysis.
[0705] These studies demonstrate a broad anti-tumor efficacy of
anti-158P3D2 antibodies on initiation and progression of bladder
and kidney cancer in xenograft mouse models. Anti-158P3D2
antibodies inhibit tumor formation of both androgen-dependent and
androgen-independent tumors as well as retarding the growth of
already established tumors and prolong the survival of treated
mice. Moreover, anti-158P3D2 mAbs demonstrate a dramatic inhibitory
effect on the spread of local prostate tumor to distal sites, even
in the presence of a large tumor burden. Thus, anti-158P3D2 mAbs
are efficacious on major clinically relevant end points (tumor
growth), prolongation of survival, and health.
Example 39
Therapeutic and Diagnostic Use of Anti-158P3D2 Antibodies in
Humans
[0706] Anti-158P3D2 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-158P3D2 mAb show strong extensive staining in
carcinoma but significantly lower or undetectable levels in normal
tissues. Detection of 158P3D2 in carcinoma and in metastatic
disease demonstrates the usefulness of the mAb as a diagnostic
and/or prognostic indicator. Anti-158P3D2 antibodies are therefore
used in diagnostic applications such as immunohistochemistry of
kidney biopsy specimens to detect cancer from suspect patients.
[0707] As determined by flow cytometry, anti-158P3D2 mAb
specifically binds to carcinoma cells. Thus, anti-158P3D2
antibodies are used in diagnostic whole body imaging applications,
such as radioimmunoscintigraphy and radioimmunotherapy, (see, e.g.,
Potamianos S., et. al. Anticancer Res 20(2A):925-948 (2000)) for
the detection of localized and metastatic cancers that exhibit
expression of 158P3D2. Shedding or release of an extracellular
domain of 158P3D2 into the extracellular milieu, such as that seen
for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology
27:563-568 (1998)), allows diagnostic detection of 158P3D2 by
anti-158P3D2 antibodies in serum and/or urine samples from suspect
patients.
[0708] Anti-158P3D2 antibodies that specifically bind 158P3D2 are
used in therapeutic applications for the treatment of cancers that
express 158P3D2. Anti-158P3D2 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-158P3D2 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., Example
38). Conjugated and unconjugated anti-158P3D2 antibodies are used
as a therapeutic modality in human clinical trials either alone or
in combination with other treatments as described in following
Examples.
Example 40
Human Clinical Trials for the Treatment and Diagnosis of Human
Carcinomas Through Use of Human Anti-158P3D2 Antibodies in vivo
[0709] Antibodies are used in accordance with the present invention
which recognize an epitope on 158P3D2, and are used in the
treatment of certain tumors such as those listedin Table I. Based
upon a number of factors, including 158P3D2 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.
[0710] I.) Adjunctive therapy: In adjunctive therapy, patients are
treated with anti-158P3D2 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-158P3D2
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-158P3D2 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).
[0711] II.) Monotherapy: In connection with the use of the
anti-158P3D2 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.
[0712] III.) Imaging Agent: Through binding a radionuclide (e.g.,
iodine or yttrium (I.sup.131, Y.sup.90) to anti-158P3D2 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 158P3D2. In connection with the use of the anti-158P3D2
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)-158P3D2 antibody is used as an imaging agent in a
Phase I human clinical trial in patients having a carcinoma that
expresses 158P3D2 (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
[0713] Dose and Route of Administration
[0714] 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-158P3D2
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-158P3D2 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-158P3D2 antibodies that are fully human
antibodies, as compared to the chimeric antibody, have slower
clearance; accordingly, dosing in patients with such fully human
anti-i 58P3D2 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.
[0715] Three distinct delivery approaches are useful for delivery
of anti-158P3D2 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.
[0716] Clinical Development Plan (CDP)
[0717] Overview: The CDP follows and develops treatments of
anti-158P3D2 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-158P3D2 antibodies. As will be appreciated, one criteria
that can be utilized in connection with enrollment of patients is
158P3D2 expression levels in their tumors as determined by
biopsy.
[0718] 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 158P3D2. Standard tests and follow-up are utilized to
monitor each of these safety concerns. Anti-158P3D2 antibodies are
found to be safe upon human administration.
Example 41
Human Clinical Trial Adjunctive Therapy with Human Anti-158P3D2
Antibody and Chemotherapeutic Agent
[0719] A phase I human clinical trial is initiated to assess the
safety of six intravenous doses of a human anti-158P3D2 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-158P3D2 antibodies when utilized as an adjunctive therapy
to an antineoplastic or chemotherapeutic agent, such as cisplatin,
topotecan, doxorubicin, adriamycin, taxol, or the like, is
assessed. The trial design includes delivery of six single doses of
an anti-158P3D2 antibody with dosage of antibody escalating from
approximately about 25 mg/m.sup.2 to about 275 mg/m.sup.2 over the
course of the treatment in accordance with the following
schedule:
2 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)
[0720] 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 158P3D2. 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.
[0721] The anti-158P3D2 antibodies are demonstrated to be safe and
efficacious, Phase II trials confirm the efficacy and refine
optimum dosing.
Example 42
Human Clinical Trial: Monotherapy with Human Anti-158P3D2
Antibody
[0722] Anti-158P3D2 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-158P3D2
antibodies.
Example 43
Human Clinical Trial: Diagnostic Imaging with Anti-158P3D2
Antibody
[0723] 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-158P3D2 antibodies as
a diagnostic imaging agent. The protocol is designed in a
substantially similar manner to those described in the art, such as
in Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991). The
antibodies are found to be both safe and efficacious when used as a
diagnostic modality.
Example 44
Homology Comparison of 158P3D2 to Known Sequences
[0724] The 158P3D2 gene is identical to a previously cloned and
sequenced gene, namely a novel protein similar to otoferlin and
dysferlin, isoform 1 (gi 7671662), showing 100% identity to that
protein (FIG. 4B). The 158P3D2 protein shows 65% homology and 45%
identity to human otoferlin long isoform (gi 10119916), and 45%
identity and 45% homology to the mouse otoferlin (gi 13994207)
(FIGS. 4C and 4D, respectively). The 158P3D2 protein consists of
328 amino acids, with calculated molecular weight of 38.4 kDa, and
pI of 8.64. 158P3D2 is a cell surface protein, with possible
localization to the endoplasmic reticulum fraction. The 158P3D2
protein contains a single transmembrane domain at aa 145. Motif
analysis revealed the presence of several known motifs, including a
C2 domains located at the amino acids 122-144 of the 158P3D2
protein, an aminoacyl-transfer RNA synthetases class II motif at aa
91-115. Pfam analysis suggests that 158P3D2 has a slight likelihood
of belonging to the chemokine receptor family (Table XXII).
[0725] C2 domains are Ca2+-binding motifs present in a variety of
proteins including phospholipases, protein kinases C and
synaptotamins (Murakami M, et al Biochim Biophys Acta. 2000,
1488:159; Marqueze B et al, Biochimie. 2000, 82:409). They are
about 116 amino-acid residues long, and function in
calcium-dependent phospholipid binding (Stahelin RV, Cho W. Biochem
J. 2001, 359:679). Since some C2-related domains are found in
proteins that do not bind calcium, C2 domains have been assigned an
additional function, namely inter-molecular association, such as
binding to inositol-1,3,4,5-tetraphosphate (Mehrotra B et al,
Biochemistry. 2000, 39:9679). C2 domains are also instrumental in
targeting proteins to specific subcellular locations. In
particular, recent studies have shown that the C2 domain of PLA
mediates the translocation of PLA from the cytosol to the golgi in
response to calcium (Evans J H et al, J. Biol. Chem. 2001,
276:30150). In addition to affecting localization and protein
association, C2 domain proteins have been reported to regulate
critical cellular functions, including proliferation, a key
component of tumoriogenesis (Koehler J A, Moran M F. Cell Growth
Differ. 2001, 12:551).
[0726] Aminoacyl-tRNA synthetases are enzymes that activate amino
acids and transfer them to specific tRNA molecules as the first
step in protein biosynthesis (Fabrega C et al, Nature. 2001,
411:110). In eukaryotes two aminoacyl-tRNA synthetases exist for
each of the 20 essential amino acid: a cytosolic form and a
mitochondrial form. The class II synthetases are specific for
alanine, asparagine, aspartic acid, glycine, histidine, lysine,
phenylalanine, proline, serine, and threonine. Since aminoacyl
transfer RNA synthetases regulate protein synthesis, it is clear
that they also regulate cell proliferation and maintain the
accuracy of protein synthesis (Jakubowski H, Goldman E. Microbiol
Rev. 1992, 56:412). This characteristic of aminoacyl transfer RNA
synthetases was used to develop reagents with anti-tumor effects in
vitro (Laske R et al, Arch Pharm. 1991, 324:153). The relevance of
aminoacyl transfer RNA synthetases to cell survival and growth was
demonstrated in cells expressing mutant lysyl-tRNA synthetase.
Mutation in lysyl-tRNA synthetases resulted in apoptosis of BHK21
cells (Fukushima et al, Genes Cells. 1996, 1:1087).
[0727] Based on the information above, 158P3D2 plays an important
role in several biological processes, including protein synthesis,
cell growth, metabolism, and survival.
[0728] Several isoforms of 158P3D2 have been identified (FIG. 1).
While both variants var2a and var5a do not contain a transmembrane
domain, var2a still maintains the C2 domain important for protein
interaction, localization and calcium binding. Variant var2b still
maintains the transmembrane domain, but fails to exhibit a
well-identified C2 domains. In addition, two variants, var3 and
var4 contain a point mutations at amino acid 103 and 102,
respectively, relative to the 158P3D2 var1 protein. These single
amino acid changes do not significantly alter the predicted
localization or motifs associated with 15 8P3D2 var1.
[0729] Accordingly, when any of the 158P3D2 variants function as
regulators of protein synthesis, cell growth, metabolism, and
survival, 158P3D2 is used for therapeutic, diagnostic, prognostic
and/or preventative purposes.
Example 45
Identification and Confirmation of Potential Signal Transduction
Pathways
[0730] 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, C2-domain
containing proteins have been reported to associate with signaling
molecules and regulate signaling pathways including mitogenic
cascades (Chow A et al, FEBS Lett. 2000;469:88; Walker E H et al,
Nature. 1999,402:313). Using immunoprecipitation and Western
blotting techniques, proteins are identified that associate with
158P3D2 and mediate signaling events. Several pathways known to
play a role in cancer biology can be regulated by 158P3D2,
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.).
[0731] To confirm that 158P3D2 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 arc listed
below.
[0732] 1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK;
growth/apoptosis/stress
[0733] 2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK;
growth/differentiation
[0734] 3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC;
growth/apoptosis/stress
[0735] 4. ARE-luc, androgen receptor; steroids/MAPK;
growth/differentiation/apoptosis
[0736] 5. p53-luc, p53; SAPK; growth/differentiation/apoptosis
[0737] 6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress
[0738] 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.
[0739] Signaling pathways activated by 158P3D2 are mapped and used
for the identification and validation of therapeutic targets. When
158P3D2 is involved in cell signaling, it is used as target for
diagnostic, prognostic, preventative and/or therapeutic
purposes.
Example 46
Involvement in Tumor Progression
[0740] Based on the reported effect of C2 domains and tRNA
synthetases on cell growth, survival, protein regulation and
signaling, the 158P3D2 gene can contribute to the growth of cancer
cells. The role of 158P3D2 in tumor growth is confirmed in a
variety of primary and transfected cell lines including, bladder
and kidney cell lines, as well as NIH 3T3 cells engineered to
stably express 158P3D2. Parental cells lacking 158P3D2 and cells
expressing 158P3D2 are evaluated for cell growth using a
well-documented proliferation assay (Fraser S P, Grimes J A,
Djamgoz M B. Prostate. 2000;44:61, Johnson D E, Ochieng J, Evans S
L. Anticancer Drugs. 1996, 7:288).
[0741] To confirm the role of 158P3D2 in the transformation
process, its effect in colony forming assays is investigated.
Parental NIH-3T3 cells lacking 158P3D2 are compared to NIH-3T3
cells expressing 158P3D2, using a soft agar assay under stringent
and more permissive conditions (Song Z. et al. Cancer Res.
2000;60:6730).
[0742] To confirm the role of 158P3D2 in invasion and metastasis of
cancer cells, a well-established assay is used, e.g., a Transwell
Insert System assay (Becton Dickinson) (Cancer Res. 1999; 59:6010).
Control cells, including bladder and kidney cell lines lacking
158P3D2 are compared to cells expressing 158P3D2. Cells are loaded
with the fluorescent dye, calcein, and plated in the top well of
the Transwell insert coated with a basement membrane analog.
Invasion is determined by fluorescence of cells in the lower
chamber relative to the fluorescence of the entire cell
population.
[0743] 158P3D2 can also play a role in cell cycle and apoptosis.
Parental cells and cells expressing 158P3D2 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 158P3D2, 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 158P3D2 can play a
critical role in regulating tumor progression and tumor load.
[0744] When 158P3D2 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 47
Involvement in Angiogenesis
[0745] 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 158P3D2 on cellular functions and protein expression,
158P3D2 plays a role in angiogenesis. In addition, recent studies
have associated human tyrosyl- and tryptophanyl-tRNA synthetases to
angiogenesis (Otani A et al, Proc Natl Acad Sci U S A. 2002,
99:178). 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 158P3D2
in angiogenesis, enhancement or inhibition, is confirmed.
[0746] For example, endothelial cells engineered to express 158P3D2
are evaluated using tube formation and proliferation assays. The
effect of 158P3D2 is also confirmed in animal models in vivo. For
example, cells either expressing or lacking 158P3D2 are implanted
subcutaneously in immunocompromised mice. Endothelial cell
migration and angiogenesis are evaluated 5-15 days later using
immunohistochemistry techniques. 158P3D2 affects angiogenesis, and
it is used as a target for diagnostic, prognostic, preventative
and/or therapeutic purposes
Example 48
Regulation of Protein Synthesis
[0747] The presence of a tRNA synthetase motif indicates that
158P3D2 regulates protein synthesis. Regulation of protein
synthesis is confirmed, e.g., by studying gene expression in cells
expressing or lacking 158P3D2. For this purpose, cells are labeled
with .sup.3H-Leucine and evaluated for the incorporation of the
isotope (Tsurusaki Y, Yamaguchi M. Int J Mol Med. 2000, 6:295). For
examples cells lacking or expressing 158P3D2 are incubated with
.sup.3H-Leucine for 6 hours in the presence of absence of stimuli
such as growth factors, serum, phorbol esters. Cells are lysed and
evaluated for .sup.3H-Leucine incorporation using a beta-counter
(cpm).
[0748] Thus, 158P3D2 regulates protein synthesis, it is used as a
target for diagnostic, prognostic, preventative and/or therapeutic
purposes.
Example 49
Protein-Protein Association
[0749] C2 domain-containing proteins have been shown to mediate
protein-protein association (Murakami M, et al Biochim Biophys
Acta. 2000, 1488:159; Chow A et al, FEBS Lett. 2000;469:88). Using
immunoprecipitation techniques as well as two yeast hybrid systems,
proteins are identified that associate with 158P3D2.
Immunoprecipitates from cells expressing 158P3D2 and cells lacking
158P3D2 are compared for specific protein-protein associations.
[0750] Studies are performed to confirm the extent of association
of 158P3D2 with effector molecules, such as signaling
intermediates, nuclear proteins, transcription factors, kinases,
phosophates, etc. Studies comparing 158P3D2 positive and 158P3D2
negative cells as well as studies comparing unstimulated/resting
cells and cells treated with epithelial cell activators, such as
cytokines, growth factors, androgen and anti-integrin Ab reveal
unique interactions.
[0751] 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 158P3D2-DNA-binding domain fusion protein and a
reporter construct. Protein-protein interaction is detected by
calorimetric reporter activity. Specific association with effector
molecules and transcription factors directs one of skill to the
mode of action of 158P3D2, 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 158P3D2.
[0752] Thus it is found that 158P3D2 associates with proteins and
small molecules. Accordingly, 158P3D2 and these proteins and small
molecules are used for diagnostic, prognostic, preventative and/or
therapeutic purposes.
[0753] 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.
[0754] The present invention is not to be limited in scope by the
embodiments disclosed herein, which are intended as single
illustrations of individual aspects of the invention, and any that
are functionally equivalent are within the scope of the invention.
Various modifications to the models and methods of the invention,
in addition to those described herein, will become apparent to
those skilled in the art from the foregoing description and
teachings, and are similarly intended to fall within the scope of
the invention. Such modifications or other embodiments can be
practiced without departing from the true scope and spirit of the
invention.
TABLES
[0755]
3TABLE I Tissues that Express 158P3D2 When Malignant Prostate
Bladder Kidney Colon Ovary Lung Breast Pancreas
[0756]
4TABLE 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
[0757]
5TABLE 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 URL
www.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
[0758]
6TABLE IV HLA Class I/II Motifs/Supermotifs TABLE IV (A): HLA Class
I Supermotifs/Motifs POSITION POSITION POSITION C Terminus 2
(Primary 3 (Primary (Primary Anchor) Anchor) Anchor) SUPERMOTIFS A1
TILVMS FWY A2 LIVMATQ IVMATL A3 VSMATLI RK A24 YFWIVLMT FIYWLM B7 P
YILFMWYA 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
[0759] Bolded residues are preferred, italicized residues are less
preferred: A peptide is considered notif-bearing if it has primary
anchors at each primary anchor position for a motif or supermotif
as specified in the above table.
7TABLE 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
[0760]
8TABLE 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 W I VSTCPALIM MH
MH deleterious R WDE DR1 preferred MFLIVWY C CH PAMQ CWD VMATSPLIC
M D AVM deleterious FD GDE DR7 preferred MFLIVWY M W A IVMSACTPL M
N IV deleterious C G GRD G DR3 MOTIFS 1.degree. anchor 1 2 3
1.degree. anchor 4 5 1.degree. anchor 6 motif a LIVMFY D KRH
preferred LIVMFAY DNQEST motif b preferred DR MFLIVWY VMSTACPLI
Supermotif
[0761]
9TABLE IV (D) HLA Class I Supermotifs SUPER- MOTIFS POSITION: 1 2 3
4 5 6 7 8 C-terminus A1 1 1 .degree. Anchor TILVMS 2 1 .degree.
Anchor FWY A2 3 1 .degree. Anchor LIVMATQ 4 1 .degree. Anchor
LIVMAT A3 preferred 5 1 .degree. Anchor VSMATLI YFW (4/5) YFW (3/5)
YFW (4/5) P (4/5) 6 1 .degree. Anchor RK deleterious DE (3/5); DE P
(5/5) (4/5) A24 7 1 .degree. Anchor YFWIVLMT 8 1 .degree. Anchor
FIYWLM B7 preferred FWY (5/5) LIVM (3/5) 9 1 .degree. Anchor P FWY
(4/5) FWY (3/5) 10 1 .degree. Anchor 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 11 1 .degree. Anchor RHK 12 1 .degree. Anchor FYLWMIVA B44 13 1
.degree. Anchor ED 14 1 .degree. Anchor FWYLIMVA B58 15 1 .degree.
Anchor ATS 16 1 .degree. Anchor FWYLIVMA B62 17 1 .degree. Anchor
QLIVMP 18 1 .degree. Anchor FWYMIVLA
[0762]
10TABLE IV (E) HLA Class I Motifs 9 or POSITION: 1 2 3 4 5 6 7 8
C-terminus C-terminus A1 9-mer preferred GFY W 19 1 .degree. Anchor
STM DEA YFW P DEQN YFW 20 1 .degree. Anchor Y deleterious DE
RHKLIVMP A G A A1 9-mer preferred GRHK ASTCLIVM 21 1 .degree.
Anchor DEAS GSTC ASTC LIVM DE 22 1 .degree. Anchor Y deleterious A
RHKDEPY DE PQN RHK PG GP FW A1 10-mer preferred YFW 23 1 .degree.
Anchor STM DEAQN A YFWQN PASTC GDE P 24 1 .degree. Anchor Y
deleterious GP RHKGLIVM DE RHK QNA RHKYFW RHK A A1 10-mer preferred
YFW STCLIVM 25 1 .degree. Anchor DEAS A YFW PG G YFW 26 1 .degree.
Anchor Y deletetious RHK RHKDEPY P G PRHK QN FW A2.1 9-mer
preferred YFW 27 1 .degree. Anchor LMIVQAT YFW STC YFW A P 28 1
.degree. Anchor VLIMAT deleterious DEP DERKH RKH DERKH A2.1 10-mer
preferred AYFW 29 1 .degree. Anchor LMIVQAT LVIM G G FYWL VIM 30 1
.degree. Anchor VLIMAT deleterious DEP DE RKHA P RKH DERKH RKH A3
preferred RHK 31 1 .degree. Anchor LMVISATFCGD YFW PRHK YFW A YFW P
32 1 .degree. Anchor KYRHFA deleterious DEP DE A11 preferred A 33 1
.degree. Anchor VTLMISAGNCDF YFW YFW A YFW YFW P 34 1 .degree.
Anchor KRYH deleterious DEP A G A24 9-mer preferred YFWRHK 35 1
.degree. Anchor YFWM STC YFW YFW 36 1 .degree. Anchor FLIW
deleterious DEG DE G QNP DERH G AQN K A24 10-mer preferred 37 1
.degree. Anchor YFWM P YFWP P 38 1 .degree. Anchor FLIW deleterious
GDE QN RHK DE A QN DEA A3101 preferred RHK 39 1 .degree. Anchor
MVTALIS YFW P YFW YFW AP 40 1 .degree. Anchor RK deleterious DEP DE
ADE DE DE DE A3301 preferred 41 1 .degree. Anchor MVALFIST YFW AYFW
42 1 .degree. Anchor RK deleterious GP DE A6801 preferred YFWSTC 43
1 .degree. Anchor AVTMSLI YFWLIV M YFW P 44 1 .degree. Anchor RK
deleterious GP DEG RHK A B0702 preferred RHKFW Y 45 1 .degree.
Anchor P RHK RHK RHK RHK PA 46 1 .degree. Anchor LMFWYAIV
deleterious DEQNP DEP DE DE GDE QN DE B3501 preferred FWYLIV M 47 1
.degree. Anchor P FWY FWY 48 1 .degree. Anchor LMFWYIVA deleterious
AGP G G B51 preferred LIVMFW Y 49 1 .degree. Anchor P FWY STC FWY G
FWY 50 1 .degree. Anchor LIVFWYAM deleterious AGPDER HKSTC DE G
DEQN GDE B5301 preferred LIVMFW Y 51 1 .degree. Anchor P FWY STC
FWY LIVMFWY FWY 52 1 .degree. Anchor IMFWYALV deleterious AGPQN G
RHKQN DE B5401 preferred FWY 53 1 .degree. Anchor P FWYL IVM LIVM
ALIVM FWYAP 54 1 .degree. Anchor ATIVLMFWY deleterious GPQNDE GDES
TC RHKDE DE QNDGE DE
[0763]
11TABLE V 158P3D2 A1, 9mers (variants 1, 2a, 3, 4 and 5a) SEQ. ID
Pos 123456789 Score NO. Table V: 158P3D2 v.1 A1-9-mers 222
FTDMGGNVY 62.500 47 TGEMSSDIY 11.250 219 DLEFTDMGG 4.500 110
ALEEAEFRQ 4.500 237 EAEFELLTV 4.500 247 EAEKRPVGK 3.600 198
AQEAQAGKK 2.700 78 TGEGNFNWR 2.250 259 QPEPLEKPS 2.250 113
EAEFRQPAV 1.800 140 SLELQLPDM 1.800 281 KTFVFFIWR 1.250 303
LTVFLLLVF 1.250 145 LPDMVRGAR 1.250 312 YTIPGQISQ 1.250 69
ETDVHFNSL 1.250 34 NTEDVVLDD 1.125 320 QVIFRPLHK 1.000 166
GAGPRCNLF 1.000 304 TVFLLLVFY 1.000 39 VLDDENPLT 1.000 188
LKEAEDVER 0.900 235 KVEAEFELL 0.900 190 EAEDVEREA 0.900 62
GLEHDKQET 0.900 51 SSDIYVKSW 0.750 2 WIDIFPQDV 0.500 257 RKQPEPLEK
0.500 142 ELQLPDMVR 0.500 283 FVFFIWRRY 0.500 121 VLVLQVWDY 0.500
156 ELCSVQLAR 0.500 154 GPELCSVQL 0.450 97 EREVSVWRR 0.450 242
LLTVEEAEK 0.400 197 EAQEAQAGK 0.400 243 LTVEEAEKR 0.250 90
RFDYLPTER 0.250 49 EMSSDIYVK 0.200 4 DIFPQDVPA 0.200 11 PAPPPVDIK
0.200 123 VLQVWDYDR 0.200 53 DIYVKSWVK 0.200 262 PLEKPSRPK 0.180 75
NSLTGEGNF 0.150 67 KQETDVHFN 0.135 126 VWDYDRISA 0.125 293
RTLVLLLLV 0.125 81 GNFNWRFVF 0.125 277 VNPLKTFVF 0.125 77 LTGEGNFNW
0.125 214 KGRPEDLEF 0.125 270 KTSFNWFVN 0.125 85 WRFVFRFDY 0.125 40
LDDENPLTG 0.125 216 RPEDLEFTD 0.113 298 LLLVLLTVF 0.100 200
EAQAGKKKR 0.100 170 RCNLFRCRR 0.100 109 FALEEAEFR 0.100 276
FVNPLKTFV 0.100 244 TVEEAEKRP 0.090 25 SYELRVVIW 0.090 193
DVEREAQEA 0.090 195 EREAQEAQA 0.090 132 ISANDFLGS 0.075 316
GQISQVIFR 0.075 105 RSGPFALEE 0.075 10 VPAPPPVDI 0.050 71 DVHFNSLTG
0.050 300 LVLLTVFLL 0.050 137 FLGSLELQL 0.050 232 LTGKVEAEF 0.050
294 TLVLLLLVL 0.050 301 VLLTVFLLL 0.050 302 LLTVFLLLV 0.050 227
GNVYILTGK 0.050 297 LLLLVLLTV 0.050 296 VLLLLVLLT 0.050 131
RISANDFLG 0.050 308 LLVFYTIPG 0.050 245 VEEAEKRPV 0.045 143
LQLPDMVRG 0.030 24 ISYELRVVI 0.030 201 AQAGKKKRK 0.030 50 MSSDIYVKS
0.030 116 FRQPAVLVL 0.025 46 LTGEMSSDI 0.025 191 AEDVEREAQ 0.025 95
PTEREVSVW 0.022 59 WVKGLEHDK 0.020 179 LRGWWPVVK 0.020 306
FLLLVFYTI 0.020 157 LCSVQLARN 0.020 230 YILTGKVEA 0.020 309
LVFYTIPGQ 0.020 299 LLVLLTVFL 0.020 17 DIKPRQPIS 0.020 295
LVLLLLVLL 0.020 158 CSVQLARNG 0.015 Table V: 158P3D2 v.2a A1-9mers
180 ETELTVAVF 45.000 203 HIDLENRFY 25.000 101 FSEPQISRG 13.500 138
KADPYVVVS 10.000 46 SLEEEFNHF 9.000 93 YPESEAVLF 4.500 6 DSDGVNLIS
3.750 205 DLENRFYSH 1.800 167 FGEILELSI 1.125 24 EAEVKGTVS 0.900
194 GSDDLIGET 0.750 95 ESEAVLFSE 0.675 57 WLNVFPLYR 0.500 35
KAVATLKIY 0.500 53 HFEDWLNVF 0.450 109 GIPQNRPIK 0.400 153
DTKERYIPK 0.250 201 ETHIDLENR 0.250 1 MDDPGDSDG 0.250 73 GGEEEGSGH
0.225 22 QGEAEVKGT 0.225 37 VATLKIYNR 0.200 30 TVSPKKAVA 0.200 130
LAPADPNGK 0.200 129 NLAPADPNG 0.200 19 IQDQGEAEV 0.150 78 GSGHLVGKF
0.150 175 ISLPAETEL 0.150 216 ANCGLASQY 0.125 134 DPNGKADPY 0.125
77 EGSGHLVGK 0.100 59 NVFPLYRGQ 0.100 162 QLNPIFGEI 0.100 143
VVVSAGRER 0.100 91 LIYPESEAV 0.100 178 PAETELTVA 0.090 170
ILELSISLP 0.090 187 VFEHDLVGS 0.090 45 RSLEEEFNH 0.075 151
RQDTKERYI 0.075 9 GVNLISMVG 0.050 56 DWLNVFPLY 0.050 36 AVATLKIYN
0.050 182 ELTVAVFEH 0.050 132 PADPNGKAD 0.050 198 LIGETHIDL 0.050
169 EILELSISL 0.050 192 LVGSDDLIG 0.050 186 AVFEHDLVG 0.050 79
SGHLVGKFK 0.050 74 GEEEGSGHL 0.045 75 EEEGSGHLV 0.045 223 QYEVWVQQG
0.045 118 LLVRVYVVK 0.040 88 GSFLIYPES 0.030 173 LSISLPAET 0.030
195 SDDLIGETH 0.025 113 NRPIKLLVR 0.025 150 ERQDTKERY 0.025 108
RGIPQNRPI 0.025 29 GTVSPKKAV 0.025 100 LFSEPQISR 0.025 4 PGDSDGVNL
0.025 48 EEEFNHFED 0.022 16 VGEIQDQGE 0.022 199 IGETHIDLE 0.022 98
AVLFSEPQI 0.020 121 RVYVVKATN 0.020 220 LASQYEVWV 0.020 26
EVKGTVSPK 0.020 117 KLLVRVYVV 0.020 27 VKGTVSPKK 0.020 215
RANCGLASQ 0.020 106 ISRGIPQNR 0.015 221 ASQYEVWVQ 0.015 211
YSHHRANCG 0.015 228 VQQGPQEPF 0.015 85 KFKGSFLIY 0.013 112
QNRPIKLLV 0.013 177 LPAETELTV 0.013 110 IPQNRPIKL 0.013 11
NLISMVGEI 0.010 144 VVSAGRERQ 0.010 90 FLIYPESEA 0.010 12 LISMVGEIQ
0.010 99 VLFSEPQIS 0.010 15 MVGEIQDQG 0.010 81 HLVGKFKGS 0.010 82
LVGKFKGSF 0.010 191 DLVGSDDLI 0.010 184 TVAVFEHDL 0.010 20
QDQGEAEVK 0.010 185 VAVFEHDLV 0.010 176 SLPAETELT 0.010 219
GLASQYEVW 0.010 97 EAVLFSEPQ 0.010 154 TKERYIPKQ 0.009 69 GQDGGGEEE
0.007 13 ISMVGEIQD 0.007 115 PIKLLVRVY 0.005 Table V: 158P3D2 v.3
A1-9mers 3 EREVSVRRR 0.450 1 PTEREVSVR 0.225 5 EVSVRRRSG 0.010 7
SVRRRSGPF 0.001 2 TEREVSVRR 0.001 4 REVSVRRRS 0.001 9 RRRSGPFAL
0.000 6 VSVRRRSGP 0.000 8 VRRRSGPFA 0.000 Table V: 158P3D2 v.4
A1-9mers 4 EREVSIWRR 0.450 2 PTEREVSIW 0.022 6 EVSIWRRSG 0.010 1
LPTEREVSI 0.005 3 TEREVSIWR 0.003 7 VSIWRRSGP 0.002 8 SIWRRSGPF
0.001 5 REVSIWRRS 0.001 9 IWRRSGPFA 0.000 Table V: 158P3D2 v.5a
A1-9mers 16 SLDPWSCSY 250.000 28 CVGPGAPSS 0.200 8 YTASLPMTS 0.125
32 GAPSSALCS 0.050 43 AMGPGRGAI 0.050 14 MTSLDPWSC 0.025 27
WCVGPGAPS 0.020 36 SALCSWPAM 0.020 49 GAICFAAAA 0.020 37 ALCSWPAMG
0.020 2 VLQVWDYTA 0.020 39 CSWPAMGPG 0.015 15 TSLDPWSCS 0.015 22
CSYQTWCVG 0.015 20 WSCSYQTWC 0.015 10 ASLPMTSLD 0.015 35 SSALCSWPA
0.015 45 GPGRGAICF 0.013 21 SCSYQTWCV 0.010 1 LVLQVWDYT 0.010 40
SWPAMGPGR 0.010 9 TASLPMTSL 0.010 11 SLPMTSLDP 0.005 31 PGAPSSALC
0.005 38 LCSWPAMGP 0.005 48 RGAICFAAA 0.005 44 MGPGRGAIC 0.005 25
QTWCVGPGA 0.005 6 WDYTASLPM 0.003 41 WPAMGPGRG 0.003 29 VGPGAPSSA
0.003 5 VWDYTASLP 0.003 30 GPGAPSSAL 0.003 33 APSSALCSW 0.003 12
LPMTSLDPW 0.003 47 GRGAICFAA 0.003 4 QVWDYTASL 0.002 24 YQTWCVGPG
0.002 3 LQVWDYTAS 0.002 7 DYTASLPMT 0.001 13 PMTSLDPWS 0.001 42
PAMGPGRGA 0.001 17 LDPWSCSYQ 0.001 18 DPWSCSYQT 0.001 34 PSSALCSWP
0.000 23 SYQTWCVGP 0.000 26 TWCVGPGAP 0.000 19 PWSCSYQTW 0.000 46
PGRGAICFA 0.000
[0764]
12TABLE VI 158P3D2 v.1 A1-10mers SEQ. ID Pos 1234567890 Score NO.
Table VI: 158P3D2 A1, 10mers (variants 1, 2a, 3, 4 and 5a) 259
QPEPLEKPSR 45.000 276 FVNPLKTFVF 5.000 166 GAGPRCNLFR 5.000 235
KVEAEFELLT 4.500 198 AQEAQAGKKK 2.700 39 VLDDENPLTG 2.500 303
LTVFLLLVFY 2.500 17 DIKPRQPISY 2.500 222 FTDMGGNVYI 2.500 78
TGEGNFNWRF 2.250 113 EAEFRQPAVL 1.800 46 LTGEMSSDIY 1.250 69
ETDVHFNSLT 1.250 47 TGEMSSDIYV 1.125 140 SLELQLPDMV 0.900 219
DLEFTDMGGN 0.900 190 EAEDVEREAQ 0.900 244 TVEEAEKRPV 0.900 51
SSDIYVKSWV 0.750 67 KQETDVHFNS 0.675 134 ANDFLGSLEL 0.625 120
AVLVLQVWDY 0.500 302 LLTVFLLLVF 0.500 10 VPAPPPVDIK 0.500 95
PTEREVSVWR 0.450 241 ELLTVEEAEK 0.400 312 YTIPGQISQV 0.250 281
KTFVFFIWRR 0.250 145 LPDMVRGARG 0.250 77 LTGEGNFNWR 0.250 12
APPPVDIKPR 0.250 154 GPELCSVQLA 0.225 216 RPEDLEFTDM 0.225 34
NTEDVVLDDE 0.225 25 SYELRVVIWN 0.225 122 LVLQVWDYDR 0.200 231
ILTGKVEAEF 0.200 197 EAQEAQAGKK 0.200 200 EAQAGKKKRK 0.200 100
VSVWRRSGPF 0.150 105 RSGPFALEEA 0.150 319 SQVIFRPLHK 0.150 80
EGNFNWRFVF 0.125 293 RTLVLLLLVL 0.125 297 LLLLVLLTVF 0.100 144
QLPDMVRGAR 0.100 242 LLTVEEAEKR 0.100 193 DVEREAQEAQ 0.090 247
EAEKRPVGKG 0.090 62 GLEHDKQETD 0.090 245 VEEAEKRPVG 0.090 110
ALEEAEFRQP 0.090 237 EAEFELLTVE 0.090 107 GPFALEEAEF 0.050 15
PVDIKPRQPI 0.050 304 TVFLLLVFYT 0.050 2 WIDIFPQDVP 0.050 76
SLTGEGNFNW 0.050 307 LLLVFYTIPG 0.050 300 LVLLTVFLLL 0.050 295
LVLLLLVLLT 0.050 301 VLLTVFLLLV 0.050 299 LLVLLTVFLL 0.050 261
EPLEKPSRPK 0.050 277 VNPLKTPVFF 0.050 109 FALEEAEFRQ 0.050 81
GNFNWRFVFR 0.050 296 VLLLLVLLTV 0.050 314 IPGQISQVIF 0.050 226
GGNVYILTGK 0.050 131 RISANDFLGS 0.050 97 EREVSVWRRS 0.045 239
EFELLTVEEA 0.045 111 LEEAEFRQPA 0.045 41 DDENPLTGEM 0.045 195
EREAQEAQAG 0.045 178 RLRGWWPVVK 0.040 24 ISYELRVVIW 0.030 139
GSLELQLPDM 0.030 318 ISQVIFRPLH 0.030 224 DMGGNVYILT 0.025 165
NGAGPRCNLF 0.025 282 TFVFFIWRRY 0.025 280 LKTFVFFIWR 0.025 82
NFNWRFVFRF 0.025 171 CNLFRCRRLR 0.025 126 VWDYDRISAN 0.025 128
DYDRISANDF 0.025 141 LELQLPDMVR 0.025 35 TEDVVLDDEN 0.025 74
FNSLTGEGNF 0.025 221 EFTDMGGNVY 0.025 294 TLVLLLLVLL 0.020 38
VVLDDENPLT 0.020 142 ELQLPDMVRG 0.020 53 DIYVKSWVKG 0.020 246
EEAEKRPVGK 0.020 187 KLKEAEDVER 0.020 272 SFNWFVNPLK 0.020 298
LLLVLLTVFL 0.020 Table VI: 158P3D2 v.2a A1-10mers 101 FSEPQISRGI
13.500 138 KADPYVVVSA 10.000 170 ILELSISLPA 4.500 6 DSDGVNLISM
3.750 203 HIDLENRFYS 2.500 129 NLAPADPNGK 2.000 19 IQDQGEAEVK 1.500
199 IGETHIDLEN 1.125 93 YPESEAVLFS 1.125 108 RGIPQNRPIK 1.000 205
DLENRFYSHH 0.900 194 GSDDLIGETH 0.750 215 RANCGLASQY 0.500 99
VLFSEPQISR 0.500 180 ETELTVAVFE 0.450 117 KLLVRVYVVK 0.400 78
GSGHLVGKFK 0.300 201 ETHIDLENRF 0.250 1 MDDPGDSDGV 0.250 73
GGEEEGSGHL 0.225 16 VGEIQDQGEA 0.225 22 QGEAEVKGTV 0.225 167
FGEILELSIS 0.225 75 EEEGSGHLVG 0.225 36 AVATLKIYNR 0.200 30
TVSPKKAVAT 0.200 91 LIYPESEAVL 0.200 178 PAETELTVAV 0.180 24
EAEVKGTVSP 0.180 175 ISLPAETELT 0.150 45 RSLEEEFNHF 0.150 95
ESEAVLFSEP 0.135 112 QNRPIKLLVR 0.125 54 FEDWLNVFPL 0.125 132
PADPNGKADP 0.100 81 HLVGKFKGSF 0.100 162 QLNPIFGEIL 0.100 59
NVFPLYRGQG 0.100 142 YVVVSAGRER 0.100 227 WVQQGPQEPF 0.100 46
SLEEEFNHFE 0.090 69 GQDGGGEEEG 0.075 140 DPYVVVSAGR 0.050 176
SLPAETELTV 0.050 197 DLIGETHIDL 0.050 35 KAVATLKIYN 0.050 29
GTVSPKKAVA 0.050 185 VAVFEHDLVG 0.050 191 DLVGSDDLIG 0.050 109
GIPQNRPIKL 0.050 148 GRERQDTKER 0.045 74 GEEEGSGHLV 0.045 48
EEEFNHFEDW 0.045 26 EVKGTVSPKK 0.040 221 ASQYEVWVQQ 0.030 34
KKAVATLKIY 0.025 195 SDDLIGETHI 0.025 77 EGSGHLVGKF 0.025 56
DWLNVFPLYR 0.025 133 ADPNGKADPY 0.025 202 THIDLENRFY 0.025 127
ATNLAPADPN 0.025 183 LTVAVFEHDL 0.025 189 EHDLVGSDDL 0.025 47
LEEEFNHFED 0.022 76 EEGSGHLVGK 0.020 186 AVFEHDLVGS 0.020 217
NCGLASQYEV 0.020 172 ELSISLPAET 0.020 97 EAVLFSEPQI 0.020 158
YIPKQLNPIF 0.020 57 WLNVFPLYRG 0.020 146 SAGRERQDTK 0.020 18
EIQDQGEAEV 0.020 219 GLASQYEVWV 0.020 151 RQDTKERYIP 0.015 13
ISMVGEIQDQ 0.015 145 VSAGRERQDT 0.015 211 YSHHRANCGL 0.015 84
GKFKGSFLIY 0.013 79 SGHLVGKFKG 0.013 114 RPIKLLVRVY 0.013 164
NPIFGEILEL 0.013 8 DGVNLISMVG 0.013 103 EPQISRGIPQ 0.013 51
FNHFEDWLNV 0.013 4 PGDSDGVNLI 0.013 179 AETELTVAVF 0.010 92
IYPESEAVLF 0.010 174 SISLPAETEL 0.010 184 TVAVFEHDLV 0.010 90
FLIYPESEAV 0.010 11 NLISMVGEIQ 0.010 98 AVLFSEPQIS 0.010 121
RVYVVKATNL 0.010 37 VATLKIYNRS 0.010 143 VVVSAGRERQ 0.010 220
LASQYEVWVQ 0.010 130 LAPADPNGKA 0.010 105 QISRGIPQNR 0.010 Table
VI: 158P3D2 v.3 A1-10mers 2 PTEREVSVRR 0.450 4 EREVSVRRRS 0.045 1
LPTEREVSVR 0.025 7 VSVRRRSGPF 0.015 6 EVSVRRRSGP 0.001 3 TEREVSVRRR
0.001 5 REVSVRRRSG 0.001 8 SVRRRSGPFA 0.000 9 VRRRSGPFAL 0.000 10
RRRSGPFALE 0.000 Table VI: 158P3D2 v.4 A1-10mers 3 PTEREVSIWR 1.125
8 VSIWRRSGPF 0.150 5 EREVSIWRRS 0.045 1 YLPTEREVSI 0.020 2
LPTEREVSIW 0.003 7 EVSIWRRSGP 0.001 4 TEREVSIWRR 0.001 6 REVSIWRRSG
0.001 9 SIWRRSGPFA 0.000 10 IWRRSGPFAL 0.000 Table VI: 158P3D2 v.5a
A1-10mers 17 SLDPWSCSYQ 5.000 16 TSLDPWSCSY 0.750 40 CSWPAMGPGR
0.300 45 MGPGRGAICF 0.125 6 VWDYTASLPM 0.125 29 CVGPGAPSSA 0.100 44
AMGPGRGAIC 0.100 11 ASLPMTSLDP 0.075 36 SSALCSWPAM 0.030 15
MTSLDPWSCS 0.025 9 YTASLPMTSL 0.025 28 WCVGPGAPSS 0.020 2
LVLQVWDYTA 0.020 37 SALCSWPAMG 0.020 21 WSCSYQTWCV 0.015 32
PGAPSSALCS 0.013 1 VLVLQVWDYT 0.010 12 SLPMTSLDPW 0.010 39
LCSWPAMGPG 0.010 3 VLQVWDYTAS 0.010 33 GAPSSALCSW 0.010 22
SCSYQTWCVG 0.010 49 RGAICFAAAA 0.005 38 ALCSWPAMGP 0.005 13
LPMTSLDPWS 0.005 31 GPGAPSSALC 0.005 23 CSYQTWCVGP 0.003 4
LQVWDYTASL 0.003 25 YQTWCVGPGA 0.003 8 DYTASLPMTS 0.003 42
WPAMGPGRGA 0.003 30 VGPGAPSSAL 0.003 35 PSSALCSWPA 0.002 18
LDPWSCSYQT 0.001 27 TWCVGPGAPS 0.001 48 GRGAICFAAA 0.001 10
TASLPMTSLD 0.001 7 WDYTASLPMT 0.001 43 PAMGPGRGAI 0.001 24
SYQTWCVGPG 0.001 41 SWPAMGPGRG 0.001 14 PMTSLDPWSC 0.001 46
GPGRGAICFA 0.000 26 QTWCVGPGAP 0.000 19 DPWSCSYQTW 0.000 34
APSSALCSWP 0.000 47 PGRGAICFAA 0.000 5 QVWDYTASLP 0.000 20
PWSCSYQTWC 0.000
[0765]
13TABLE VII 158P3D2 A2, 9mers (variants 1, 2a, 3, 4 and 5a) SEQ. ID
Pos 123456789 Score NO. Table VII: 158P3D2 v.1 A2-9mers 302
LLTVFLLLV 1033.404 297 LLLLVLLTV 1006.209 286 FIWRRYWRT 440.113 306
FLLLVFYTI 337.376 301 VLLTVFLLL 255.302 299 LLVLLTVFL 199.738 300
LVLLTVFLL 156.843 276 FVNPLKTFV 153.971 296 VLLLLVLLT 107.808 137
FLGSLELQL 98.267 2 WIDIFPQDV 66.867 38 VVLDDENPL 48.205 48
GEMSSDIYV 27.521 31 VIWNTEDVV 27.109 295 LVLLLLVLL 27.042 313
TIPGQISQV 21.996 39 VLDDENPLT 20.776 294 TLVLLLLVL 20.145 230
YILTGKVEA 11.626 144 QLPDMVRGA 9.370 293 RTLVLLLLV 8.221 30
VVIWNTEDV 5.069 141 LELQLPDMV 4.168 236 VEAEFELLT 3.838 178
RLRGWWPVV 3.684 94 LPTEREVSV 3.165 180 RGWWPVVKL 2.662 228
NVYILTGKV 2.532 305 VFLLLVFYT 2.388 279 PLKTFVFFI 2.240 121
VLVLQVWDY 2.185 240 FELLTVEEA 1.853 133 SANDFLGSL 1.382 124
LQVWDYDRI 1.322 224 DMGGNVYIL 1.091 118 QPAVLVLQV 1.044 46
LTGEMSSDI 1.010 83 FNWRFVFRF 0.941 27 ELRVVIWNT 0.733 140 SLELQLPDM
0.731 234 GKVEAEFEL 0.706 55 YVKSWVKGL 0.692 114 AEFRQPAVL 0.630 24
ISYELRVVI 0.623 52 SDIYVKSWV 0.531 62 GLEHDKQET 0.477 177 RRLRGWWPV
0.456 22 QPISYELRV 0.454 298 LLLVLLTVF 0.442 159 SVQLARNGA 0.435 76
SLTGEGNFN 0.410 235 KVEAEFELL 0.390 183 WPVVKLKEA 0.343 269
PKTSFNWFV 0.333 26 YELRVVIWN 0.312 304 TVFLLLVFY 0.305 186
VKLKEAEDV 0.298 223 TDMGGNVYI 0.295 307 LLLVFYTIP 0.219 4 DIFPQDVPA
0.190 165 NGAGPRCNL 0.139 272 SFNWFVNPL 0.130 308 LLVFYTIPG 0.127
225 MGGNVYILT 0.124 10 VPAPPPVDI 0.116 112 EEAEFRQPA 0.113 135
NDFLGSLEL 0.110 143 LQLPDMVRG 0.109 281 KTFVFFIWR 0.106 171
CNLFRCRRL 0.103 8 QDVPAPPPV 0.097 318 ISQVIFRPL 0.090 87 FVFRFDYLP
0.084 86 RFVFRFDYL 0.076 93 YLPTEREVS 0.069 80 EGNFNWRFV 0.064 131
RISANDFLG 0.059 290 RYWRTLVLL 0.057 314 IPGQISQVI 0.047 77
LTGEGNFNW 0.042 79 GEGNFNWRF 0.041 23 PISYELRVV 0.040 70 TDVHFNSLT
0.039 109 FALEEAEFR 0.039 283 FVFFIWRRY 0.038 122 LVLQVWDYD 0.038
106 SGPFALEEA 0.037 68 QETDVHFNS 0.034 168 GPRCNLFRC 0.033 292
WRTLVLLLL 0.031 245 VEEAEKRPV 0.029 319 SQVIFRPLH 0.029 231
ILTGKVEAE 0.029 317 QISQVIFRP 0.027 120 AVLVLQVWD 0.027 215
GRPEDLEFT 0.026 242 LLTVEEAEK 0.025 123 VLQVWDYDR 0.025 16
VDIKPRQPI 0.025 258 KQPEPLEKP 0.024 Table VII: 158P3D2 v.2a
A2-9mers 117 KLLVRVYVV 849.359 91 LIYPESEAV 25.492 90 FLIYPESEA
22.853 198 LIGETHIDL 20.473 158 YIPKQLNPI 15.177 220 LASQYEVWV
9.032 184 TVAVFEHDL 7.103 179 AETELTVAV 5.545 19 IQDQGEAEV 4.795
176 SLPAETELT 3.651 98 AVLFSEPQI 3.378 169 EILELSISL 3.342 116
IKLLVRVYV 3.342 177 LPAETELTV 3.165 11 NLISMVGEI 3.119 162
QLNPIFGEI 2.577 123 YVVKATNLA 2.000 218 CGLASQYEV 1.680 57
WLNVFPLYR 1.433 52 NHFEDWLNV 1.246 114 RPIKLLVRV 1.044 29 GTVSPKKAV
0.966 175 ISLPAETEL 0.877 185 VAVFEHDLV 0.805 23 GEAEVKGTV 0.721
171 LELSISLPA 0.608 165 PIFGEILEL 0.550 151 RQDTKERYI 0.465 191
DLVGSDDLI 0.383 84 GKFKGSFLI 0.311 161 KQLNPIFGE 0.261 55 EDWLNVFPL
0.246 137 GKADPYVVV 0.244 110 IPQNRPIKL 0.237 99 VLFSEPQIS 0.192
163 LNPIFGEIL 0.181 30 TVSPKKAVA 0.178 39 TLKIYNRSL 0.150 5
GDSDGVNLI 0.137 119 LVRVYVVKA 0.129 28 KGTVSPKKA 0.114 155
KERYIPKQL 0.110 111 PQNRPIKLL 0.110 146 SAGRERQDT 0.104 204
IDLENRFYS 0.085 173 LSISLPAET 0.083 31 VSPKKAVAT 0.083 8 DGVNLISMV
0.078 182 ELTVAVFEH 0.075 129 NLAPADPNG 0.075 135 PNGKADPYV 0.055
34 KKAVATLKI 0.051 83 VGKFKGSFL 0.046 45 RSLEEEFNH 0.043 102
SEPQISRGI 0.041 186 AVFEHDLVG 0.041 46 SLEEEFNHF 0.037 36 AVATLKIYN
0.036 112 QNRPIKLLV 0.035 222 SQYEVWVQQ 0.034 125 VKATNLAPA 0.027
14 SMVGEIQDQ 0.025 194 GSDDLIGET 0.024 105 QISRGIPQN 0.024 41
KIYNRSLEE 0.023 219 GLASQYEVW 0.022 15 MVGEIQDQG 0.022 121
RVYVVKATN 0.021 167 FGEILELSI 0.020 131 APADPNGKA 0.017 51
FNHFEDWLN 0.017 139 ADPYVVVSA 0.016 7 SDGVNLISM 0.016 118 LLVRVYVVK
0.016 212 SHHRANCGL 0.015 74 GEEEGSGHL 0.014 206 LENRFYSHH 0.014
108 RGIPQNRPI 0.014 17 GEIQDQGEA 0.013 50 EFNHFEDWL 0.011 32
SPKKAVATL 0.011 92 IYPESEAVL 0.008 61 FPLYRGQGG 0.008 22 QGEAEVKGT
0.007 136 NGKADPYVV 0.007 75 EEEGSGHLV 0.006 228 VQQGPQEPF 0.006
227 WVQQGPQEP 0.006 181 TELTVAVFE 0.006 38 ATLKIYNRS 0.006 82
LVGKFKGSF 0.005 122 VYVVKATNL 0.005 81 HLVGKFKGS 0.005 86 FKGSFLIYP
0.005 192 LVGSDDLIG 0.005 120 VRVYVVKAT 0.004 196 DDLIGETHI 0.004
170 ILELSISLP 0.004 2 DDPGDSDGV 0.004 35 KAVATLKIY 0.003 Table VII:
158P3D2 v.3 A2-9mers 9 RRRSGPFAL 0.001 8 VRRRSGPFA 0.000 4
REVSVRRRS 0.000 6 VSVRRRSGP 0.000 5 EVSVRRRSG 0.000 2 TEREVSVRR
0.000 7 SVRRRSGPF 0.000 1 PTEREVSVR 0.000 3 EREVSVRRR 0.000 Table
VII: 158P3D2 v.4 A2-9mers 1 LPTEREVSI 0.475 8 SIWRRSGPF 0.011 3
TEREVSIWR 0.000 5 REVSIWRRS 0.000 9 IWRRSGPFA 0.000 7 VSIWRRSGP
0.000 6 EVSIWRRSG 0.000 2 PTEREVSIW 0.000 4 EREVSIWRR 0.000 Table
VII: 158P3D2 v.5a A2-9mers 4 QVWDYTASL 63.609 1 LVLQVWDYT 18.791 2
VLQVWDYTA 8.446 21 SCSYQTWCV 3.405 43 AMGPGRGAI 0.980 14 MTSLDPWSC
0.880 20 WSCSYQTWC 0.820 9 TASLPMTSL 0.682 25 QTWCVGPGA 0.573 36
SALCSWPAM 0.434 49 GAICFAAAA 0.262 35 SSALCSWPA 0.243 30 GPGAPSSAL
0.139 6 WDYTASLPM 0.102 37 ALCSWPAMG 0.075 48 RGAICFAAA 0.062 29
VGPGAPSSA 0.055 18 DPWSCSYQT 0.030 16 SLDPWSCSY 0.030 44 MGPGRGAIC
0.023 3 LQVWDYTAS 0.019 11 SLPMTSLDP 0.015 15 TSLDPWSCS 0.013 24
YQTWCVGPG 0.010 28 CVGPGAPSS 0.007 13 PMTSLDPWS 0.007 8 YTASLPMTS
0.005 47 GRGAICFAA 0.004 12 LPMTSLDPW 0.003 27 WCVGPGAPS 0.002 39
CSWPAMGPG 0.001 42 PAMGPGRGA 0.001 33 APSSALCSW 0.001 22 CSYQTWCVG
0.001 32 GAPSSALCS 0.001 31 PGAPSSALC 0.001 46 PGRGAICFA 0.001 45
GPGRGAICF 0.000 10 ASLPMTSLD 0.000 41 WPAMGPGRG 0.000 17 LDPWSCSYQ
0.000 7 DYTASLPMT 0.000 38 LCSWPAMGP 0.000 34 PSSALCSWP 0.000 23
SYQTWCVGP 0.000 40 SWPAMGPGR 0.000 5 VWDYTASLP 0.000 19 PWSCSYQTW
0.000 26 TWCVGPGAP 0.000
[0766]
14TABLE VIII 158P3D2 A2, 10mers (variants 1, 2a, 3, 4 and 5a) SEQ.
ID Pos 1234567890 Score NO. Table VIII: 158P3D2 v.1 A2-10mers 301
VLLTVFLLLV 3823.593 296 VLLLLVLLTV 1006.209 298 LLLVLLTVFL 739.032
299 LLVLLTVFLL 484.457 93 YLPTEREVSV 319.939 304 TVFLLLVFYT 177.011
278 NPLKTFVFFI 70.254 294 TLVLLLLVLL 49.134 26 YELRVVIWNT 42.542
286 FIWRRYWRTL 38.130 300 LVLLTVFLLL 22.339 236 VEAEFELLTV 21.680
101 SVWRRSGPFA 19.844 31 VIWNTEDVVL 16.993 38 VVLDDENPLT 16.816 87
FVFRFDYLPT 16.647 117 RQPAVLVLQV 16.219 125 QVWDYDRISA 14.793 123
VLQVWDYDRI 13.036 312 YTIPGQISQV 10.220 295 LVLLLLVLLT 9.433 63
LEHDKQETDV 9.426 21 RQPISYELRV 7.052 114 AEFRQPAVLV 5.004 271
TSFNWFVNPL 4.510 68 QETDVHFNSL 3.236 29 RVVIWNTEDV 2.982 61
KGLEHDKQET 2.583 79 GEGNFNWRFV 2.529 268 RPKTSFNWFV 2.491 140
SLELQLPDMV 2.181 30 VVIWNTEDVV 2.078 273 FNWFVNPLKT 1.857 222
FTDMGGNVYI 1.466 143 LQLPDMVRGA 1.457 275 WFVNPLKTFV 1.222 139
GSLELQLPDM 1.132 317 QISQVIFRPL 1.116 220 LEFTDMGGNV 1.106 293
RTLVLLLLVL 1.035 51 SSDIYVKSWV 0.999 309 LVFYTIPGQI 0.746 224
DMGGNVYILT 0.605 306 FLLLVFYTIP 0.593 313 TIPGQISQVI 0.588 153
RGPELCSVQL 0.572 235 KVEAEFELLT 0.555 307 LLLVFYTIPG 0.469 297
LLLLVLLTVF 0.442 167 AGPRCNLFRC 0.433 76 SLTGEGNFNW 0.432 120
AVLVLQVWDY 0.416 112 EEAEFRQPAV 0.416 244 TVEEAEKRPV 0.319 91
FDYLPTEREV 0.284 189 KEAEDVEREA 0.277 172 NLFRCRRLRG 0.276 132
ISANDFLGSL 0.269 285 FFIWRRYWRT 0.268 85 WRFVFRFDYL 0.259 1
MWIDIFPQDV 0.256 148 MVRGARGPEL 0.242 45 PLTGEMSSDI 0.230 39
VLDDENPLTG 0.208 185 VVKLKEAEDV 0.177 281 KTFVFFIWRR 0.176 151
GARGPELCSV 0.169 47 TGEMSSDIYV 0.160 137 FLGSLELQLP 0.158 37
DVVLDDENPL 0.140 164 RNGAGPRCNL 0.139 231 ILTGKVEAEF 0.127 283
FVFFIWRRYW 0.122 302 LLTVFLLLVF 0.119 121 VLVLQVWDYD 0.116 234
GKVEAEFELL 0.113 258 KQPEPLEKPS 0.108 223 TDMGGNVYIL 0.104 292
WRTLVLLLLV 0.102 305 VFLLLVFYTI 0.087 22 QPISYELRVV 0.086 109
FALEEAEFRQ 0.084 214 KGRPEDLEFT 0.080 276 FVNPLKTFVF 0.071 9
DVPAPPPVDI 0.068 7 PQDVPAPPPV 0.062 227 GNVYILTGKV 0.059 308
LLVFYTIPGQ 0.058 290 RYWRTLVLLL 0.057 134 ANDFLGSLEL 0.056 194
VEREAQEAQA 0.051 111 LEEAEFRQPA 0.040 230 YILTGKVEAE 0.039 19
KPRQPISYEL 0.037 105 RSGPFALEEA 0.037 158 CSVQLARNGA 0.032 233
TGKVEAEFEL 0.028 129 YDRISANDFL 0.028 170 RCNLFRCRRL 0.028 177
RRLRGWWPVV 0.025 Table VIII: 158P3D2 v.2a A2-10mers 219 GLASQYEVWV
382.536 90 FLIYPESEAV 156.770 176 SLPAETELTV 69.552 118 LLVRVYVVKA
19.425 82 LVGKFKGSFL 17.477 162 QLNPIFGEIL 16.308 54 FEDWLNVFPL
10.196 91 LIYPESEAVL 6.551 121 RVYVVKATNL 5.981 51 FNHFEDWLNV 3.550
161 KQLNPIFGEI 3.383 184 TVAVFEHDLV 2.982 18 EIQDQGEAEV 2.941 174
SISLPAETEL 2.937 109 GIPQNRPIKL 2.937 183 LTVAVFEHDL 1.917 197
DLIGETHIDL 1.602 28 KGTVSPKKAV 1.589 49 EEFNHFEDWL 1.180 57
WLNVFPLYRG 0.788 30 TVSPKKAVAT 0.652 211 YSHHRANCGL 0.641 116
IKLLVRVYVV 0.573 172 ELSISLPAET 0.559 31 VSPKKAVATL 0.545 110
IPQNRPIKLL 0.545 170 ILELSISLPA 0.541 21 DQGEAEVKGT 0.534 217
NCGLASQYEV 0.454 168 GEILELSISL 0.415 74 GEEEGSGHLV 0.355 164
NPIFGEILEL 0.321 222 SQYEVWVQQG 0.228 186 AVFEHDLVGS 0.228 138
KADPYVVVSA 0.222 7 SDGVNLISMV 0.222 38 ATLKIYNRSL 0.220 177
LPAETELTVA 0.213 119 LVRVYVVKAT 0.194 134 DPNGKADPYV 0.187 111
PQNRPIKLLV 0.155 175 ISLPAETELT 0.150 10 VNLISMVGEI 0.128 117
KLLVRVYVVK 0.119 193 VGSDDLIGET 0.101 99 VLFSEPQISR 0.094 145
VSAGRERQDT 0.083 46 SLEEEFNHFE 0.082 181 TELTVAVFEH 0.072 124
VVKATNLAPA 0.059 166 IFGEILELSI 0.050 3 DPGDSDGVNL 0.043 115
PIKLLVRVYV 0.041 1 MDDPGDSDGV 0.032 29 GTVSPKKAVA 0.028 14
SMVGEIQDQG 0.026 41 KIYNRSLEEE 0.026 83 VGKFKGSFLI 0.024 113
NRPIKLLVRV 0.022 35 KAVATLKIYN 0.020 158 YIPKQLNPIF 0.019 198
LIGETHIDLE 0.016 130 LAPADPNGKA 0.015 129 NLAPADPNGK 0.015 227
WVQQGPQEPF 0.015 45 RSLEEEFNHF 0.014 89 SFLIYPESEA 0.013 27
VKGTVSPKKA 0.012 209 RFYSHHRANC 0.011 97 EAVLFSEPQI 0.011 98
AVLFSEPQIS 0.010 136 NGKADPYVVV 0.010 15 MVGEIQDQGE 0.009 123
YVVKATNLAP 0.006 195 SDDLIGETHI 0.006 179 AETELTVAVF 0.006 188
FEHDLVGSDD 0.005 169 EILELSISLP 0.005 192 LVGSDDLIGE 0.005 204
IDLENRFYSH 0.005 73 GGEEEGSGHL 0.005 203 HIDLENRFYS 0.004 171
LELSISLPAE 0.004 135 PNGKADPYVV 0.004 101 FSEPQISRGI 0.004 22
QGEAEVKGTV 0.004 12 LISMVGEIQD 0.003 157 RYIPKQLNPI 0.003 59
NVFPLYRGQG 0.003 9 GVNLISMVGE 0.003 36 AVATLKIYNR 0.003 11
NLISMVGEIQ 0.003 79 SGHLVGKFKG 0.003 37 VATLKIYNRS 0.003 87
KGSFLIYPES 0.003 220 LASQYEVWVQ 0.002 23 GEAEVKGTVS 0.002 191
DLVGSDDLIG 0.002 6 DSDGVNLISM 0.002 105 QISRGIPQNR 0.002 Table
VIII: 158P3D2 v.3 A2-10mers 8 SVRRRSGPFA 0.182 9 VRRRSGPFAL 0.002 1
LPTEREVSVR 0.001 5 REVSVRRRSG 0.000 7 VSVRRRSGPF 0.000 6 EVSVRRRSGP
0.000 3 TEREVSVRRR 0.000 10 RRRSGPFALE 0.000 2 PTEREVSVRR 0.000 4
EREVSVRRRS 0.000 Table VIII: 158P3D2 v.4 A2-10mers 1 YLPTEREVSI
47.991 9 SIWRRSGPFA 31.184 2 LPTEREVSIW 0.003 10 IWRRSGPFAL 0.002 4
TEREVSIWRR 0.002 6 REVSIWRRSG 0.000 8 VSIWRRSGPF 0.000 7 EVSIWRRSGP
0.000 3 PTEREVSIWR 0.000 5 EREVSIWRRS 0.000 Table VIII: 158P3D2
v.5a A2-10mers 1 VLVLQVWDYT 58.040 21 WSCSYQTWCV 15.664 4
LQVWDYTASL 3.682 9 YTASLPMTSL 3.139 2 LVLQVWDYTA 2.734 25
YQTWCVGPGA 2.317 44 AMGPGRGAIC 1.471 14 PMTSLDPWSC 0.592 29
CVGPGAPSSA 0.435 46 GPGRGAICFA 0.410 7 WDYTASLPMT 0.350 30
VGPGAPSSAL 0.237 3 VLQVWDYTAS 0.190 49 RGAICFAAAA 0.123 12
SLPMTSLDPW 0.084 36 SSALCSWPAM 0.055 5 QVWDYTASLP 0.044 17
SLDPWSCSYQ 0.033 31 GPGAPSSALC 0.032 42 WPAMGPGRGA 0.030 18
LDPWSCSYQT 0.018 13 LPMTSLDPWS 0.017 38 ALCSWPAMGP 0.015 16
TSLDPWSCSY 0.007 35 PSSALCSWPA 0.005 37 SALCSWPAMG 0.004 15
MTSLDPWSCS 0.003 33 GAPSSALCSW 0.002 28 WCVGPGAPSS 0.002 43
PAMGPGRGAI 0.002 48 GRGAICFAAA 0.001 45 MGPGRGAICF 0.001 40
CSWPAMGPGR 0.001 34 APSSALCSWP 0.001 6 VWDYTASLPM 0.000 19
DPWSCSYQTW 0.000 11 ASLPMTSLDP 0.000 22 SCSYQTWCVG 0.000 47
PGRGAICFAA 0.000 23 CSYQTWCVGP 0.000 39 LCSWPAMGPG 0.000 26
QTWCVGPGAP 0.000 10 TASLPMTSLD 0.000 20 PWSCSYQTWC 0.000 32
PGAPSSALCS 0.000 27 TWCVGPGAPS 0.000 24 SYQTWCVGPG 0.000 41
SWPAMGPGRG 0.000 8 DYTASLPMTS 0.000
[0767]
15TABLE IX 158P3D2 A3, 9mers (variants 1, 2a, 3, 4 and 5a) SEQ. ID
Pos 123456789 Score NO. Table IX: 158P3D2 v.1 A3-9-mers 281
KTFVFFIWR 54.000 121 VLVLQVWDY 54.000 123 VLQVWDYDR 36.000 49
EMSSDIYVK 27.000 242 LLTVEEAEK 20.000 306 FLLLVFYTI 12.150 53
DIYVKSWVK 9.000 301 VLLTVFLLL 8.100 320 QVIFRPLHK 6.000 298
LLLVLLTVF 4.500 142 ELQLPDMVR 3.600 156 ELCSVQLAR 3.600 316
GQISQVIFR 3.240 59 WVKGLEHDK 3.000 304 TVFLLLVFY 3.000 294
TLVLLLLVL 2.700 224 DMGGNVYIL 2.430 172 NLFRCRRLR 2.000 302
LLTVFLLLV 1.800 279 PLKTFVFFI 1.620 297 LLLLVLLTV 1.350 137
FLGSLELQL 1.200 181 GWWPVVKLK 1.013 299 LLVLLTVFL 0.900 296
VLLLLVLLT 0.900 178 RLRGWWPVV 0.900 300 LVLLTVFLL 0.810 81
GNFNWRFVF 0.540 235 KVEAEFELL 0.540 83 FNWRFVFRF 0.540 303
LTVFLLLVF 0.450 243 LTVEEAEKR 0.450 201 AQAGKKKRK 0.450 227
GNVYILTGK 0.405 62 GLEHDKQET 0.300 273 FNWFVNPLK 0.300 262
PLEKPSRPK 0.300 283 FVFFIWRRY 0.300 101 SVWRRSGPF 0.300 140
SLELQLPDM 0.300 55 YVKSWVKGL 0.270 27 ELRVVIWNT 0.203 222 FTDMGGNVY
0.200 85 WRFVFRFDY 0.180 308 LLVFYTIPG 0.180 198 AQEAQAGKK 0.180 79
GEGNFNWRF 0.162 286 FIWRRYWRT 0.150 232 LTGKVEAEF 0.150 295
LVLLLLVLL 0.135 11 PAPPPVDIK 0.135 21 RQPISYELR 0.120 170 RCNLFRCRR
0.120 31 VIWNTEDVV 0.100 39 VLDDENPLT 0.100 278 NPLKTFVFF 0.090 187
KLKEAEDVE 0.090 231 ILTGKVEAE 0.090 265 KPSRPKTSF 0.090 87
FVFRFDYLP 0.090 110 ALEEAEFRQ 0.090 307 LLLVFYTIP 0.090 38
VVLDDENPL 0.090 166 GAGPRCNLF 0.090 109 FALEEAEFR 0.090 197
EAQEAQAGK 0.090 282 TFVFFIWRR 0.081 179 LRGWWPVVK 0.060 257
RKQPEPLEK 0.060 144 QLPDMVRGA 0.060 268 RPKTSFNWF 0.060 247
EAEKRPVGK 0.060 2 WIDIFPQDV 0.060 46 LTGEMSSDI 0.045 293 RTLVLLLLV
0.045 4 DIFPQDVPA 0.045 77 LTGEGNFNW 0.045 313 TIPGQISQV 0.045 93
YLPTEREVS 0.040 230 YILTGKVEA 0.030 76 SLTGEGNFN 0.030 228
NVYILTGKV 0.030 57 KSWVKGLEH 0.030 276 FVNPLKTFV 0.030 30 VVIWNTEDV
0.030 199 QEAQAGKKK 0.030 69 ETDVHFNSL 0.027 319 SQVIFRPLH 0.027
168 GPRCNLFRC 0.027 124 LQVWDYDRI 0.027 96 TEREVSVWR 0.027 24
ISYELRVVI 0.022 159 SVQLARNGA 0.020 161 QLARNGAGP 0.020 285
FFIWRRYWR 0.018 250 KRPVGKGRK 0.018 214 KGRPEDLEF 0.018 78
TGEGNFNWR 0.018 154 GPELCSVQL 0.018 22 QPISYELRV 0.018 Table IX:
158P3D2 v.2a A3-9mers 118 LLVRVYVVK 45.000 57 WLNVFPLYR 24.000 46
SLEEEFNHF 9.000 117 KLLVRVYVV 8.100 109 GIPQNRPIK 6.000 26
EVKGTVSPK 2.700 162 QLNPIFGEI 1.215 153 DTKERYIPK 0.900 11
NLISMVGEI 0.810 219 GLASQYEVW 0.600 182 ELTVAVFEH 0.540 205
DLENRFYSH 0.540 90 FLIYPESEA 0.450 191 DLVGSDDLI 0.405 130
LAPADPNGK 0.200 99 VLFSEPQIS 0.200 37 VATLKIYNR 0.180 184 TVAVFEHDL
0.180 82 LVGKFKGSF 0.180 39 TLKIYNRSL 0.180 119 LVRVYVVKA 0.180 198
LIGETHIDL 0.180 91 LIYPESEAV 0.150 165 PIFGEILEL 0.135 228
VQQGPQEPF 0.135 81 HLVGKFKGS 0.135 35 KAVATLKIY 0.135 85 KFKGSFLIY
0.108 176 SLPAETELT 0.100 201 ETHIDLENR 0.090 98 AVLFSEPQI 0.090
180 ETELTVAVF 0.090 158 YIPKQLNPI 0.090 169 EILELSISL 0.081 14
SMVGEIQDQ 0.068 143 VVVSAGRER 0.060 41 KIYNRSLEE 0.060 106
ISRGIPQNR 0.045 203 HIDLENRFY 0.040 29 GTVSPKKAV 0.034 20 QDQGEAEVK
0.030 27 VKGTVSPKK 0.030 147 AGRERQDTK 0.030 123 YVVKATNLA 0.030
129 NLAPADPNG 0.030 170 ILELSISLP 0.030 186 AVFEHDLVG 0.030 30
TVSPKKAVA 0.030 84 GKFKGSFLI 0.027 78 GSGHLVGKF 0.027 93 YPESEAVLF
0.020 159 IPKQLNPIF 0.020 161 KQLNPIFGE 0.018 100 LFSEPQISR 0.018 9
GVNLISMVG 0.018 32 SPKKAVATL 0.018 134 DPNGKADPY 0.018 138
KADPYVVVS 0.016 79 SGHLVGKFK 0.015 121 RVYVVKATN 0.015 197
DLIGETHID 0.013 77 EGSGHLVGK 0.013 115 PIKLLVRVY 0.012 216
ANCGLASQY 0.012 113 NRPIKLLVR 0.012 110 IPQNRPIKL 0.012 53
HFEDWLNVF 0.009 172 ELSISLPAE 0.009 56 DWLNVFPLY 0.008 55 EDWLNVFPL
0.008 207 ENRFYSHHR 0.007 45 RSLEEEFNH 0.007 175 ISLPAETEL 0.007
222 SQYEVWVQQ 0.007 183 LTVAVFEHD 0.007 149 RERQDTKER 0.006 220
LASQYEVWV 0.006 19 IQDQGEAEV 0.006 177 LPAETELTV 0.006 5 GDSDGVNLI
0.005 114 RPIKLLVRV 0.005 38 ATLKIYNRS 0.005 15 MVGEIQDQG 0.005 88
GSFLIYPES 0.005 155 KERYIPKQL 0.004 36 AVATLKIYN 0.004 43 YNRSLEEEF
0.004 124 VVKATNLAP 0.004 192 LVGSDDLIG 0.004 34 KKAVATLKI 0.004
163 LNPIFGEIL 0.004 202 THIDLENRF 0.003 33 PKKAVATLK 0.003 185
VAVFEHDLV 0.003 52 NHFEDWLNV 0.003 105 QISRGIPQN 0.003 12 LISMVGEIQ
0.003 62 PLYRGQGGQ 0.003 174 SISLPAETE 0.003 69 GQDGGGEEE 0.003
Table IX: 158P3D2 v.3 A3-9mers 1 PTEREVSVR 0.060 7 SVRRRSGPF 0.060
2 TEREVSVRR 0.027 9 RRRSGPFAL 0.002 3 EREVSVRRR 0.000 8 VRRRSGPFA
0.000 6 VSVRRRSGP 0.000 5 EVSVRRRSG 0.000 4 REVSVRRRS 0.000 Table
IX: 158P3D2 v.4 A3-9mers 8 SIWRRSGPF 0.300 3 TEREVSIWR 0.054 1
LPTEREVSI 0.009 4 EREVSIWRR 0.005 2 PTEREVSIW 0.003 9 IWRRSGPFA
0.000 6 EVSIWRRSG 0.000 7 VSIWRRSGP 0.000 5 REVSIWRRS 0.000 Table
IX: 158P3D2 v.5a A3-9mers 16 SLDPWSCSY 18.000 2 VLQVWDYTA 1.800 4
QVWDYTASL 0.900 43 AMGPGRGAI 0.270 45 GPGRGAICF 0.120 25 QTWCVGPGA
0.075 37 ALCSWPAMG 0.060 11 SLPMTSLDP 0.040 14 MTSLDPWSC 0.030 30
GPGAPSSAL 0.027 49 GAICFAAAA 0.027 1 LVLQVWDYT 0.022 9 TASLPMTSL
0.013 21 SCSYQTWCV 0.006 28 CVGPGAPSS 0.006 12 LPMTSLDPW 0.005 18
DPWSCSYQT 0.005 8 YTASLPMTS 0.004 13 PMTSLDPWS 0.004 40 SWPAMGPGR
0.004 33 APSSALCSW 0.003 20 WSCSYQTWC 0.003 35 SSALCSWPA 0.003 36
SALCSWPAM 0.003 47 GRGAICFAA 0.003 32 GAPSSALCS 0.002 6 WDYTASLPM
0.002 3 LQVWDYTAS 0.002 27 WCVGPGAPS 0.001 38 LCSWPAMGP 0.001 48
RGAICFAAA 0.001 24 YQTWCVGPG 0.001 22 CSYQTWCVG 0.001 15 TSLDPWSCS
0.000 39 CSWPAMGPG 0.000 29 VGPGAPSSA 0.000 44 MGPGRGAIC 0.000 10
ASLPMTSLD 0.000 42 PAMGPGRGA 0.000 23 SYQTWCVGP 0.000 41 WPAMGPGRG
0.000 46 PGRGAICFA 0.000 7 DYTASLPMT 0.000 31 PGAPSSALC 0.000 5
VWDYTASLP 0.000 17 LDPWSCSYQ 0.000 19 PWSCSYQTW 0.000 34 PSSALCSWP
0.000 26 TWCVGPGAP 0.000
[0768]
16TABLE X 158P3D2 A3, 10mers (variants 1, 2a, 3, 4 and 5a) SEQ. ID
Pos 1234567890 Score NO. Table X: 158P3D2 v.1 A3-10mers 178
RLRGWWPVVK 90.000 281 KTFVFFIWRR 40.500 187 KLKEAEDVER 18.000 241
ELLTVEEAEK 9.000 299 LLVLLTVFLL 8.100 302 LLTVFLLLVF 6.000 122
LVLQVWDYDR 5.400 120 AVLVLQVWDY 5.400 297 LLLLVLLTVF 4.500 231
ILTGKVEAEF 4.500 242 LLTVEEAEKR 4.000 301 VLLTVFLLLV 2.700 144
QLPDMVRGAR 1.800 319 SQVIFRPLHK 1.800 296 VLLLLVLLTV 1.350 294
TLVLLLLVLL 1.350 10 VPAPPPVDIK 1.350 48 GEMSSDIYVK 1.215 161
QLARNGAGPR 1.200 298 LLLVLLTVFL 0.900 77 LTGEGNFNWR 0.900 276
FVNPLKTFVF 0.900 76 SLTGEGNFNW 0.900 300 LVLLTVFLLL 0.810 123
VLQVWDYDRI 0.600 303 LTVFLLLVFY 0.450 304 TVFLLLVFYT 0.450 81
GNFNWRFVFR 0.360 17 DIKPRQPISY 0.360 166 GAGPRCNLFR 0.360 31
VIWNTEDVVL 0.300 107 GPFALEEAEF 0.300 46 LTGEMSSDIY 0.300 198
AQEAQAGKKK 0.300 279 PLKTFVFFIW 0.270 278 NPLKTFVFFI 0.243 180
RGWWPVVKLK 0.225 93 YLPTEREVSV 0.200 140 SLELQLPDMV 0.200 172
NLFRCRRLRG 0.200 125 QVWDYDRISA 0.200 307 LLLVFYTIPG 0.180 235
KVEAEFELLT 0.180 96 TEREVSVWRR 0.162 226 GGNVYILTGK 0.135 293
RTLVLLLLVL 0.135 309 LVFYTIPGQI 0.135 224 DMGGNVYILT 0.135 271
TSFNWFVNPL 0.135 313 TIPGQISQVI 0.135 256 GRKQPEPLEK 0.120 87
FVFRFDYLPT 0.100 101 SVWRRSGPFA 0.100 52 SDIYVKSWVK 0.090 295
LVLLLLVLLT 0.090 148 MVRGARGPEL 0.090 306 FLLLVFYTIP 0.090 45
PLTGEMSSDI 0.090 286 FIWRRYWRTL 0.090 19 KPRQPISYEL 0.081 280
LKTFVFFIWR 0.072 62 GLEHDKQETD 0.060 259 QPEPLEKPSR 0.060 284
VFFIWRRYWR 0.060 196 REAQEAQAGK 0.060 82 NFNWRFVFRF 0.054 141
LELQLPDMVR 0.054 121 VLVLQVWDYD 0.045 308 LLVFYTIPGQ 0.045 12
APPPVDIKPR 0.045 39 VLDDENPLTG 0.040 84 NWRFVFRFDY 0.036 168
GPRCNLFRCR 0.036 117 RQPAVLVLQV 0.036 21 RQPISYELRV 0.036 312
YTIPGQISQV 0.034 272 SFNWFVNPLK 0.030 58 SWVKGLEHDK 0.030 200
EAQAGKKKRK 0.030 30 VVIWNTEDVV 0.030 283 FVFFIWRRYW 0.030 137
FLGSLELQLP 0.030 222 FTDMGGNVYI 0.030 29 RVVIWNTEDV 0.030 95
PTEREVSVWR 0.030 37 DVVLDDENPL 0.027 78 TGEGNFNWRF 0.027 9
DVPAPPPVDI 0.027 317 QISQVIFRPL 0.027 270 KTSFNWFVNP 0.027 246
EEAEKRPVGK 0.027 197 EAQEAQAGKK 0.027 131 RTSANDFLGS 0.024 24
ISYELRVVIW 0.022 261 EPLEKPSRPK 0.020 314 IPGQISQVIF 0.020 202
QAGKKKRKQR 0.020 89 FRFDYLPTER 0.020 185 VVKLKEAEDV 0.020 316
GQISQVIFRP 0.018 Table X: 158P3D2 v.2a A3-10mers 117 KLLVRVYVVK
135.000 99 VLFSEPQISR 60.000 129 NLAPADPNGK 30.000 81 HLVGKFKGSF
4.050 162 QLNPIFGEIL 2.700 118 LLVRVYVVKA 2.700 219 GLASQYEVWV
1.800 36 AVATLKIYNR 1.800 26 EVKGTVSPKK 1.350 197 DLIGETHIDL 0.810
170 ILELSISLPA 0.600 105 QISRGIPQNR 0.600 19 IQDQGEAEVK 0.600 91
LIYPESEAVL 0.450 176 SLPAETELTV 0.400 84 GKFKGSFLIY 0.360 109
GIPQNRPIKL 0.360 90 FLIYPESEAV 0.300 121 RVYVVKATNL 0.300 32
SPKKAVATLK 0.300 227 WVQQGPQEPF 0.300 25 AEVKGTVSPK 0.270 78
GSGHLVGKFK 0.225 158 YIPKQLNPIF 0.200 146 SAGRERQDTK 0.200 205
DLENRFYSHH 0.180 183 LTVAVFEHDL 0.135 57 WLNVFPLYRG 0.135 161
KQLNPIFGEI 0.109 46 SLEEEFNHFE 0.090 140 DPYVVVSAGR 0.090 45
RSLEEEFNHF 0.068 52 NHFEDWLNVF 0.068 14 SMVGEIQDQG 0.068 142
YVVVSAGRER 0.060 82 LVGKFKGSFL 0.060 174 SISLPAETEL 0.060 200
GETHIDLENR 0.054 108 RGIPQNRPIK 0.045 41 KIYNRSLEEE 0.045 186
AVFEHDLVGS 0.045 11 NLISMVGEIQ 0.045 29 GTVSPKKAVA 0.045 222
SQYEVWVQQG 0.041 138 KADPYVVVSA 0.041 215 RANCGLASQY 0.040 152
QDTKERYIPK 0.040 112 QNRPIKLLVR 0.036 206 LENRFYSHHR 0.036 172
ELSISLPAET 0.030 201 ETHIDLENRF 0.030 124 VVKATNLAPA 0.030 182
ELTVAVFEHD 0.027 164 NPIFGEILEL 0.027 76 EEGSGHLVGK 0.027 191
DLVGSDDLIG 0.027 55 EDWLNVFPLY 0.027 179 AETELTVAVF 0.027 119
LVRVYVVKAT 0.022 39 TLKIYNRSLE 0.020 184 TVAVFEHDLV 0.020 114
RPIKLLVRVY 0.018 168 GEILELSISL 0.016 54 FEDWLNVFPL 0.016 30
TVSPKKAVAT 0.015 38 ATLKIYNRSL 0.013 59 NVFPLYRGQG 0.013 203
HIDLENRFYS 0.012 149 RERQDTKERY 0.012 56 DWLNVFPLYR 0.011 34
KKAVATLKIY 0.009 31 VSPKKAVATL 0.009 9 GVNLISMVGE 0.009 181
TELTVAVFEH 0.008 49 EEFNHFEDWL 0.008 165 PIFGEILELS 0.007 110
IPQNRPIKLL 0.007 148 GRERQDTKER 0.006 123 YVVKATNLAP 0.006 192
LVGSDDLIGE 0.006 217 NCGLASQYEV 0.006 98 AVLFSEPQIS 0.006 18
EIQDQGEAEV 0.006 88 GSFLIYPESE 0.005 198 LIGETHIDLE 0.005 177
LPAETELTVA 0.005 194 GSDDLIGETH 0.005 204 IDLENRFYSH 0.004 12
LISMVGEIQD 0.004 133 ADPNGKADPY 0.004 92 IYPESEAVLF 0.003 15
MVGEIQDQGE 0.003 225 EVWVQQGPQE 0.003 115 PIKLLVRVYV 0.003 62
PLYRGQGGQD 0.003 211 YSHHRANCGL 0.003 143 VVVSAGRERQ 0.003 116
IKLLVRVYVV 0.003 69 GQDGGGEEEG 0.003 97 EAVLFSEPQI 0.003 Table X:
158P3D2 v.3 A3-10mers 1 LPTEREVSVR 0.180 2 PTEREVSVRR 0.030 8
SVRRRSGPFA 0.020 3 TEREVSVRRR 0.005 7 VSVRRRSGPF 0.005 9 VRRRSGPFAL
0.002 6 EVSVRRRSGP 0.001 10 RRRSGPFALE 0.000 5 REVSVRRRSG 0.000 4
EREVSVRRRS 0.000 Table X: 158P3D2 v.4 A3-10mers 1 YLPTEREVSI 0.600
9 SIWRRSGPFA 0.100 4 TEREVSIWRR 0.081 3 PTEREVSIWR 0.060 2
LPTEREVSIW 0.009 8 VSIWRRSGPF 0.005 10 IWRRSGPFAL 0.002 7
EVSIWRRSGP 0.001 6 REVSIWRRSG 0.000 5 EREVSIWRRS 0.000 Table X:
158P3D2 v.5a A3-10mers 44 AMGPGRGAIC 0.300 12 SLPMTSLDPW 0.300 2
LVLQVWDYTA 0.270 1 VLVLQVWDYT 0.225 40 CSWPAMGPGR 0.150 16
TSLDPWSCSY 0.090 4 LQVWDYTASL 0.081 9 YTASLPMTSL 0.068 38
ALCSWPAMGP 0.060 14 PMTSLDPWSC 0.060 3 VLQVWDYTAS 0.040 29
CVGPGAPSSA 0.030 17 SLDPWSCSYQ 0.030 5 QVWDYTASLP 0.010 25
YQTWCVGPGA 0.009 46 GPGRGAICFA 0.009 33 GAPSSALCSW 0.009 45
MGPGRGAICF 0.006 31 GPGAPSSALC 0.006 19 DPWSCSYQTW 0.003 15
MTSLDPWSCS 0.003 21 WSCSYQTWCV 0.003 48 GRGAICFAAA 0.002 26
QTWCVGPGAP 0.002 23 CSYQTWCVGP 0.002 30 VGPGAPSSAL 0.001 36
SSALCSWPAM 0.001 28 WCVGPGAPSS 0.001 37 SALCSWPAMG 0.001 7
WDYTASLPMT 0.001 49 RGAICFAAAA 0.001 13 LPMTSLDPWS 0.001 11
ASLPMTSLDP 0.000 43 PAMGPGRGAI 0.000 6 VWDYTASLPM 0.000 18
LDPWSCSYQT 0.000 42 WPAMGPGRGA 0.000 35 PSSALCSWPA 0.000 34
APSSALCSWP 0.000 22 SCSYQTWCVG 0.000 10 TASLPMTSLD 0.000 47
PGRGAICFAA 0.000 39 LCSWPAMGPG 0.000 20 PWSCSYQTWC 0.000 27
TWCVGPGAPS 0.000 8 DYTASLPMTS 0.000 24 SYQTWCVGPG 0.000 32
PGAPSSALCS 0.000 41 SWPAMGPGRG 0.000
[0769]
17TABLE XI 158P3D2 A11, 9mers (variants 1, 2a, 3, 4 and 5a) SEQ. ID
Pos 123456789 Score NO. Table XI: 158P3D2 v.1 A11-9mers 320
QVIFRPLHK 6.000 281 KTFVFFIWR 2.400 59 WVKGLEHDK 2.000 316
GQISQVIFR 1.080 198 AQEAQAGKK 0.600 53 DIYVKSWVK 0.480 242
LLTVEEAEK 0.400 21 RQPISYELR 0.360 243 LTVEEAEKR 0.300 201
AQAGKKKRK 0.300 49 EMSSDIYVK 0.240 227 GNVYILTGK 0.180 123
VLQVWDYDR 0.160 257 RKQPEPLEK 0.120 90 RFDYLPTER 0.120 282
TFVFFIWRR 0.120 170 RCNLFRCRR 0.120 285 FFIWRRYWR 0.120 293
RTLVLLLLV 0.090 300 LVLLTVFLL 0.090 273 FNWFVNPLK 0.080 181
GWWPVVKLK 0.060 250 KRPVGKGRK 0.060 109 FALEEAEFR 0.060 247
EAEKRPVGK 0.060 197 EAQEAQAGK 0.060 235 KVEAEFELL 0.060 142
ELQLPDMVR 0.048 156 ELCSVQLAR 0.048 145 LPDMVRGAR 0.040 304
TVFLLLVFY 0.040 82 NFNWRFVFR 0.040 101 SVWRRSGPF 0.040 228
NVYILTGKV 0.040 162 LARNGAGPR 0.040 295 LVLLLLVLL 0.030 77
LTGEGNFNW 0.030 199 QEAQAGKKK 0.030 303 LTVFLLLVF 0.030 38
VVLDDENPL 0.030 30 VVIWNTEDV 0.030 290 RYWRTLVLL 0.024 276
FVNPLKTFV 0.020 179 LRGWWPVVK 0.020 11 PAPPPVDIK 0.020 159
SVQLARNGA 0.020 172 NLFRCRRLR 0.016 204 GKKKRKQRR 0.012 306
FLLLVFYTI 0.012 301 VLLTVFLLL 0.012 121 VLVLQVWDY 0.012 96
TEREVSVWR 0.012 178 RLRGWWPVV 0.012 297 LLLLVLLTV 0.012 294
TLVLLLLVL 0.012 232 LTGKVEAEF 0.010 222 FTDMGGNVY 0.010 55
YVKSWVKGL 0.010 46 LTGEMSSDI 0.010 29 RVVIWNTED 0.009 124 LQVWDYDRI
0.009 270 KTSFNWFVN 0.009 86 RFVFRFDYL 0.009 319 SQVIFRPLH 0.009
302 LLTVFLLLV 0.008 87 FVFRFDYLP 0.008 137 FLGSLELQL 0.008 167
AGPRCNLFR 0.008 31 VIWNTEDVV 0.008 81 GNFNWRFVF 0.007 48 GEMSSDIYV
0.007 208 RKQRRRKGR 0.006 206 KKRKQRRRK 0.006 154 GPELCSVQL 0.006
230 YILTGKVEA 0.006 22 QPISYELRV 0.006 299 LLVLLTVFL 0.006 193
DVEREAQEA 0.006 298 LLLVLLTVF 0.006 265 KPSRPKTSF 0.006 166
GAGPRCNLF 0.006 200 EAQAGKKKR 0.006 175 RCRRLRGWW 0.006 268
RPKTSFNWF 0.006 262 PLEKPSRPK 0.004 25 SYELRVVIW 0.004 2 WIDIFPQDV
0.004 78 TGEGNFNWR 0.004 188 LKEAEDVER 0.004 309 LVFYTIPGQ 0.004
118 QPAVLVLQV 0.004 313 TIPGQISQV 0.004 283 FVFFIWRRY 0.004 310
VFYTIPGQI 0.004 140 SLELQLPDM 0.004 131 RISANDFLG 0.004 79
GEGNFNWRF 0.004 312 YTIPGQISQ 0.003 278 NPLKTFVFF 0.003 120
AVLVLQVWD 0.003 Table XI: 158P3D2 v.2a A11-9mers 109 GIPQNRPIK
1.200 153 DTKERYIPK 0.600 118 LLVRVYVVK 0.600 26 EVKGTVSPK 0.600
130 LAPADPNGK 0.200 57 WLNVFPLYR 0.160 37 VATLKIYNR 0.080 100
LFSEPQISR 0.080 201 ETHIDLENR 0.060 143 VVVSAGRER 0.060 117
KLLVRVYVV 0.036 98 AVLFSEPQI 0.030 123 YVVKATNLA 0.030 29 GTVSPKKAV
0.022 27 VKGTVSPKK 0.020 30 TVSPKKAVA 0.020 184 TVAVFEHDL 0.020 147
AGRERQDTK 0.020 82 LVGKFKGSF 0.020 20 QDQGEAEVK 0.020 119 LVRVYVVKA
0.020 149 RERQDTKER 0.018 141 PYVVVSAGR 0.012 85 KFKGSFLIY 0.012
219 GLASQYEVW 0.012 121 RVYVVKATN 0.012 9 GVNLISMVG 0.012 79
SGHLVGKFK 0.010 114 RPIKLLVRV 0.009 161 KQLNPIFGE 0.008 186
AVFEHDLVG 0.008 113 NRPIKLLVR 0.008 91 LIYPESEAV 0.008 198
LIGETHIDL 0.008 228 VQQGPQEPF 0.006 19 IQDQGEAEV 0.006 90 FLIYPESEA
0.006 11 NLISMVGEI 0.006 77 EGSGHLVGK 0.006 122 VYVVKATNL 0.006 41
KIYNRSLEE 0.005 35 KAVATLKIY 0.005 124 VVKATNLAP 0.004 46 SLEEEFNHF
0.004 177 LPAETELTV 0.004 158 YIPKQLNPI 0.004 106 ISRGIPQNR 0.004
192 LVGSDDLIG 0.004 36 AVATLKIYN 0.004 110 IPQNRPIKL 0.004 92
IYPESEAVL 0.004 162 QLNPIFGEI 0.004 84 GKFKGSFLI 0.004 157
RYIPKQLNP 0.004 169 EILELSISL 0.004 182 ELTVAVFEH 0.004 185
VAVFEHDLV 0.003 180 ETELTVAVF 0.003 142 YVVVSAGRE 0.003 45
RSLEEEFNH 0.003 17 GEIQDQGEA 0.003 207 ENRFYSHHR 0.002 205
DLENRFYSH 0.002 33 PKKAVATLK 0.002 144 VVSAGRERQ 0.002 159
IPKQLNPIF 0.002 53 HFEDWLNVF 0.002 32 SPKKAVATL 0.002 227 WVQQGPQEP
0.002 131 APADPNGKA 0.002 220 LASQYEVWV 0.002 15 MVGEIQDQG 0.002 93
YPESEAVLF 0.002 151 RQDTKERYI 0.002 69 GQDGGGEEE 0.002 66 GQGGQDGGG
0.002 23 GEAEVKGTV 0.002 74 GEEEGSGHL 0.002 171 LELSISLPA 0.002 191
DLVGSDDLI 0.002 165 PIFGEILEL 0.002 183 LTVAVFEHD 0.002 38
ATLKIYNRS 0.002 225 EVWVQQGPQ 0.001 34 KKAVATLKI 0.001 222
SQYEVWVQQ 0.001 127 ATNLAPADP 0.001 155 KERYIPKQL 0.001 99
VLFSEPQIS 0.001 112 QNRPIKLLV 0.001 52 NHFEDWLNV 0.001 126
KATNLAPAD 0.001 138 KADPYVVVS 0.001 78 GSGHLVGKF 0.001 14 SMVGEIQDQ
0.001 218 CGLASQYEV 0.001 73 GGEEEGSGH 0.001 215 RANCGLASQ 0.001
164 NPIFGEILE 0.001 5 GDSDGVNLI 0.001 Table XI: 158P3D2 v.3
A11-9mers 1 PTEREVSVR 0.020 7 SVRRRSGPF 0.020 2 TEREVSVRR 0.012 9
RRRSGPFAL 0.002 8 VRRRSGPFA 0.000 3 EREVSVRRR 0.000 5 EVSVRRRSG
0.000 6 VSVRRRSGP 0.000 4 REVSVRRRS 0.000 Table XI: 158P3D2 v.4
A11-9mers 3 TEREVSIWR 0.024 8 SIWRRSGPF 0.008 4 EREVSIWRR 0.002 1
LPTEREVSI 0.002 2 PTEREVSIW 0.001 9 IWRRSGPFA 0.000 6 EVSIWRRSG
0.000 7 VSIWRRSGP 0.000 5 REVSIWRRS 0.000 Table XI: 158P3D2 v.5a
A11-9mers 4 QVWDYTASL 0.040 25 QTWCVGPGA 0.020 45 GPGRGAICF 0.012
49 GAICFAAAA 0.009 2 VLQVWDYTA 0.008 30 GPGAPSSAL 0.006 16
SLDPWSCSY 0.004 21 SCSYQTWCV 0.004 43 AMGPGRGAI 0.004 40 SWPAMGPGR
0.004 12 LPMTSLDPW 0.004 1 LVLQVWDYT 0.003 36 SALCSWPAM 0.003 14
MTSLDPWSC 0.002 9 TASLPMTSL 0.002 33 APSSALCSW 0.002 28 CVGPGAPSS
0.002 8 YTASLPMTS 0.002 47 GRGAICFAA 0.002 32 GAPSSALCS 0.001 3
LQVWDYTAS 0.001 11 SLPMTSLDP 0.001 6 WDYTASLPM 0.001 24 YQTWCVGPG
0.001 48 RGAICFAAA 0.001 37 ALCSWPAMG 0.000 38 LCSWPAMGP 0.000 23
SYQTWCVGP 0.000 35 SSALCSWPA 0.000 27 WCVGPGAPS 0.000 18 DPWSCSYQT
0.000 41 WPAMGPGRG 0.000 29 VGPGAPSSA 0.000 7 DYTASLPMT 0.000 22
CSYQTWCVG 0.000 13 PMTSLDPWS 0.000 39 CSWPAMGPG 0.000 42 PAMGPGRGA
0.000 15 TSLDPWSCS 0.000 10 ASLPMTSLD 0.000 26 TWCVGPGAP 0.000 20
WSCSYQTWC 0.000 5 VWDYTASLP 0.000 46 PGRGAICFA 0.000 19 PWSCSYQTW
0.000 17 LDPWSCSYQ 0.000 44 MGPGRGAIC 0.000 34 PSSALCSWP 0.000 31
PGAPSSALC 0.000
[0770]
18TABLE XII 158P3D2 A11, 10mers (variants 1, 2a, 3, 4 and 5a) SEQ.
ID Pos 1234567890 Score NO. Table XII: 158P3D2 v.1 A11-10mers 281
KTFVFFIWRR 2.400 319 SQVIFRPLHK 1.800 122 LVLQVWDYDR 1.200 178
RLRGWWPVVK 1.200 48 GEMSSDIYVK 0.720 198 AQEAQAGKKK 0.300 166
GAGPRCNLFR 0.240 187 KLKEAEDVER 0.240 272 SFNWFVNPLK 0.200 10
VPAPPPVDIK 0.200 77 LTGEGNFNWR 0.200 241 ELLTVEEAEK 0.180 196
REAQEAQAGK 0.180 284 VFFIWRRYWR 0.160 256 GRKQPEPLEK 0.120 29
RVVIWNTEDV 0.090 293 RTLVLLLLVL 0.090 125 QVWDYDRISA 0.080 144
QLPDMVRGAR 0.080 161 QLARNGAGPR 0.080 242 LLTVEEAEKR 0.080 226
GGNVYILTGK 0.060 180 RGWWPVVKLK 0.060 52 SDIYVKSWVK 0.060 120
AVLVLQVWDY 0.060 300 LVLLTVFLLL 0.060 197 EAQEAQAGKK 0.060 276
FVNPLKTFVF 0.060 81 GNFNWRFVFR 0.048 290 RYWRTLVLLL 0.048 101
SVWRRSGPFA 0.040 259 QPEPLEKPSR 0.040 309 LVFYTIPGQI 0.040 21
RQPISYELRV 0.036 141 LELQLPDMVR 0.036 117 RQPAVLVLQV 0.036 58
SWVKGLEHDK 0.030 200 EAQAGKKKRK 0.030 30 VVIWNTEDVV 0.030 96
TEREVSVWRR 0.024 12 APPPVDIKPR 0.020 148 MVRGARGPEL 0.020 202
QAGKKKRKQR 0.020 185 VVKLKEAEDV 0.020 95 PTEREVSVWR 0.020 299
LLVLLTVFLL 0.018 246 EEAEKRPVGK 0.018 303 LTVFLLLVFY 0.015 312
YTIPGQISQV 0.015 168 GPRCNLFRCR 0.012 235 KVEAEFELLT 0.012 19
KPRQPISYEL 0.012 304 TVFLLLVFYT 0.012 296 VLLLLVLLTV 0.012 107
GPFALEEAEF 0.012 76 SLTGEGNFNW 0.012 301 VLLTVFLLLV 0.012 268
RPKTSFNWFV 0.012 222 FTDMGGNVYI 0.010 46 LTGEMSSDIY 0.010 37
DVVLDDENPL 0.009 261 EPLEKPSRPK 0.009 278 NPLKTFVFFI 0.009 316
GQISQVIFRP 0.008 280 LKTFVFFIWR 0.008 87 FVFRFDYLPT 0.008 302
LLTVFLLLVF 0.008 89 FRFDYLPTER 0.008 31 VIWNTEDVVL 0.008 207
KRKQRRRKGR 0.006 205 KKKRKQRRRK 0.006 216 RPEDLEFTDM 0.006 249
EKRPVGKGRK 0.006 294 TLVLLLLVLL 0.006 305 VFLLLVFYTI 0.006 82
NFNWRFVFRF 0.006 297 LLLLVLLTVF 0.006 199 QEAQAGKKKR 0.006 295
LVLLLLVLLT 0.006 154 GPELCSVQLA 0.006 151 GARGPELCSV 0.006 9
DVPAPPPVDI 0.006 298 LLLVLLTVFL 0.006 248 AEKRPVGKGR 0.006 229
VYILTGKVEA 0.006 67 KQETDVHFNS 0.005 123 VLQVWDYDRI 0.004 93
YLPTEREVSV 0.004 283 FVFFIWRRYW 0.004 203 AGKKKRKQRR 0.004 231
ILTGKVEAEF 0.004 108 PFALEEAEFR 0.004 140 SLELQLPDMV 0.004 313
TIPGQISQVI 0.004 155 PELCSVQLAR 0.004 38 VVLDDENPLT 0.003 275
WFVNPLKTFV 0.003 270 KTSFNWFVNP 0.003 54 IYVKSWVKGL 0.003 131
RISANDFLGS 0.002 Table XII: 158P3D2 v.2a A11-10-mers 117 KLLVRVYVVK
1.800 36 AVATLKIYNR 0.800 19 IQDQGEAEVK 0.600 26 EVKGTVSPKK 0.600
129 NLAPADPNGK 0.400 99 VLFSEPQISR 0.320 32 SPKKAVATLK 0.200 146
SAGRERQDTK 0.200 121 RVYVVKATNL 0.120 108 RGIPQNRPIK 0.090 25
AEVKGTVSPK 0.090 105 QISRGIPQNR 0.080 142 YVVVSAGRER 0.060 29
GTVSPKKAVA 0.045 152 QDTKERYIPK 0.040 200 GETHIDLENR 0.036 78
GSGHLVGKFK 0.030 161 KQLNPIFGEI 0.027 140 DPYVVVSAGR 0.024 109
GIPQNRPIKL 0.024 227 WVQQGPQEPF 0.020 82 LVGKFKGSFL 0.020 184
TVAVFEHDLV 0.020 124 VVKATNLAPA 0.020 76 EEGSGHLVGK 0.018 157
RYIPKQLNPI 0.018 112 QNRPIKLLVR 0.016 183 LTVAVFEHDL 0.015 219
GLASQYEVWV 0.012 206 LENRFYSHHR 0.012 170 ILELSISLPA 0.008 176
SLPAETELTV 0.008 91 LIYPESEAVL 0.008 148 GRERQDTKER 0.006 164
NPIFGEILEL 0.006 122 VYVVKATNLA 0.006 9 GVNLISMVGE 0.006 118
LLVRVYVVKA 0.006 81 HLVGKFKGSF 0.006 138 KADPYVVVSA 0.006 215
RANCGLASQY 0.006 123 YVVKATNLAP 0.006 90 FLIYPESEAV 0.006 168
GEILELSISL 0.005 59 NVFPLYRGQG 0.004 186 AVFEHDLVGS 0.004 42
IYNRSLEEEF 0.004 217 NCGLASQYEV 0.004 166 IFGEILELSI 0.004 192
LVGSDDLIGE 0.004 174 SISLPAETEL 0.004 158 YIPKQLNPIF 0.004 162
QLNPIFGEIL 0.004 92 IYPESEAVLF 0.004 151 RQDTKERYIP 0.004 197
DLIGETHIDL 0.004 56 DWLNVFPLYR 0.004 143 VVVSAGRERQ 0.003 201
ETHIDLENRF 0.003 89 SFLIYPESEA 0.003 98 AVLFSEPQIS 0.003 181
TELTVAVFEH 0.003 41 KIYNRSLEEE 0.002 84 GKFKGSFLIY 0.002 130
LAPADPNGKA 0.002 30 TVSPKKAVAT 0.002 15 MVGEIQDQGE 0.002 177
LPAETELTVA 0.002 66 GQGGQDGGGE 0.002 35 KAVATLKIYN 0.002 69
GQDGGGEEEG 0.002 54 FEDWLNVFPL 0.002 74 GEEEGSGHLV 0.002 149
RERQDTKERY 0.002 38 ATLKIYNRSL 0.002 203 HIDLENRFYS 0.001 85
KFKGSFLIYP 0.001 209 RFYSHHRANC 0.001 222 SQYEVWVQQG 0.001 111
PQNRPIKLLV 0.001 225 EVWVQQGPQE 0.001 18 EIQDQGEAEV 0.001 205
DLENRFYSHH 0.001 110 IPQNRPIKLL 0.001 119 LVRVYVVKAT 0.001 127
ATNLAPADPN 0.001 45 RSLEEEFNHF 0.001 114 RPIKLLVRVY 0.001 97
EAVLFSEPQI 0.001 57 WLNVFPLYRG 0.001 51 FNHFEDWLNV 0.001 12
LISMVGEIQD 0.001 14 SMVGEIQDQG 0.001 116 IKLLVRVYVV 0.001 73
GGEEEGSGHL 0.001 44 NRSLEEEFNH 0.001 10 VNLISMVGEI 0.001 126
KATNLAPADP 0.001 194 GSDDLIGETH 0.001 83 VGKFKGSFLI 0.001 Table
XII: 158P3D2 v.3 A11-10mers 1 LPTEREVSVR 0.040 8 SVRRRSGPFA 0.020 2
PTEREVSVRR 0.020 3 TEREVSVRRR 0.001 6 EVSVRRRSGP 0.001 9 VRRRSGPFAL
0.001 7 VSVRRRSGPF 0.000 10 RRRSGPFALE 0.000 5 REVSVRRRSG 0.000 4
EREVSVRRRS 0.000 Table XII: 158P3D2 v.4 A11-10mers 3 PTEREVSIWR
0.040 4 TEREVSIWRR 0.024 9 SIWRRSGPFA 0.008 1 YLPTEREVSI 0.004 2
LPTEREVSIW 0.002 7 EVSIWRRSGP 0.001 10 IWRRSGPFAL 0.001 8
VSIWRRSGPF 0.000 6 REVSIWRRSG 0.000 5 EREVSIWRRS 0.000 Table XII:
158P3D2 v.5a A11-10mers 2 LVLQVWDYTA 0.060 29 CVGPGAPSSA 0.020 9
YTASLPMTSL 0.010 4 LQVWDYTASL 0.009 40 CSWPAMGPGR 0.008 46
GPGRGAICFA 0.006 25 YQTWCVGPGA 0.006 33 GAPSSALCSW 0.006 5
QVWDYTASLP 0.004 12 SLPMTSLDPW 0.004 26 QTWCVGPGAP 0.002 19
DPWSCSYQTW 0.001 15 MTSLDPWSCS 0.001 38 ALCSWPAMGP 0.001 1
VLVLQVWDYT 0.001 48 GRGAICFAAA 0.001 49 RGAICFAAAA 0.001 31
GPGAPSSALC 0.001 45 MGPGRGAICF 0.000 6 VWDYTASLPM 0.000 21
WSCSYQTWCV 0.000 43 PAMGPGRGAI 0.000 44 AMGPGRGAIC 0.000 3
VLQVWDYTAS 0.000 13 LPMTSLDPWS 0.000 24 SYQTWCVGPG 0.000 17
SLDPWSCSYQ 0.000 16 TSLDPWSCSY 0.000 37 SALCSWPAMG 0.000 28
WCVGPGAPSS 0.000 8 DYTASLPMTS 0.000 34 APSSALCSWP 0.000 22
SCSYQTWCVG 0.000 10 TASLPMTSLD 0.000 30 VGPGAPSSAL 0.000 36
SSALCSWPAM 0.000 42 WPAMGPGRGA 0.000 39 LCSWPAMGPG 0.000 14
PMTSLDPWSC 0.000 11 ASLPMTSLDP 0.000 47 PGRGAICFAA 0.000 23
CSYQTWCVGP 0.000 7 WDYTASLPMT 0.000 18 LDPWSCSYQT 0.000 35
PSSALCSWPA 0.000 41 SWPAMGPGRG 0.000 27 TWCVGPGAPS 0.000 32
PGAPSSALCS 0.000 20 PWSCSYQTWC 0.000
[0771]
19TABLE XIII 158P3D2 A24, 9mers (variants 1, 2a, 3, 4 and 5a) SEQ.
ID Pos 123456789 Score NO. Table XIII: 158P3D2 v.1 A24-9mers 268
RPKTSFNWF 120.000 265 KPSRPKTSF 40.000 278 NPLKTFVFF 20.000 214
KGRPEDLEF 9.000 314 IPGQISQVI 8.000 94 LPTEREVSV 8.000 10 VPAPPPVDI
8.000 154 GPELCSVQL 6.000 168 GPRCNLFRC 6.000 255 KGRKQPEPL 6.000
133 SANDFLGSL 6.000 318 ISQVIFRPL 5.000 75 NSLTGEGNF 5.000 118
QPAVLVLQV 4.000 24 ISYELRVVI 4.000 22 QPISYELRV 4.000 38 VVLDDENPL
3.000 55 YVKSWVKGL 3.000 175 RCRRLRGWW 3.000 166 GAGPRCNLF 3.000
180 RGWWPVVKL 2.000 183 WPVVKLKEA 2.000 283 FVFFIWRRY 2.000 304
TVFLLLVFY 2.000 121 VLVLQVWDY 2.000 44 NPLTGEMSS 2.000 19 KPRQPISYE
1.200 178 RLRGWWPVV 1.200 299 LLVLLTVFL 1.000 165 NGAGPRCNL 1.000
224 DMGGNVYIL 1.000 277 VNPLKTFVF 1.000 298 LLLVLLTVF 1.000 294
TLVLLLLVL 1.000 137 FLGSLELQL 1.000 171 CNLFRCRRL 1.000 101
SVWRRSGPF 1.000 81 GNFNWRFVF 1.000 300 LVLLTVFLL 1.000 50 MSSDIYVKS
1.000 83 FNWRFVFRF 1.000 232 LTGKVEAEF 1.000 303 LTVFLLLVF 1.000
301 VLLTVFLLL 1.000 295 LVLLLLVLL 1.000 77 LTGEGNFNW 1.000 235
KVEAEFELL 0.900 151 GARGPELCS 0.900 46 LTGEMSSDI 0.800 51 SSDIYVKSW
0.750 132 ISANDFLGS 0.750 222 FTDMGGNVY 0.600 47 TGEMSSDIY 0.600
259 QPEPLEKPS 0.600 140 SLELQLPDM 0.600 212 RRKGRPEDL 0.600 124
LQVWDYDRI 0.600 293 RTLVLLLLV 0.400 306 FLLLVFYTI 0.400 251
RPVGKGRKQ 0.400 6 FPQDVPAPP 0.400 261 EPLEKPSRP 0.400 129 YDRISANDF
0.300 291 YWRTLVLLL 0.300 17 DIKPRQPIS 0.300 27 ELRVVIWNT 0.300 287
IWRRYWRTL 0.300 69 ETDVHFNSL 0.300 103 WRRSGPFAL 0.300 237
EAEFELLTV 0.270 216 RPEDLEFTD 0.240 164 RNGAGPRCN 0.200 234
GKVEAEFEL 0.200 30 VVIWNTEDV 0.200 313 TIPGQISQV 0.200 18 IKPRQPISY
0.200 150 RGARGPELC 0.200 297 LLLLVLLTV 0.200 42 DENPLTGEM 0.200
107 GPFALEEAE 0.200 290 RYWRTLVLL 0.200 302 LLTVFLLLV 0.200 12
APPPVDIKP 0.200 31 VIWNTEDVV 0.200 276 FVNPLKTFV 0.200 228
NVYILTGKV 0.200 125 QVWDYDRIS 0.200 86 RFVFRFDYL 0.200 144
QLPDMVRGA 0.200 66 DKQETDVHF 0.200 80 EGNFNWRFV 0.200 85 WRFVFRFDY
0.200 289 RRYWRTLVL 0.200 270 KTSFNWFVN 0.200 113 EAEFRQPAV 0.180
190 EAEDVEREA 0.180 76 SLTGEGNFN 0.150 266 PSRPKTSFN 0.150 32
IWNTEDVVL 0.150 119 PAVLVLQVW 0.150 Table XIII: 158P3D2 v.2a
A24-9mers 92 IYPESEAVL 360.000 122 VYVVKATNL 300.000 50 EFNHFEDWL
30.000 53 HFEDWLNVF 21.600 169 EILELSISL 8.640 175 ISLPAETEL 7.920
110 IPQNRPIKL 6.600 163 LNPIFGEIL 6.000 46 SLEEEFNHF 5.184 210
FYSHHRANC 5.000 198 LIGETHIDL 4.800 83 VGKFKGSFL 4.000 32 SPKKAVATL
4.000 39 TLKIYNRSL 4.000 184 TVAVFEHDL 4.000 108 RGIPQNRPI 3.600
162 QLNPIFGEI 3.326 228 VQQGPQEPF 3.000 180 ETELTVAVF 3.000 93
YPESEAVLF 3.000 43 YNRSLEEEF 2.640 78 GSGHLVGKF 2.640 159 IPKQLNPIF
2.400 82 LVGKFKGSF 2.000 151 RQDTKERYI 2.000 167 FGEILELSI 1.800
157 RYIPKQLNP 1.800 158 YIPKQLNPI 1.800 11 NLISMVGEI 1.650 191
DLVGSDDLI 1.500 98 AVLFSEPQI 1.500 85 KFKGSFLIY 1.200 155 KERYIPKQL
1.120 209 RFYSHHRAN 1.000 223 QYEVWVQQG 0.900 166 IFGEILELS 0.840
42 IYNRSLEEE 0.825 187 VFEHDLVGS 0.750 74 GEEEGSGHL 0.720 190
HDLVGSDDL 0.600 111 PQNRPIKLL 0.600 202 THIDLENRF 0.518 63
LYRGQGGQD 0.500 165 PIFGEILEL 0.440 4 PGDSDGVNL 0.400 55 EDWLNVFPL
0.400 212 SHHRANCGL 0.400 114 RPIKLLVRV 0.360 117 KLLVRVYVV 0.300
35 KAVATLKIY 0.300 121 RVYVVKATN 0.280 38 ATLKIYNRS 0.252 56
DWLNVFPLY 0.252 138 KADPYVVVS 0.240 34 KKAVATLKI 0.220 28 KGTVSPKKA
0.220 102 SEPQISRGI 0.210 173 LSISLPAET 0.198 81 HLVGKFKGS 0.180
123 YVVKATNLA 0.180 8 DGVNLISMV 0.180 112 QNRPIKLLV 0.168 218
CGLASQYEV 0.165 90 FLIYPESEA 0.165 194 GSDDLIGET 0.158 88 GSFLIYPES
0.154 31 VSPKKAVAT 0.150 185 VAVFEHDLV 0.150 24 EAEVKGTVS 0.150 29
GTVSPKKAV 0.150 196 DDLIGETHI 0.150 134 DPNGKADPY 0.150 176
SLPAETELT 0.150 128 TNLAPADPN 0.150 22 QGEAEVKGT 0.150 5 GDSDGVNLI
0.144 6 DSDGVNLIS 0.140 131 APADPNGKA 0.132 36 AVATLKIYN 0.120 99
VLFSEPQIS 0.120 30 TVSPKKAVA 0.120 146 SAGRERQDT 0.120 91 LIYPESEAV
0.120 177 LPAETELTV 0.120 3 DPGDSDGVN 0.120 216 ANCGLASQY 0.120 119
LVRVYVVKA 0.110 19 IQDQGEAEV 0.110 141 PYVVVSAGR 0.105 220
LASQYEVWV 0.100 136 NGKADPYVV 0.100 51 FNHFEDWLN 0.100 84 GKFKGSFLI
0.100 219 GLASQYEVW 0.100 71 DGGGEEEGS 0.100 105 QISRGIPQN 0.100
203 HIDLENRFY 0.100 89 SFLIYPESE 0.075 60 VFPLYRGQG 0.075 100
LFSEPQISR 0.060 Table XIII: 158P3D2 v.3 A24-9mers 7 SVRRRSGPF 2.000
9 RRRSGPFAL 0.800 4 REVSVRRRS 0.042 6 VSVRRRSGP 0.015 8 VRRRSGPFA
0.010 5 EVSVRRRSG 0.010 2 TEREVSVRR 0.002 3 EREVSVRRR 0.002 1
PTEREVSVR 0.002 Table XIII: 158P3D2 v.4 A24-9mers 8 SIWRRSGPF 2.000
1 LPTEREVSI 1.200 9 IWRRSGPFA 0.100 5 REVSIWRRS 0.042 7 VSIWRRSGP
0.015 2 PTEREVSIW 0.015 6 EVSIWRRSG 0.010 3 TEREVSIWR 0.002 4
EREVSIWRR 0.002 Table XIII: 158P3D2 v.5a A24-9mers 7 DYTASLPMT
5.000 4 QVWDYTASL 4.800 30 GPGAPSSAL 4.000 9 TASLPMTSL 4.000 45
GPGRGAICF 2.000 43 AMGPGRGAI 1.200 23 SYQTWCVGP 0.750 36 SALCSWPAM
0.750 48 RGAICFAAA 0.240 1 LVLQVWDYT 0.210 15 TSLDPWSCS 0.180 29
VGPGAPSSA 0.150 27 WCVGPGAPS 0.150 3 LQVWDYTAS 0.150 12 LPMTSLDPW
0.150 32 GAPSSALCS 0.150 49 GAICFAAAA 0.150 44 MGPGRGAIC 0.150 2
VLQVWDYTA 0.150 25 QTWCVGPGA 0.140 8 YTASLPMTS 0.120 16 SLDPWSCSY
0.120 28 CVGPGAPSS 0.120 14 MTSLDPWSC 0.100 33 APSSALCSW 0.100 35
SSALCSWPA 0.100 21 SCSYQTWCV 0.100 18 DPWSCSYQT 0.100 20 WSCSYQTWC
0.100 6 WDYTASLPM 0.050 10 ASLPMTSLD 0.018 40 SWPAMGPGR 0.015 11
SLPMTSLDP 0.015 42 PAMGPGRGA 0.015 47 GRGAICFAA 0.014 19 PWSCSYQTW
0.012 13 PMTSLDPWS 0.012 31 PGAPSSALC 0.012 39 CSWPAMGPG 0.012 24
YQTWCVGPG 0.010 41 WPAMGPGRG 0.010 5 VWDYTASLP 0.010 22 CSYQTWCVG
0.010 46 PGRGAICFA 0.010 37 ALCSWPAMG 0.010 26 TWCVGPGAP 0.010 38
LCSWPAMGP 0.010 17 LDPWSCSYQ 0.002 34 PSSALCSWP 0.001
[0772]
20TABLE XIV 158P3D2 A24, 10mers (variants 1, 2a, 3, 4 and 5a) SEQ.
ID Pos 1234567890 Score NO. Table XIV: 158P3D2 v.1 A24-10mers 290
RYWRTLVLLL 480.000 54 IYVKSWVKGL 300.000 128 DYDRISANDF 120.000 136
DFLGSLELQL 36.000 115 EFRQPAVLVL 20.000 82 NFNWRFVFRF 15.000 153
RGPELCSVQL 14.400 293 RTLVLLLLVL 14.400 305 VFLLLVFYTI 12.600 19
KPRQPISYEL 12.320 170 RCNLFRCRRL 12.000 25 SYELRVVIWN 10.500 300
LVLLTVFLLL 10.080 92 DYLPTEREVS 9.000 229 VYILTGKVEA 8.250 164
RNGAGPRCNL 8.000 37 DVVLDDENPL 7.200 294 TLVLLLLVLL 7.200 298
LLLVLLTVFL 7.200 317 QISQVIFRPL 6.720 299 LLVLLTVFLL 6.000 113
EAEFRQPAVL 6.000 291 YWRTLVLLLL 5.600 271 TSFNWFVNPL 4.800 134
ANDFLGSLEL 4.400 233 TGKVEAEFEL 4.400 148 MVRGARGPEL 4.400 31
VIWNTEDVVL 4.000 286 FIWRRYWRTL 4.000 132 ISANDFLGSL 4.000 102
VWRRSGPFAL 4.000 277 VNPLKTFVFF 3.600 276 FVNPLKTFVF 3.600 297
LLLLVLLTVF 3.600 231 ILTGKVEAEF 3.080 80 EGNFNWRFVF 3.000 78
TGEGNFNWRF 3.000 100 VSVWRRSGPF 3.000 313 TIPGQISQVI 2.520 302
LLTVFLLLVF 2.400 165 NGAGPRCNLF 2.400 107 GPFALEEAEF 2.200 216
RPEDLEFTDM 2.160 314 IPGQISQVIF 2.000 74 FNSLTGEGNF 2.000 274
NWFVNPLKTF 2.000 9 DVPAPPPVDI 1.500 278 NPLKTFVFFI 1.500 123
VLQVWDYDRI 1.500 309 LVFYTJPGQI 1.400 282 TFVFFIWRRY 1.050 222
FTDMGGNVYI 1.000 275 WFVNPLKTFV 0.900 139 GSLELQLPDM 0.900 234
GKVEAEFELL 0.864 239 EFELLTVEEA 0.825 211 RRRKGRPEDL 0.800 289
RRYWRTLVLL 0.800 285 FFIWRRYWRT 0.750 73 HFNSLTGEGN 0.750 221
EFTDMGGNVY 0.720 68 QETDVHFNSL 0.691 223 TDMGGNVYIL 0.600 310
VFYTIPGQIS 0.600 311 FYTIPGQISQ 0.500 173 LFRCRRLRGW 0.500 85
WRFVFRFDYL 0.480 61 KGLEHDKQET 0.475 213 RKGRPEDLEF 0.440 179
LRGWWPVVKL 0.440 267 SRPKTSFNWF 0.432 258 KQPEPLEKPS 0.432 67
KQETDVHFNS 0.420 129 YDRISANDFL 0.400 254 GKGRKQPEPL 0.400 288
WRRYWRTLVL 0.400 117 RQPAVLVLQV 0.360 29 RVVIWNTEDV 0.300 235
KVEAEFELLT 0.300 264 EKPSRPKTSF 0.300 21 RQPISYELRV 0.300 105
RSGPFALEEA 0.264 214 KGRPEDLEFT 0.240 131 RISANDFLGS 0.240 1
MWIDIFPQDV 0.216 296 VLLLLVLLTV 0.210 268 RPKTSFNWFV 0.200 265
KPSRPKTSFN 0.200 65 HDKQETDVHF 0.200 150 RGARGPELCS 0.200 227
GNVYILTGKV 0.198 154 GPELCSVQLA 0.180 303 LTVFLLLVFY 0.180 38
VVLDDENPLT 0.180 312 YTIPGQISQV 0.180 158 CSVQLARNGA 0.180 143
LQLPDMVRGA 0.180 295 LVLLLLVLLT 0.180 244 TVEEAEKRPV 0.180 75
NSLTGEGNFN 0.180 Table XIV: 158P3D2 v.2a A24-10mers 157 RYIPKQLNPI
216.000 42 IYNRSLEEEF 198.000 92 IYPESEAVLF 180.000 45 RSLEEEFNHF
10.368 122 VYVVKATNLA 9.000 121 RVYVVKATNL 8.000 166 IFGEILELSI
7.200 73 GGEEEGSGHL 7.200 162 QLNPIFGEIL 7.200 164 NPIFGEILEL 6.600
109 GIPQNRPIKL 6.600 31 VSPKKAVATL 6.000 38 ATLKIYNRSL 6.000 110
IPQNRPIKLL 6.000 183 LTVAVFEHDL 6.000 197 DLIGETHIDL 6.000 161
KQLNPIFGEI 5.544 3 DPGDSDGVNL 4.800 91 LIYPESEAVL 4.800 174
SISLPAETEL 4.400 211 YSHHRANCGL 4.000 82 LVGKFKGSFL 4.000 158
YIPKQLNPIF 3.600 227 WVQQGPQEPF 3.000 81 HLVGKFKGSF 3.000 201
ETHIDLENRF 2.880 77 EGSGHLVGKF 2.640 101 FSEPQISRGI 2.520 10
VNLISMVGEI 1.650 97 EAVLFSEPQI 1.500 223 QYEVWVQQGP 1.260 209
RFYSHHRANC 1.000 83 VGKFKGSFLI 1.000 154 TKERYIPKQL 0.840 89
SFLIYPESEA 0.825 50 EFNHFEDWLN 0.750 168 GEILELSISL 0.720 63
LYRGQGGQDG 0.600 210 FYSHHRANCG 0.600 6 DSDGVNLISM 0.500 189
EHDLVGSDDL 0.400 49 EEFNHFEDWL 0.400 54 FEDWLNVFPL 0.400 114
RPIKLLVRVY 0.360 215 RANCGLASQY 0.360 35 KAVATLKIYN 0.360 138
KADPYVVVSA 0.336 87 KGSFLIYPES 0.308 52 NHFEDWLNVF 0.288 179
AETELTVAVF 0.240 199 IGETHIDLEN 0.231 22 QGEAEVKGTV 0.210 170
ILELSISLPA 0.210 28 KGTVSPKKAV 0.200 18 EIQDQGEAEV 0.198 150
ERQDTKERYI 0.180 175 ISLPAETELT 0.180 98 AVLFSEPQIS 0.180 37
VATLKIYNRS 0.168 130 LAPADPNGKA 0.165 16 VGEIQDQGEA 0.165 118
LLVRVYVVKA 0.165 193 VGSDDLIGET 0.158 167 FGEILELSIS 0.150 90
FLIYPESEAV 0.150 29 GTVSPKKAVA 0.150 93 YPESEAVLFS 0.150 190
HDLVGSDDLI 0.150 218 CGLASQYEVW 0.150 127 ATNLAPADPN 0.150 176
SLPAETELTV 0.150 134 DPNGKADPYV 0.150 119 LVRVYVVKAT 0.140 172
ELSISLPAET 0.132 30 TVSPKKAVAT 0.120 145 VSAGRERQDT 0.120 4
PGDSDGVNLI 0.120 21 DQGEAEVKGT 0.120 186 AVFEHDLVGS 0.120 177
LPAETELTVA 0.120 217 NCGLASQYEV 0.110 53 HFEDWLNVFP 0.108 107
SRGIPQNRPI 0.100 184 TVAVFEHDLV 0.100 124 VVKATNLAPA 0.100 207
ENRFYSHHRA 0.100 203 HIDLENRFYS 0.100 136 NGKADPYVVV 0.100 51
FNHFEDWLNV 0.100 85 KFKGSFLIYP 0.100 195 SDDLIGETHI 0.100 219
GLASQYEVWV 0.100 43 YNRSLEEEFN 0.100 60 VFPLYRGQGG 0.090 187
VFEHDLVGSD 0.090 141 PYVVVSAGRE 0.075 100 LFSEPQISRG 0.060 117
KLLVRVYVVK 0.042 108 RGIPQNRPIK 0.036 65 RGQGGQDGGG 0.030 Table
XIV: 158P3D2 v.3 A24-10mers 7 VSVRRRSGPF 3.000 9 VRRRSGPFAL 0.400 8
SVRRRSGPFA 0.100 4 EREVSVRRRS 0.021 1 LPTEREVSVR 0.012 6 EVSVRRRSGP
0.010 5 REVSVRRRSG 0.003 10 RRRSGPFALE 0.002 2 PTEREVSVRR 0.002 3
TEREVSVRRR 0.001 Table XIV: 158P3D2 v.4 A24-10mers 10 IWRRSGPFAL
4.000 8 VSIWRRSGPF 3.000 1 YLPTEREVSI 1.500 2 LPTEREVSIW 0.120 9
SIWRRSGPFA 0.100 5 EREVSIWRRS 0.021 7 EVSIWRRSGP 0.010 6 REVSIWRRSG
0.003 3 PTEREVSIWR 0.002 4 TEREVSIWRR 0.001 Table XIV: 158P3D2 v.5a
A24-10mers 8 DYTASLPMTS 6.000 4 LQVWDYTASL 6.000 30 VGPGAPSSAL
6.000 9 YTASLPMTSL 4.000 45 MGPGRGAICF 3.000 24 SYQTWCVGPG 0.750 36
SSALCSWPAM 0.500 6 VWDYTASLPM 0.500 1 VLVLQVWDYT 0.210 49
RGAICFAAAA 0.200 16 TSLDPWSCSY 0.180 13 LPMTSLDPWS 0.180 43
PAMGPGRGAI 0.150 3 VLQVWDYTAS 0.150 12 SLPMTSLDPW 0.150 28
WCVGPGAPSS 0.150 33 GAPSSALCSW 0.150 2 LVLQVWDYTA 0.150 25
YQTWCVGPGA 0.140 19 DPWSCSYQTW 0.120 29 CVGPGAPSSA 0.120 44
AMGPGRGAIC 0.120 27 TWCVGPGAPS 0.100 42 WPAMGPGRGA 0.100 15
MTSLDPWSCS 0.100 46 GPGRGAICFA 0.100 21 WSCSYQTWCV 0.100 31
GPGAPSSALC 0.100 11 ASLPMTSLDP 0.018 18 LDPWSCSYQT 0.015 37
SALCSWPAMG 0.015 41 SWPAMGPGRG 0.015 47 PGRGAICFAA 0.014 34
APSSALCSWP 0.012 40 CSWPAMGPGR 0.012 48 GRGAICFAAA 0.012 32
PGAPSSALCS 0.012 17 SLDPWSCSYQ 0.012 5 QVWDYTASLP 0.012 7
WDYTASLPMT 0.010 39 LCSWPAMGPG 0.010 23 CSYQTWCVGP 0.010 20
PWSCSYQTWC 0.010 14 PMTSLDPWSC 0.010 26 QTWCVGPGAP 0.010 10
TASLPMTSLD 0.010 35 PSSALCSWPA 0.010 38 ALCSWPAMGP 0.010 22
SCSYQTWCVG 0.010
[0773]
21TABLE XV 158P3D2 B7, 9mers (variants 1, 2a, 3, 4 and 5a) SEQ. ID
Pos 123456789 Score NO. Table XV: 158P3D2 v.1 B7-9mers 255
KGRKQPEPL 40.000 154 GPELCSVQL 24.000 300 LVLLTVFLL 20.000 55
YVKSWVKGL 20.000 168 GPRCNLFRC 20.000 295 LVLLLLVLL 20.000 38
VVLDDENPL 20.000 133 SANDFLGSL 12.000 10 VPAPPPVDI 12.000 165
NGAGPRCNL 9.000 314 IPGQISQVI 8.000 180 RGWWPVVKL 6.000 235
KVEAEFELL 6.000 294 TLVLLLLVL 4.000 94 LPTEREVSV 4.000 22 QPISYELRV
4.000 301 VLLTVFLLL 4.000 291 YWRTLVLLL 4.000 318 ISQVIFRPL 4.000
103 WRRSGPFAL 4.000 299 LLVLLTVFL 4.000 118 QPAVLVLQV 4.000 137
FLGSLELQL 4.000 287 IWRRYWRTL 4.000 171 CNLFRCRRL 4.000 224
DMGGNVYIL 4.000 19 KPRQPISYE 3.000 178 RLRGWWPVV 2.000 183
WPVVKLKEA 2.000 114 AEFRQPAVL 1.200 69 ETDVHFNSL 1.200 276
FVNPLKTFV 1.000 27 ELRVVIWNT 1.000 30 VVIWNTEDV 1.000 228 NVYILTGKV
1.000 151 GARGPELCS 0.900 159 SVQLARNGA 0.750 148 MVRGARGPE 0.750
24 ISYELRVVI 0.600 265 KPSRPKTSF 0.600 12 APPPVDIKP 0.600 292
WRTLVLLLL 0.400 32 IWNTEDVVL 0.400 289 RRYWRTLVL 0.400 149
VRGARGPEL 0.400 46 LTGEMSSDI 0.400 306 FLLLVFYTI 0.400 272
SFNWFVNPL 0.400 234 GKVEAEFEL 0.400 278 NPLKTFVFF 0.400 130
DRISANDFL 0.400 86 RFVFRFDYL 0.400 135 NDFLGSLEL 0.400 44 NPLTGEMSS
0.400 212 RRKGRPEDL 0.400 268 RPKTSFNWF 0.400 290 RYWRTLVLL 0.400
116 FRQPAVLVL 0.400 124 LQVWDYDRI 0.400 140 SLELQLPDM 0.300 288
WRRYWRTLV 0.300 162 LARNGAGPR 0.300 115 EFRQPAVLV 0.300 175
RCRRLRGWW 0.300 214 KGRPEDLEF 0.200 80 EGNFNWRFV 0.200 302
LLTVFLLLV 0.200 297 LLLLVLLTV 0.200 261 EPLEKPSRP 0.200 107
GPFALEEAE 0.200 31 VIWNTEDVV 0.200 313 TIPGQISQV 0.200 251
RPVGKGRKQ 0.200 293 RTLVLLLLV 0.200 6 FPQDVPAPP 0.200 237 EAEFELLTV
0.180 113 EAEFRQPAV 0.180 193 DVEREAQEA 0.150 120 AVLVLQVWD 0.150
259 QPEPLEKPS 0.120 223 TDMGGNVYI 0.120 283 FVFFIWRRY 0.100 106
SGPFALEEA 0.100 101 SVWRRSGPF 0.100 150 RGARGPELC 0.100 304
TVFLLLVFY 0.100 42 DENPLTGEM 0.100 296 VLLLLVLLT 0.100 125
QVWDYDRIS 0.100 225 MGGNVYILT 0.100 144 QLPDMVRGA 0.100 102
VWRRSGPFA 0.100 4 DIFPQDVPA 0.100 88 VFRFDYLPT 0.100 209 KQRRRKGRP
0.100 286 FIWRRYWRT 0.100 230 YILTGKVEA 0.100 16 VDIKPRQPI 0.090
145 LPDMVRGAR 0.090 190 EAEDVEREA 0.090 Table XV: 158P3D2 v.2a
B7-9-mers 32 SPKKAVATL 80.000 110 IPQNRPIKL 80.000 184 TVAVFEHDL
20.000 131 APADPNGKA 9.000 98 AVLFSEPQI 6.000 119 LVRVYVVKA 5.000
198 LIGETHIDL 4.000 175 ISLPAETEL 4.000 169 EILELSISL 4.000 114
RPIKLLVRV 4.000 83 VGKFKGSFL 4.000 163 LNPIFGEIL 4.000 39 TLKIYNRSL
4.000 177 LPAETELTV 4.000 155 KERYIPKQL 4.000 112 QNRPIKLLV 2.000
220 LASQYEVWV 0.600 111 PQNRPIKLL 0.600 185 VAVFEHDLV 0.600 123
YVVKATNLA 0.500 30 TVSPKKAVA 0.500 146 SAGRERQDT 0.450 190
HDLVGSDDL 0.400 92 IYPESEAVL 0.400 212 SHHRANCGL 0.400 122
VYVVKATNL 0.400 55 EDWLNVFPL 0.400 191 DLVGSDDLI 0.400 165
PIFGEILEL 0.400 50 EFNHFEDWL 0.400 159 IPKQLNPIF 0.400 108
RGIPQNRPI 0.400 162 QLNPIFGEI 0.400 134 DPNGKADPY 0.400 3 DPGDSDGVN
0.400 11 NLISMVGEI 0.400 158 YIPKQLNPI 0.400 29 GTVSPKKAV 0.300 103
EPQISRGIP 0.300 147 AGRERQDTK 0.300 36 AVATLKIYN 0.300 61 FPLYRGQGG
0.200 164 NPIFGEILE 0.200 136 NGKADPYVV 0.200 8 DGVNLISMV 0.200 218
CGLASQYEV 0.200 43 YNRSLEEEF 0.200 117 KLLVRVYVV 0.200 91 LIYPESEAV
0.200 140 DPYVVVSAG 0.200 186 AVFEHDLVG 0.150 90 FLIYPESEA 0.150 93
YPESEAVLF 0.120 74 GEEEGSGHL 0.120 167 FGEILELSI 0.120 151
RQDTKERYI 0.120 4 PGDSDGVNL 0.120 207 ENRFYSHHR 0.100 28 KGTVSPKKA
0.100 213 HHRANCGLA 0.100 173 LSISLPAET 0.100 176 SLPAETELT 0.100 7
SDGVNLISM 0.100 31 VSPKKAVAT 0.100 121 RVYVVKATN 0.100 106
ISRGIPQNR 0.100 82 LVGKFKGSF 0.100 144 VVSAGRERQ 0.075 38 ATLKIYNRS
0.060 179 AETELTVAV 0.060 216 ANCGLASQY 0.060 35 KAVATLKIY 0.060 19
IQDQGEAEV 0.060 143 VVVSAGRER 0.050 192 LVGSDDLIG 0.050 142
YVVVSAGRE 0.050 9 GVNLISMVG 0.050 124 VVKATNLAP 0.050 59 NVFPLYRGQ
0.050 26 EVKGTVSPK 0.050 225 EVWVQQGPQ 0.050 227 WVQQGPQEP 0.050 15
MVGEIQDQG 0.050 34 KKAVATLKI 0.040 5 GDSDGVNLI 0.040 196 DDLIGETHI
0.040 84 GKFKGSFLI 0.040 102 SEPQISRGI 0.040 116 IKLLVRVYV 0.030
128 TNLAPADPN 0.030 126 KATNLAPAD 0.030 221 ASQYEVWVQ 0.030 130
LAPADPNGK 0.030 228 VQQGPQEPF 0.030 37 VATLKIYNR 0.030 137
GKADPYVVV 0.030 139 ADPYVVVSA 0.030 13 ISMVGEIQD 0.030 215
RANCGLASQ 0.030 127 ATNLAPADP 0.030 Table XV: 158P3D2 v.3 B7-9mers
9 RRRSGPFAL 4.000 7 SVRRRSGPF 1.000 8 VRRRSGPFA 0.100 5 EVSVRRRSG
0.075 6 VSVRRRSGP 0.015 2 TEREVSVRR 0.010 4 REVSVRRRS 0.003 3
EREVSVRRR 0.000 1 PTEREVSVR 0.000 Table XV: 158P3D2 v.4 B7-9mers 1
LPTEREVSI 8.000 9 IWRRSGPFA 0.100 6 EVSIWRRSG 0.075 8 SIWRRSGPF
0.020 7 VSIWRRSGP 0.015 3 TEREVSIWR 0.010 5 REVSIWRRS 0.002 2
PTEREVSIW 0.001 4 EREVSIWRR 0.000 Table XV: 158P3D2 v.5a -B7-9-mers
30 GPGAPSSAL 120.000 4 QVWDYTASL 20.000 9 TASLPMTSL 18.000 36
SALCSWPAM 3.000 18 DPWSCSYQT 2.000 43 AMGPGRGAI 1.800 12 LPMTSLDPW
1.200 33 APSSALCSW 1.200 1 LVLQVWDYT 0.500 45 GPGRGAICF 0.400 49
GAICFAAAA 0.300 41 WPAMGPGRG 0.200 21 SCSYQTWCV 0.200 42 PAMGPGRGA
0.135 48 RGAICFAAA 0.100 6 WDYTASLPM 0.100 2 VLQVWDYTA 0.100 35
SSALCSWPA 0.100 28 CVGPGAPSS 0.100 46 PGRGAICFA 0.100 20 WSCSYQTWC
0.100 29 VGPGAPSSA 0.100 25 QTWCVGPGA 0.100 14 MTSLDPWSC 0.100 44
MGPGRGAIC 0.100 32 GAPSSALCS 0.060 15 TSLDPWSCS 0.030 27 WCVGPGAPS
0.030 10 ASLPMTSLD 0.030 37 ALCSWPAMG 0.030 8 YTASLPMTS 0.020 3
LQVWDYTAS 0.020 38 LCSWPAMGP 0.015 11 SLPMTSLDP 0.010 31 PGAPSSALC
0.010 22 CSYQTWCVG 0.010 39 CSWPAMGPG 0.010 47 GRGAICFAA 0.010 24
YQTWCVGPG 0.010 7 DYTASLPMT 0.010 16 SLDPWSCSY 0.006 13 PMTSLDPWS
0.002 17 LDPWSCSYQ 0.001 40 SWPAMGPGR 0.001 23 SYQTWCVGP 0.001 26
TWCVGPGAP 0.001 34 PSSALCSWP 0.001 5 VWDYTASLP 0.000 19 PWSCSYQTW
0.000
[0774]
22TABLE XVI 158P3D2 B7, 10mers (variants 1, 2a, 3, 4 and 5a) SEQ.
ID Pos 1234567890 Score NO. Table XVI: 158P3D2 v.1 B7-10mers 19
KPRQPISYEL 800.000 148 MVRGARGPEL 200.000 37 DVVLDDENPL 20.000 300
LVLLTVFLLL 20.000 164 RNGAGPRCNL 9.000 278 NPLKTFVFFI 8.000 151
GARGPELCSV 6.000 216 RPEDLEFTDM 6.000 31 VIWNTEDVVL 4.000 298
LLLVLLTVFL 4.000 294 TLVLLLLVLL 4.000 129 YDRISANDFL 4.000 132
ISANDFLGSL 4.000 288 WRRYWRTLVL 4.000 170 RCNLFRCRRL 4.000 22
QPISYELRVV 4.000 115 EFRQPAVLVL 4.000 153 RGPELCSVQL 4.000 293
RTLVLLLLVL 4.000 291 YWRTLVLLLL 4.000 286 FIWRRYWRTL 4.000 271
TSFNWFVNPL 4.000 233 TGKVEAEFEL 4.000 268 RPKTSFNWFV 4.000 299
LLVLLTVFLL 4.000 211 RRRKGRPEDL 4.000 102 VWRRSGPFAL 4.000 317
QISQVIFRPL 4.000 134 ANDFLGSLEL 3.600 113 EAEFRQPAVL 3.600 9
DVPAPPPVDI 3.000 162 LARNGAGPRC 3.000 309 LVFYTIPGQI 2.000 168
GPRCNLFRCR 2.000 223 TDMGGNVYIL 1.200 30 VVIWNTEDVV 1.000 29
RVVIWNTEDV 1.000 214 KGRPEDLEFT 1.000 185 VVKLKEAEDV 1.000 139
GSLELQLPDM 1.000 125 QVWDYDRISA 0.750 12 APPPVDIKPR 0.600 154
GPELCSVQLA 0.600 179 LRGWWPVVKL 0.600 295 LVLLLLVLLT 0.500 38
VVLDDENPLT 0.500 87 FVFRFDYLPT 0.500 101 SVWRRSGPFA 0.500 304
TVFLLLVFYT 0.500 54 IYVKSWVKGL 0.400 313 TIPGQISQVI 0.400 289
RRYWRTLVLL 0.400 136 DFLGSLELQL 0.400 234 GKVEAEFELL 0.400 254
GKGRKQPEPL 0.400 118 QPAVLVLQVW 0.400 314 IPGQISQVIF 0.400 68
QETDVHFNSL 0.400 107 GPFALEEAEF 0.400 123 VLQVWDYDRI 0.400 290
RYWRTLVLLL 0.400 94 LPTEREVSVW 0.400 265 KPSRPKTSFN 0.400 85
WRFVFRFDYL 0.400 261 EPLEKPSRPK 0.300 10 VPAPPPVDIK 0.300 120
AVLVLQVWDY 0.300 167 AGPRCNLFRC 0.300 287 IWRRYWRTLV 0.300 244
TVEEAEKRPV 0.300 251 RPVGKGRKQP 0.300 6 FPQDVPAPPP 0.300 296
VLLLLVLLTV 0.200 117 RQPAVLVLQV 0.200 44 NPLTGEMSSD 0.200 176
CRRLRGWWPV 0.200 183 WPVVKLKEAE 0.200 301 VLLTVFLLLV 0.200 227
GNVYILTGKV 0.200 21 RQPISYELRV 0.200 312 YTIPGQISQV 0.200 93
YLPTEREVSV 0.200 235 KVEAEFELLT 0.150 158 CSVQLARNGA 0.150 283
FVFFIWRRYW 0.150 255 KGRKQPEPLE 0.150 15 PVDIKPRQPI 0.135 222
FTDMGGNVYI 0.120 209 KQRRRKGRPE 0.100 105 RSGPFALEEA 0.100 27
ELRVVIWNTE 0.100 273 FNWFVNPLKT 0.100 143 LQLPDMVRGA 0.100 175
RCRRLRGWWP 0.100 276 FVNPLKTFVF 0.100 61 KGLEHDKQET 0.100 224
DMGGNVYILT 0.100 178 RLRGWWPVVK 0.100 194 VEREAQEAQA 0.100 114
AEFRQPAVLV 0.090 Table XVI: 158P3D2 v.2a B7-10mers 110 IPQNRPIKLL
120.000 164 NPIFGEILEL 80.000 3 DPGDSDGVNL 80.000 121 RVYVVKATNL
20.000 82 LVGKFKGSFL 20.000 38 ATLKIYNRSL 12.000 119 LVRVYVVKAT
5.000 91 LIYPESEAVL 4.000 197 DLIGETHIDL 4.000 211 YSHHRANCGL 4.000
31 VSPKKAVATL 4.000 162 QLNPIFGEIL 4.000 174 SISLPAETEL 4.000 134
DPNGKADPYV 4.000 109 GIPQNRPIKL 4.000 183 LTVAVFEHDL 4.000 177
LPAETELTVA 2.000 73 GGEEEGSGHL 1.200 97 EAVLFSEPQI 1.200 184
TVAVFEHDLV 1.000 207 ENRFYSHHRA 1.000 131 APADPNGKAD 0.600 124
VVKATNLAPA 0.500 30 TVSPKKAVAT 0.500 130 LAPADPNGKA 0.450 83
VGKFKGSFLI 0.400 161 KQLNPIFGEI 0.400 10 VNLISMVGEI 0.400 114
RPIKLLVRVY 0.400 168 GEILELSISL 0.400 49 EEFNHFEDWL 0.400 98
AVLFSEPQIS 0.300 147 AGRERQDTKE 0.300 136 NGKADPYVVV 0.300 6
DSDGVNLISM 0.300 186 AVFEHDLVGS 0.300 28 KGTVSPKKAV 0.300 32
SPKKAVATLK 0.200 219 GLASQYEVWV 0.200 61 FPLYRGQGGQ 0.200 18
EIQDQGEAEV 0.200 217 NCGLASQYEV 0.200 103 EPQISRGIPQ 0.200 51
FNHFEDWLNV 0.200 140 DPYVVVSAGR 0.200 90 FLIYPESEAV 0.200 159
IPKQLNPIFG 0.200 176 SLPAETELTV 0.200 43 YNRSLEEEFN 0.200 106
ISRGIPQNRP 0.150 36 AVATLKIYNR 0.150 145 VSAGRERQDT 0.150 227
WVQQGPQEPF 0.150 93 YPESEAVLFS 0.120 154 TKERYIPKQL 0.120 189
EHDLVGSDDL 0.120 54 FEDWLNVFPL 0.120 101 FSEPQISRGI 0.120 29
GTVSPKKAVA 0.100 112 QNRPIKLLVR 0.100 175 ISLPAETELT 0.100 21
DQGEAEVKGT 0.100 193 VGSDDLIGET 0.100 118 LLVRVYVVKA 0.100 172
ELSISLPAET 0.100 127 ATNLAPADPN 0.090 138 KADPYVVVSA 0.090 143
VVVSAGRERQ 0.075 59 NVFPLYRGQG 0.075 35 KAVATLKIYN 0.060 215
RANCGLASQY 0.060 37 VATLKIYNRS 0.060 22 QGEAEVKGTV 0.060 192
LVGSDDLIGE 0.050 142 YVVVSAGRER 0.050 144 VVSAGRERQD 0.050 9
GVNLISMVGE 0.050 225 EVWVQQGPQE 0.050 123 YVVKATNLAP 0.050 26
EVKGTVSPKK 0.050 15 MVGEIQDQGE 0.050 107 SRGIPQNRPI 0.040 166
IFGEILELSI 0.040 157 RYIPKQLNPI 0.040 190 HDLVGSDDLI 0.040 150
ERQDTKERYI 0.040 220 LASQYEVWVQ 0.030 216 ANCGLASQYE 0.030 126
KATNLAPADP 0.030 146 SAGRERQDTK 0.030 13 ISMVGEIQDQ 0.030 221
ASQYEVWVQQ 0.030 185 VAVFEHDLVG 0.030 115 PIKLLVRVYV 0.030 155
KERYIPKQLN 0.030 16 VGEIQDQGEA 0.030 170 ILELSISLPA 0.030 158
YIPKQLNPIF 0.020 7 SDGVNLISMV 0.020 149 RERQDTKERY 0.020 Table XVI:
158P3D2 v.3 B7-10mers 8 SVRRRSGPFA 5.000 9 VRRRSGPFAL 4.000 1
LPTEREVSVR 0.200 6 EVSVRRRSGP 0.075 7 VSVRRRSGPF 0.020 10
RRRSGPFALE 0.015 3 TEREVSVRRR 0.010 5 REVSVRRRSG 0.002 4 EREVSVRRRS
0.001 2 PTEREVSVRR 0.000 Table XVI: 158P3D2 v.4 B7-10mers 10
IWRRSGPFAL 4.000 1 YLPTEREVSI 0.400 2 LPTEREVSIW 0.400 9 SIWRRSGPFA
0.100 7 EVSIWRRSGP 0.075 8 VSIWRRSGPF 0.020 4 TEREVSIWRR 0.010 6
REVSIWRRSG 0.002 5 EREVSIWRRS 0.001 3 PTEREVSIWR 0.000 Table XVI:
158P3D2 v.5a B7-10mers 9 YTASLPMTSL 6.000 30 VGPGAPSSAL 6.000 4
LQVWDYTASL 4.000 42 WPAMGPGRGA 3.000 46 GPGRGAICFA 2.000 31
GPGAPSSALC 2.000 13 LPMTSLDPWS 1.200 36 SSALCSWPAM 1.000 34
APSSALCSWP 0.600 43 PAMGPGRGAI 0.540 29 CVGPGAPSSA 0.500 2
LVLQVWDYTA 0.500 19 DPWSCSYQTW 0.400 44 AMGPGRGAIC 0.300 21
WSCSYQTWCV 0.200 1 VLVLQVWDYT 0.100 25 YQTWCVGPGA 0.100 49
RGAICFAAAA 0.100 47 PGRGAICFAA 0.100 33 GAPSSALCSW 0.060 5
QVWDYTASLP 0.050 38 ALCSWPAMGP 0.045 15 MTSLDPWSCS 0.030 11
ASLPMTSLDP 0.030 10 TASLPMTSLD 0.030 37 SALCSWPAMG 0.030 6
VWDYTASLPM 0.030 45 MGPGRGAICF 0.020 16 TSLDPWSCSY 0.020 12
SLPMTSLDPW 0.020 28 WCVGPGAPSS 0.020 3 VLQVWDYTAS 0.020 48
GRGAICFAAA 0.010 22 SCSYQTWCVG 0.010 23 CSYQTWCVGP 0.010 18
LDPWSCSYQT 0.010 14 PMTSLDPWSC 0.010 7 WDYTASLPMT 0.010 35
PSSALCSWPA 0.010 39 LCSWPAMGPG 0.010 26 QTWCVGPGAP 0.010 40
CSWPAMGPGR 0.010 27 TWCVGPGAPS 0.003 17 SLDPWSCSYQ 0.003 8
DYTASLPMTS 0.002 32 PGAPSSALCS 0.002 24 SYQTWCVGPG 0.001 41
SWPAMGPGRG 0.001 20 PWSCSYQTWC 0.001
[0775]
23TABLE XVII 158P3D2 B35, 9mers (variants 1, 2a, 3, 4 and 5a) SEQ.
ID Pos 123456789 Score NO. Table XVII: 158P3D2 v.1 B35-9mers 268
RPKTSFNWF 120.000 265 KPSRPKTSF 40.000 278 NPLKTFVFF 20.000 214
KGRPEDLEF 9.000 314 IPGQISQVI 8.000 94 LPTEREVSV 8.000 10 VPAPPPVDI
8.000 154 GPELCSVQL 6.000 168 GPRCNLFRC 6.000 255 KGRKQPEPL 6.000
133 SANDFLGSL 6.000 318 ISQVIFRPL 5.000 75 NSLTGEGNF 5.000 118
QPAVLVLQV 4.000 24 ISYELRVVI 4.000 22 QPISYELRV 4.000 38 VVLDDENPL
3.000 55 YVKSWVKGL 3.000 175 RCRRLRGWW 3.000 166 GAGPRCNLF 3.000
180 RGWWPVVKL 2.000 183 WPVVKLKEA 2.000 283 FVFFIWRRY 2.000 304
TVFLLLVFY 2.000 121 VLVLQVWDY 2.000 44 NPLTGEMSS 2.000 19 KPRQPISYE
1.200 178 RLRGWWPVV 1.200 299 LLVLLTVFL 1.000 165 NGAGPRCNL 1.000
224 DMGGNVYIL 1.000 277 VNPLKTFVF 1.000 298 LLLVLLTVF 1.000 294
TLVLLLLVL 1.000 137 FLGSLELQL 1.000 171 CNLFRCRRL 1.000 101
SVWRRSGPF 1.000 81 GNFNWRFVF 1.000 300 LVLLTVFLL 1.000 50 MSSDIYVKS
1.000 83 FNWRFVFRF 1.000 232 LTGKVEAEF 1.000 303 LTVFLLLVF 1.000
301 VLLTVFLLL 1.000 295 LVLLLLVLL 1.000 77 LTGEGNFNW 1.000 235
KVEAEFELL 0.900 151 GARGPELCS 0.900 46 LTGEMSSDI 0.800 51 SSDIYVKSW
0.750 132 ISANDFLGS 0.750 222 FTDMGGNVY 0.600 47 TGEMSSDIY 0.600
259 QPEPLEKPS 0.600 140 SLELQLPDM 0.600 212 RRKGRPEDL 0.600 124
LQVWDYDRI 0.600 293 RTLVLLLLV 0.400 306 FLLLVFYTI 0.400 251
RPVGKGRKQ 0.400 6 FPQDVPAPP 0.400 261 EPLEKPSRP 0.400 129 YDRISANDF
0.300 291 YWRTLVLLL 0.300 17 DIKPRQPIS 0.300 27 ELRVVIWNT 0.300 287
IWRRYWRTL 0.300 69 ETDVHFNSL 0.300 103 WRRSGPFAL 0.300 237
EAEFELLTV 0.270 216 RPEDLEFTD 0.240 164 RNGAGPRCN 0.200 234
GKVEAEFEL 0.200 30 VVIWNTEDV 0.200 313 TIPGQISQV 0.200 18 IKPRQPISY
0.200 150 RGARGPELC 0.200 297 LLLLVLLTV 0.200 42 DENPLTGEM 0.200
107 GPFALEEAE 0.200 290 RYWRTLVLL 0.200 302 LLTVFLLLV 0.200 12
APPPVDIKP 0.200 31 VIWNTEDVV 0.200 276 FVNPLKTFV 0.200 228
NVYILTGKV 0.200 125 QVWDYDRIS 0.200 86 RFVFRFDYL 0.200 144
QLPDMVRGA 0.200 66 DKQETDVHF 0.200 80 EGNFNWRFV 0.200 85 WRFVFRFDY
0.200 289 RRYWRTLVL 0.200 270 KTSFNWFVN 0.200 113 EAEFRQPAV 0.180
190 EAEDVEREA 0.180 76 SLTGEGNFN 0.150 266 PSRPKTSFN 0.150 32
IWNTEDVVL 0.150 119 PAVLVLQVW 0.150 Table XVII: 158P3D2 v.2a
B35-9-mers 32 SPKKAVATL 60.000 159 IPKQLNPIF 60.000 134 DPNGKADPY
40.000 110 IPQNRPIKL 20.000 35 KAVATLKIY 12.000 93 YPESEAVLF 9.000
177 LPAETELTV 8.000 114 RPIKLLVRV 8.000 78 GSGHLVGKF 5.000 175
ISLPAETEL 5.000 131 APADPNGKA 4.000 3 DPGDSDGVN 4.000 39 TLKIYNRSL
3.000 83 VGKFKGSFL 3.000 43 YNRSLEEEF 3.000 198 LIGETHIDL 2.000 216
ANCGLASQY 2.000 169 EILELSISL 2.000 85 KFKGSFLIY 1.200 228
VQQGPQEPF 1.000 184 TVAVFEHDL 1.000 82 LVGKFKGSF 1.000 163
LNPIFGEIL 1.000 136 NGKADPYVV 0.900 203 HIDLENRFY 0.900 185
VAVFEHDLV 0.900 46 SLEEEFNHF 0.900 108 RGIPQNRPI 0.800 155
KERYIPKQL 0.600 112 QNRPIKLLV 0.600 220 LASQYEVWV 0.600 115
PIKLLVRVY 0.600 88 GSFLIYPES 0.500 219 GLASQYEVW 0.500 173
LSISLPAET 0.500 31 VSPKKAVAT 0.500 146 SAGRERQDT 0.450 158
YIPKQLNPI 0.400 11 NLISMVGEI 0.400 191 DLVGSDDLI 0.400 98 AVLFSEPQI
0.400 117 KLLVRVYVV 0.400 162 QLNPIFGEI 0.400 150 ERQDTKERY 0.400
194 GSDDLIGET 0.300 91 LIYPESEAV 0.300 119 LVRVYVVKA 0.300 45
RSLEEEFNH 0.300 180 ETELTVAVF 0.300 151 RQDTKERYI 0.240 140
DPYVVVSAG 0.200 121 RVYVVKATN 0.200 202 THIDLENRF 0.200 28
KGTVSPKKA 0.200 7 SDGVNLISM 0.200 56 DWLNVFPLY 0.200 8 DGVNLISMV
0.200 164 NPIFGEILE 0.200 218 CGLASQYEV 0.200 29 GTVSPKKAV 0.200 61
FPLYRGQGG 0.200 92 IYPESEAVL 0.200 103 EPQISRGIP 0.200 138
KADPYVVVS 0.180 165 PIFGEILEL 0.150 106 ISRGIPQNR 0.150 176
SLPAETELT 0.150 6 DSDGVNLIS 0.150 51 FNHFEDWLN 0.150 99 VLFSEPQIS
0.150 71 DGGGEEEGS 0.150 167 FGEILELSI 0.120 212 SHHRANCGL 0.100 50
EFNHFEDWL 0.100 36 AVATLKIYN 0.100 90 FLIYPESEA 0.100 81 HLVGKFKGS
0.100 122 VYVVKATNL 0.100 105 QISRGIPQN 0.100 190 HDLVGSDDL 0.100
38 ATLKIYNRS 0.100 123 YVVKATNLA 0.100 111 PQNRPIKLL 0.100 128
TNLAPADPN 0.100 55 EDWLNVFPL 0.100 30 TVSPKKAVA 0.100 24 EAEVKGTVS
0.090 34 KKAVATLKI 0.080 5 GDSDGVNLI 0.080 221 ASQYEVWVQ 0.075 52
NHFEDWLNV 0.060 126 KATNLAPAD 0.060 215 RANCGLASQ 0.060 153
DTKERYIPK 0.060 147 AGRERQDTK 0.060 53 HFEDWLNVF 0.060 19 IQDQGEAEV
0.060 74 GEEEGSGHL 0.060 49 EEFNHFEDW 0.050 145 VSAGRERQD 0.050
Table XVII: 158P3D2 v.3 B35-9-mers 7 SVRRRSGPF 3.000 9 RRRSGPFAL
0.600 6 VSVRRRSGP 0.050 8 VRRRSGPFA 0.030 4 REVSVRRRS 0.020 5
EVSVRRRSG 0.010 2 TEREVSVRR 0.006 1 PTEREVSVR 0.000 3 EREVSVRRR
0.000 Table XVII: 158P3D2 v.4 B35-9-mers 1 LPTEREVSI 16.000 8
SIWRRSGPF 1.000 7 VSIWRRSGP 0.050 9 IWRRSGPFA 0.030 2 PTEREVSIW
0.022 5 REVSIWRRS 0.020 6 EVSIWRRSG 0.010 3 TEREVSIWR 0.006 4
EREVSIWRR 0.000 Table XVII: 158P3D2 v.5a B35-9-mers 45 GPGRGAICF
20.000 30 GPGAPSSAL 20.000 33 APSSALCSW 10.000 12 LPMTSLDPW 10.000
36 SALCSWPAM 6.000 9 TASLPMTSL 3.000 4 QVWDYTASL 2.000 18 DPWSCSYQT
2.000 15 TSLDPWSCS 1.000 16 SLDPWSCSY 0.600 20 WSCSYQTWC 0.500 35
SSALCSWPA 0.500 43 AMGPGRGAI 0.400 49 GAICFAAAA 0.300 32 GAPSSALCS
0.300 6 WDYTASLPM 0.200 41 WPAMGPGRG 0.200 21 SCSYQTWCV 0.200 48
RGAICFAAA 0.200 3 LQVWDYTAS 0.150 14 MTSLDPWSC 0.150 1 LVLQVWDYT
0.100 2 VLQVWDYTA 0.100 27 WCVGPGAPS 0.100 25 QTWCVGPGA 0.100 29
VGPGAPSSA 0.100 44 MGPGRGAIC 0.100 28 CVGPGAPSS 0.100 8 YTASLPMTS
0.100 10 ASLPMTSLD 0.050 22 CSYQTWCVG 0.050 39 CSWPAMGPG 0.050 42
PAMGPGRGA 0.030 46 PGRGAICFA 0.030 13 PMTSLDPWS 0.010 37 ALCSWPAMG
0.010 38 LCSWPAMGP 0.010 7 DYTASLPMT 0.010 11 SLPMTSLDP 0.010 24
YQTWCVGPG 0.010 31 PGAPSSALC 0.010 47 GRGAICFAA 0.010 34 PSSALCSWP
0.005 19 PWSCSYQTW 0.005 17 LDPWSCSYQ 0.001 26 TWCVGPGAP 0.001 23
SYQTWCVGP 0.001 40 SWPAMGPGR 0.001 5 VWDYTASLP 0.000
[0776]
24TABLE XVIII 158P3D2 B35, 10mers (variants 1, 2a, 3, 4 and 5a)
SEQ. ID Pos 1234567890 Score NO. Table XVIII: 158P3D2 v.1
B35-10mers 19 KPRQPISYEL 120.000 216 RPEDLEFTDM 72.000 94
LPTEREVSVW 30.000 107 GPFALEEAEF 30.000 268 RPKTSFNWFV 24.000 139
GSLELQLPDM 20.000 314 IPGQISQVIF 20.000 118 QPAVLVLQVW 10.000 278
NPLKTFVFFI 8.000 17 DIKPRQPISY 6.000 22 QPISYELRVV 6.000 271
TSFNWFVNPL 5.000 24 ISYELRVVIW 5.000 50 MSSDIYVKSW 5.000 132
ISANDFLGSL 5.000 100 VSVWRRSGPF 5.000 46 LTGEMSSDIY 4.000 153
RGPELCSVQL 4.000 265 KPSRPKTSFN 4.000 148 MVRGARGPEL 3.000 233
TGKVEAEFEL 3.000 151 GARGPELCSV 2.700 164 RNGAGPRCNL 2.000 120
AVLVLQVWDY 2.000 293 RTLVLLLLVL 2.000 303 LTVFLLLVFY 2.000 170
RCNLFRCRRL 2.000 37 DVVLDDENPL 1.500 31 VIWNTEDVVL 1.500 298
LLLVLLTVFL 1.000 317 QISQVIFRPL 1.000 294 TLVLLLLVLL 1.000 286
FIWRRYWRTL 1.000 299 LLVLLTVFLL 1.000 300 LVLLTVFLLL 1.000 277
VNPLKTFVFF 1.000 105 RSGPFALEEA 1.000 302 LLTVFLLLVF 1.000 74
FNSLTGEGNF 1.000 231 ILTGKVEAEF 1.000 80 EGNFNWRFVF 1.000 297
LLLLVLLTVF 1.000 165 NGAGPRCNLF 1.000 276 FVNPLKTFVF 1.000 113
EAEFRQPAVL 0.900 185 VVKLKEAEDV 0.900 214 KGRPEDLEFT 0.900 162
LARNGAGPRC 0.900 75 NSLTGEGNFN 0.750 266 PSRPKTSFNW 0.750 123
VLQVWDYDRI 0.600 154 GPELCSVQLA 0.600 84 NWRFVFRFDY 0.600 211
RRRKGRPEDL 0.600 61 KGLEHDKQET 0.600 168 GPRCNLFRCR 0.600 158
CSVQLARNGA 0.500 283 FVFFIWRRYW 0.500 76 SLTGEGNFNW 0.500 9
DVPAPPPVDI 0.400 261 EPLEKPSRPK 0.400 29 RVVIWNTEDV 0.400 21
RQPISYELRV 0.400 251 RPVGKGRKQP 0.400 309 LVFYTIPGQI 0.400 6
FPQDVPAPPP 0.400 258 KQPEPLEKPS 0.400 117 RQPAVLVLQV 0.400 313
TIPGQISQVI 0.400 221 EFTDMGGNVY 0.400 213 RKGRPEDLEF 0.300 125
QVWDYDRISA 0.300 129 YDRISANDFL 0.300 102 VWRRSGPFAL 0.300 115
EFRQPAVLVL 0.300 288 WRRYWRTLVL 0.300 134 ANDFLGSLEL 0.300 78
TGEGNFNWRF 0.300 38 VVLDDENPLT 0.300 234 GKVEAEFELL 0.300 65
HDKQETDVHF 0.300 291 YWRTLVLLLL 0.300 12 APPPVDIKPR 0.300 131
RISANDFLGS 0.300 44 NPLTGEMSSD 0.300 51 SSDIYVKSWV 0.300 290
RYWRTLVLLL 0.200 282 TFVFFIWRRY 0.200 183 WPVVKLKEAE 0.200 10
VPAPPPVDIK 0.200 68 QETDVHFNSL 0.200 227 GNVYILTGKV 0.200 93
YLPTEREVSV 0.200 30 VVIWNTEDVV 0.200 296 VLLLLVLLTV 0.200 150
RGARGPELCS 0.200 301 VLLTVFLLLV 0.200 289 RRYWRTLVLL 0.200 312
YTIPGQISQV 0.200 187 KLKEAEDVER 0.180 Table XVIII: 158P3D2 v.2a
B35-10mers 114 RPIKLLVRVY 80.000 3 DPGDSDGVNL 60.000 45 RSLEEEFNHF
30.000 164 NPIFGEILEL 30.000 110 IPQNRPIKLL 20.000 215 RANCGLASQY
12.000 177 LPAETELTVA 6.000 211 YSHHRANCGL 5.000 31 VSPKKAVATL
5.000 134 DPNGKADPYV 4.000 6 DSDGVNLISM 3.000 121 RVYVVKATNL 2.000
83 VGKFKGSFLI 1.200 97 EAVLFSEPQI 1.200 149 RERQDTKERY 1.200 201
ETHIDLENRF 1.000 91 LIYPESEAVL 1.000 81 HLVGKFKGSF 1.000 158
YIPKQLNPIF 1.000 77 EGSGHLVGKF 1.000 227 WVQQGPQEPF 1.000 109
GIPQNRPIKL 1.000 162 QLNPIFGEIL 1.000 197 DLIGETHIDL 1.000 183
LTVAVFEHDL 1.000 82 LVGKFKGSFL 1.000 174 SISLPAETEL 1.000 38
ATLKIYNRSL 1.000 161 KQLNPIFGEI 0.800 145 VSAGRERQDT 0.750 175
ISLPAETELT 0.750 32 SPKKAVATLK 0.600 93 YPESEAVLFS 0.600 202
THIDLENRFY 0.600 159 IPKQLNPIFG 0.600 35 KAVATLKIYN 0.600 73
GGEEEGSGHL 0.600 101 FSEPQISRGI 0.600 136 NGKADPYVVV 0.600 218
CGLASQYEVW 0.500 43 YNRSLEEEFN 0.450 131 APADPNGKAD 0.400 18
EIQDQGEAEV 0.400 28 KGTVSPKKAV 0.400 10 VNLISMVGEI 0.400 34
KKAVATLKIY 0.400 92 IYPESEAVLF 0.300 130 LAPADPNGKA 0.300 37
VATLKIYNRS 0.300 186 AVFEHDLVGS 0.300 124 VVKATNLAPA 0.300 51
FNHFEDWLNV 0.300 90 FLIYPESEAV 0.300 184 TVAVFEHDLV 0.300 207
ENRFYSHHRA 0.300 21 DQGEAEVKGT 0.300 119 LVRVYVVKAT 0.300 179
AETELTVAVF 0.200 61 FPLYRGQGGQ 0.200 176 SLPAETELTV 0.200 193
VGSDDLIGET 0.200 217 NCGLASQYEV 0.200 52 NHFEDWLNVF 0.200 133
ADPNGKADPY 0.200 219 GLASQYEVWV 0.200 140 DPYVVVSAGR 0.200 87
KGSFLIYPES 0.200 55 EDWLNVFPLY 0.200 84 GKFKGSFLIY 0.200 103
EPQISRGIPQ 0.200 138 KADPYVVVSA 0.180 106 ISRGIPQNRP 0.150 98
AVLFSEPQIS 0.150 29 GTVSPKKAVA 0.100 49 EEFNHFEDWL 0.100 118
LLVRVYVVKA 0.100 127 ATNLAPADPN 0.100 172 ELSISLPAET 0.100 42
IYNRSLEEEF 0.100 30 TVSPKKAVAT 0.100 168 GEILELSISL 0.100 166
IFGEILELSI 0.080 157 RYIPKQLNPI 0.080 150 ERQDTKERYI 0.080 13
ISMVGEIQDQ 0.075 115 PIKLLVRVYV 0.060 155 KERYIPKQLN 0.060 153
DTKERYIPKQ 0.060 126 KATNLAPADP 0.060 147 AGRERQDTKE 0.060 22
QGEAEVKGTV 0.060 173 LSISLPAETE 0.050 78 GSGHLVGKFK 0.050 88
GSFLIYPESE 0.050 221 ASQYEVWVQQ 0.050 220 LASQYEVWVQ 0.045 167
FGEILELSIS 0.045 16 VGEIQDQGEA 0.045 107 SRGIPQNRPI 0.040 190
HDLVGSDDLI 0.040 Table XVIII: 158P3D2 v.3 B35-10mers 7 VSVRRRSGPF
5.000 1 LPTEREVSVR 0.600 8 SVRRRSGPFA 0.300 9 VRRRSGPFAL 0.300 6
EVSVRRRSGP 0.010 10 RRRSGPFALE 0.006 3 TEREVSVRRR 0.006 4
EREVSVRRRS 0.003 5 REVSVRRRSG 0.002 2 PTEREVSVRR 0.000 Table XVIII:
158P3D2 v.4 B35-10mers 2 LPTEREVSIW 30.000 8 VSIWRRSGPF 5.000 1
YLPTEREVSI 0.400 10 IWRRSGPFAL 0.300 9 SIWRRSGPFA 0.100 7
EVSIWRRSGP 0.010 4 TEREVSIWRR 0.006 5 EREVSIWRRS 0.003 6 REVSIWRRSG
0.002 3 PTEREVSIWR 0.000 Table XVIII: 158P3D2 v.5a B35-10mers 16
TSLDPWSCSY 20.000 19 DPWSCSYQTW 10.000 36 SSALCSWPAM 10.000 31
GPGAPSSALC 2.000 42 WPAMGPGRGA 2.000 46 GPGRGAICFA 2.000 13
LPMTSLDPWS 2.000 33 GAPSSALCSW 1.500 30 VGPGAPSSAL 1.000 21
WSCSYQTWCV 1.000 9 YTASLPMTSL 1.000 45 MGPGRGAICF 1.000 4
LQVWDYTASL 1.000 12 SLPMTSLDPW 0.500 49 RGAICFAAAA 0.200 34
APSSALCSWP 0.200 3 VLQVWDYTAS 0.150 43 PAMGPGRGAI 0.120 15
MTSLDPWSCS 0.100 1 VLVLQVWDYT 0.100 44 AMGPGRGAIC 0.100 25
YQTWCVGPGA 0.100 2 LVLQVWDYTA 0.100 29 CVGPGAPSSA 0.100 28
WCVGPGAPSS 0.100 6 VWDYTASLPM 0.060 40 CSWPAMGPGR 0.050 35
PSSALCSWPA 0.050 11 ASLPMTSLDP 0.050 23 CSYQTWCVGP 0.050 47
PGRGAICFAA 0.030 37 SALCSWPAMG 0.030 10 TASLPMTSLD 0.030 5
QVWDYTASLP 0.020 14 PMTSLDPWSC 0.015 27 TWCVGPGAPS 0.010 26
QTWCVGPGAP 0.010 7 WDYTASLPMT 0.010 48 GRGAICFAAA 0.010 22
SCSYQTWCVG 0.010 39 LCSWPAMGPG 0.010 38 ALCSWPAMGP 0.010 18
LDPWSCSYQT 0.010 8 DYTASLPMTS 0.010 32 PGAPSSALCS 0.010 17
SLDPWSCSYQ 0.003 24 SYQTWCVGPG 0.001 20 PWSCSYQTWC 0.001 41
SWPAMGPGRG 0.001
[0777]
25TABLE XIXA MHC Class I Analysis of 158P3D2 (9-mers) Table XIXA,
part 1: MHC Class I nonamer analysis of 158P3D2 v.1 (aa 1-328)
Listed are scores which correlate with the ligation strength to a
defined HLA type for a sequence of amino acids. The algorithms used
are based on the book "MHC Ligands and Peptide Motifs" by H. G.
Rammensee, J. Bachmann and S. Stevanovic. The probability of being
processed and presented is given in order to predict T-cell
epitopes Pos 1 2 3 4 5 6 7 8 9 score HLA-A*0201 nonamers 297 L L L
L V L L T V 31 299 L L V L L T V F L 27 302 L L T V F L L L V 27
294 T L V L L L L V L 26 133 S A N D F L G S L 25 295 L V L L L L V
L L 25 224 D M G G N V Y I L 24 301 V L L T V F L L L 24 306 F L L
L V F Y T I .24 313 T I P G Q I S Q V 24 137 F L G S L E L Q L 23
178 R L R G W W P V V 23 296 V L L L L V L L T 23 230 Y I L T G K V
E A 22 293 R T L V L L L L V 22 300 L V L L T V F L L 22 31 V I W N
T E D V V 20 140 S L E L Q L P D M 20 144 Q L P D M V R G A 20 152
A R G P E L C S V 20 180 R G W W P V V K L 20 228 N V Y I L T G K V
20 2 W I D I F P Q D V 19 30 V V I W N T E D V 19 38 V V L D D E N
P L 19 55 Y V K S W V K G L 19 231 I L T G K V E A E 19 272 S F N W
F V N P L 19 276 F V N P L K T F V 19 279 P L K T F V F F I 19 298
L L L V L L T V F 19 23 P I S Y E L R V V 18 116 P R Q P A V L V L
18 118 Q P A V L V L Q V 18 291 Y W R T L V L L L 18 39 V L D D E N
P L T 17 94 L P T E R E V S V 17 290 R Y W R T L V L L 17 4 D I F P
Q D V P A 16 10 V P A P P P V D I 16 24 I S Y E L R V V I 16 46 L T
G E M S S D I 16 62 G L E H D K Q E T 16 135 N D F L G S L E L 16
237 E A E F E L L T V 16 27 E L R V V I W N T 15 32 I W N T E D V V
L 15 92 D Y L P T E R E V 15 114 A E F R Q P A V L 15 121 V L V L Q
V W D Y 15 141 L E L Q L P D M V 15 161 Q L A R N G A G P 15 165 N
G A G P R C N L 15 223 T D M G G N V Y I 15 234 G K V E A E F E L
15 242 L L T V E E A E K 15 287 I W R R Y W R T L 15 307 L L L V F
Y T I P 15 HLA-A1 nonamers 222 F T D M G G N V Y 36 34 N T E D V V
L D D 25 47 T G E M S S D I Y 25 18 I K P R Q P I S Y 21 121 V L V
L Q V W D Y 20 69 E T D V F I F N S L 19 51 S S D I Y V K S W 18 95
P T E R E V S V W 18 312 Y T I P G Q I S Q 18 HLA-A26 nonamers 69 E
T D V H F N S L 30 304 T V F L L L V F Y 28 55 Y V K S W V K G L 25
303 L T V F L L L V F 25 295 L V L L L L V L L 24 121 V L V L Q V W
D Y 23 232 L T G K V E A E F 23 283 P V F F I W R R Y 23 298 L L L
V L L T V F 23 4 D I F P Q D V P A 22 140 S L E L Q L P D M 22 235
K V E A E F E L L 22 300 L V L L T V F L L 22 222 F T D M G G N V Y
21 294 T L V L L L L V L 21 17 D I K P R Q P I S 20 66 D K Q E T D
V H F 20 101 S V W R R S G P F 20 224 D M G G N V Y I L 20 275 W F
V N P L K T F 20 301 V L L T V F L L L 20 313 T I P G Q I S Q V 20
27 E L R V V I W N T 19 38 V V L D D E N P L 19 108 P F A L E E A E
F 19 136 D F L G S L E L Q 19 137 F L G S L E L Q L 19 9 D V P A P
P P V D 18 42 D E N P L T G E M 18 86 R F V F R F D Y L 18 193 D V
E R E A Q E A 18 272 S F N W F V N P L 18 299 L L V L L T V F L 18
309 L V F Y T I P G Q 18 37 D V V L D D E N P 17 53 D I Y V K S W V
K 17 99 E V S V W R R S G 17 130 D R I S A N D F L 17 45 P L T G E
M S S D 16 71 D V H F N S L T G 16 156 E L C S V Q L A R 16 219 D L
E P T D M G G 16 231 I L T G K V E A E 16 268 R P K T S F N W F 16
278 N P L K T F V F F 16 281 K T F V F F I W R 16 317 Q I S Q V I F
R P 16 34 N T E D V V L D D 15 83 F N W R F V F R F 15 95 P T E R E
V S V W 15 142 E L Q L P D M V R 15 144 Q L P D M V R G A 15 239 E
F E L L T V E E 15 241 E L L T V E E A E 15 286 F I W R R Y W R T
15 293 R T L V L L L L V 15 312 Y T I P G Q I S Q 15 HLA-A3
nonamers 320 Q V I F R P L H K 31 53 D I Y V K S W V K 27 59 W V K
G L E H D K 23 178 R L R G W W P V V 23 242 L L T V E E A E K 23
161 Q L A R N G A G P 22 101 S V W R R S G P F 21 257 R K Q P E P L
E K 21 297 L L L L V L L T V 21 298 L L L V L L T V F 21 304 T V F
L L L V F Y 21 120 A V L V L Q VW D 20 142 E L Q L P D M V R 20 262
P L E K P S R P K 20 156 E L C S V Q L A R 19 179 L R G W W P V V K
19 187 K L K E A E D V E 19 198 A Q E A Q A G K K 19 172 N L F R C
R R L R 18 294 T L V L L L L V L 18 306 F L L L V F Y T I 18 9 D V
P A P P P V D 17 45 P L T G E M S S D 17 71 D V H F N S L T G 17
121 V L V L Q V W D Y 17 148 M V R G A R G P E 17 201 A Q A G K K K
R K 17 206 K K R K Q R R R K 17 247 E A E K R P V G K 17 289 R R Y
W R T L V L 17 295 L V L L L L V L L 17 15 P V D I K P R Q P 16 24
I S Y E L R V V I 16 29 R V V I W N T E D 16 76 S L T G E G N F N
16 137 F L G S L E L Q L 16 185 V V K L K E A E D 16 193 D V E R E
A Q E A 16 199 Q E A Q A G K K K 16 214 K G R P E D L E F 16 228 N
V Y I L T G K V 16 230 Y I L T G K V E A 16 231 I L T G K V E A E
16 250 K R P V G K G R K 16 252 P V G K G R K Q P 16 283 F V F F I
W R R Y 16 296 V L L L L V L L T 16 301 V L L T V F L L L 16 313 T
I P G Q I S Q V 16 4 D I F P Q D Y P A 15 93 Y L P T E R E V S 15
222 F T D M G G N V Y 15 235 K V E A E F E L L 15 299 L L V L L T V
F L 15 HLA-B*0702 nonamers 10 V P A P P P V D I 23 265 K P S R P K
T S F 23 154 G P E L C S V Q L 22 278 N P L K T F V F F 21 118 Q P
A V L V L Q V 20 314 I P G Q I S Q V I 19 22 Q P I S Y E L R V 18
94 L P T E R E V S V 18 268 R P K T S F N W F 18 165 N G A G P R C
N L 17 180 R G W W P V V K L 17 19 K P R Q P I S Y E 16 183 W P V V
K L K E A 16 116 F R Q P A V L V L 15 255 K G R K Q P E P L 15 289
R R Y W R T L V L 15 291 Y W R T L V L L L 15 32 I W N T E D V V L
14 114 A E F R Q P A V L 14 115 E F R Q P A V L V 14 149 V R G A R
G P E L 14 224 D M G G N V Y I L 14 251 R P V G K G R K Q 14 299 L
L V L L T V F L 14 12 A P P P V D I K P 13 69 E T D V H F N S L 13
103 W R R S G P F A L 13 137 F L G S L E L Q L 13 145 L P D M V R G
A R 13 178 R L R G W W P V V 13 212 R R K G R P E D L 13 235 K V E
A E F E L L 13 287 I W R R Y W R T L 13 290 R Y W R T L V L L 13
294 T L V L L L L V L 13 301 V L L T V F L L L 13 318 I S Q V I F R
P L 13 6 F P O D V P A P P 12 16 V D I K P R Q P I 12 86 R F V F R
F D Y L 12 107 G P F A L E E A E 12 135 N D F L G S L E L 12 168 G
P R C N L F R C 12 214 K G R P E D L E F 12 259 Q P E P L E K P S
12 272 S F N W F V N P L 12 292 W R T L V L L L L 12 295 L V L L L
L V L L 12 13 P P P V D I K P R 11 14 P P V D I K P R Q 11 20 P R Q
P I S Y E L 11 24 I S Y E L R V V I 11 38 V V L D D E N P L 11 55 Y
V K S W V K G L 11 88 V F R F D Y L P T 11 102 V W R R S G P F A 11
130 D R I S A N D F L 11 216 R P E D L E F T D 11 223 T D M G G N V
Y I 11 261 E P L E K P S R P 11 300 L V L L T V F L L 11 HLA-B*08
nonamers 212 R R K G R P E D L 28 185 V V K L K E A E D 23 279 P L
K T F V F F I 23 17 D I K P R Q P I S 22 55 Y V K S W V K G L 22
268 R P K T S F N W F 22 203 A G K K K R K Q R 21 149 V R G A R G P
E L 20 205 K K K R K Q R R R 20 261 E P L E K P S R P 20 154 G P E
L C S V Q L 19 166 G A G P R C N L F 19 183 W P V V K L K E A 19
204 G K K K R K Q R R 19 231 I L T G K V E A E 19 253 V G K G R K Q
P E 19 86 R F V F R F D Y L 18 171 C N L F R C R R L 18 187 K L K E
A E D V E 18 207 K R K Q R R R K G 18 277 V N P L K T F V F 18 289
R R Y W R T L V L 18 299 L L V L L T V F L 18 94 L P T E R E V S V
17 103 W R R S G P F A L 17 137 F L G S L E L Q L 17 287 I W R R Y
W R T L 17 291 Y W R T L V L L L 17 294 T L V L L L L V L 17 301 V
L L T V F L L L 17 27 E L R V V I W N T 16 101 S V W R R S G P F 16
133 S A N D F L G S L 16 210 Q R R R K G R P E 16 251 R P V G K G R
K Q 16 255 K G R K Q P E P L 16 266 P S R P K T S F N 16 53 D I Y V
K S W V K 15 113 E A E F R Q P A V 15 176 C R R L R G W W P 15 247
E A E K R P V G K 15 10 V P A P P P V D I 14 173 L F R C R R L R G
14 202 Q A G K K K R K Q 14 209 K Q R R R K G R P 14 234 G K V E A
E F E L 14 246 E E A E K R P V G 14 306 F L L L V F Y T I 14
HLA-B*1510 nonamers 32 I W N T E D V V L 16 116 F R Q P A V L V L
15 287 I W R R Y W R T L 15 318 I S Q V I F R P L 15 154 G P E L G
S V Q L 14 165 N G A G P R C N L 14 171 C N L F R C R R L 14 180 R
G W W P V V K L 14 20 P R Q P I S Y E L 13 103 W R R S G P F A L 13
114 A E F R Q P A V L 13 224 D M G G N V Y I L 13 234 G K V E A E F
E L 13 294 T L V L L L L V L 13 55 Y V K S W V K G L 12 64 E H D K
Q E T D V 12 69 E T D V H F N S L 12 135 N D F L G S L E L 12 149 V
R G A R G P E L 12 212 R R K G R P E D L 12 255 K G R K Q P E P L
12 289 R R Y W R T L V L 12 290 R Y W R T L V L L 12 291 Y W R T L
V L L L 12 295 L V L L L L V L L 12 299 L L V L L T V F L 12 38 V V
L D D E N P L 11 133 S A N D F L G S L 11 235 K V E A E F E L L 11
272 S F N W F V N P L 11 300 L V L L T V F L L 11 72 V H F N S L T
G E 10 81 G N F N W R F V F 10 86 R F V F R F D Y L 10 130 D R I S
A N D F L 10 137 F L G S L E L Q L 10 292 W R T L V L L L L 10 301
V L L T V F L L L 10 42 D E N P L T G E M 9 66 D K Q E T D V H F 9
79 G E G N F N W R F 9 83 F N W R F V F R F 9 166 G A G P R C N L F
9 214 K G R P E D L E F 9 265 K P S R P K T S F 9 278 N P L K T F V
F F 9 298 L L L V L L T V F 9 24 I S Y E L R V V I 8 108 P F A L E
E A E F 8 140 S L E L Q L P D M 8 232 L T G K V E A E F 8 275 W F V
N P L K T F 8 277 V N P L K T F V F 8 303 L T V F L L L V F 8 315 P
G Q I S Q V I F 8 HLA-B*2705 nonamers 289 R R Y W R T L V L 28 250
K R P V G K G R K 27 212 R R K G R P E D L 26 20 P R Q P I S Y E L
25 97 E R E V S V W R R 25 116 F R Q P A V L V L 24 292 W R T L V L
L L L 24 130 D R I S A N D P L 23 103 W R R S G P F A L 22 149 V R
G A R G P E L 22 179 L R G W W P V V K 22 85 W R F V F R F D Y 21
169 P R C N L F R C R 21 211 R R R K G R P E D 20 135 N D P L G S L
E L 19 177 R R L R G W W P V 19 180 R G W W P V V K L 19 90 R F D Y
L P T E R 18 104 R R S G P F A L E 18 204 G K K K R K Q R R 18 227
G N V Y I L T G K 18 257 R K Q P E P L E K 18 79 G E G N F N W R F
17 81 G N F N W R F V F 17 154 G P E L C S V Q L 17 170 R C N L F R
C R R 17 201 A Q A G K K K R K 17 205 K K K R K Q R R R 17 214 K G
R P E D L E F 17 256 G R K Q P E P L E 17 265 K P S R P K T S F 17
282 T F V F F I W R R 17 298 L L L V L L T V F 17 316 G Q I S Q V I
F R 17 53 D I Y V K S W V K 16 75 N S L T G E G N F 16 89 F R F D Y
L P T E 16 114 A E F R Q P A V L 16 163 A R N G A G P R C 16 181 G
W W P V V K L K 16 206 KK R K Q R R R K 16 207 K R K Q R R R K G 16
234 G K V E A E F E L 16 243 L T V E E A E K R 16 268 R P K T S F N
W F 16 281 K T F V F F I W R 16 290 R Y W R T L V L L 16 294 T L V
L L L L V L 16 295 L V L L L L V L L 16 21 R Q P I S Y E L R 15 32
I W N T E D V V L 15 49 E M S S D I Y V K 15 86 R F V F R F D Y L
15 109 F A L E E A E F R 15 142 E L Q L P D M V R 15 152 A R G P E
L C S V 15 165 N G A G P R C N L 15 166 G A G P R C N L F 15 200 E
A Q A G K K K R 15 203 A G K K K R K Q R 15 215 G R P E D L E F T
15 232 L T G K V E A E F 15 260 P E P L E K P S R 15 267 S R P K T
S F N W 15 278 N P L K T F V F F 15 303 L T V F L L L V F 15 304 T
V F L L L V F Y 15 28 L R V V I W N T E 14 96 T E R E V S V W R 14
108 P F A L E E A E F 14 156 E L C S V Q L A R 14 171 C N L F R C R
R L 14 188 L K E A E D V E R 14 197 E A Q E A Q A G K 14 198 A Q E
A Q A G K K 14 199 Q E A Q A G K K K 14 208 R K Q R R R K G R 14
224 D M G G N V Y I L 14 255 K G R K Q P E P L 14 262 P L E K P S R
P K 14 275 W F V N P L K T F 14 283 F V F F I W R R Y 14 299 L L V
L L T V F L 14 300 L V L L T V F L L 14 301 V L L T V F L L L 14
315 P G Q I S Q V I F 14 HLA-B*2709 nonamers 289 R R Y W R T L V L
27 177 R R L R G W W P V 24 212 R R K G R P E D L 24 20 P R Q P I S
Y E L 23 116 F R Q P A V L V L 23
130 D R I S A N D F L 22 292 W R T L V L L L L 22 103 W R R S G P F
A L 21 149 V R G A R G P E L 21 152 A R G P E L C S V 20 288 W R R
Y W R T L V 18 180 R G W W P V V K L 16 86 R F V F R F D Y L 15 154
G P E L C S V Q L 15 211 R R R K G R P E D 15 290 R Y W R T L V L L
15 293 R T L V L L L L V 15 104 R R S G P F A L E 14 215 G R P E D
L E F T 14 234 G K V E A E F E L 14 256 G R K Q P E P L E 14 38 V V
L D D E N P L 13 81 G N F N W R F V F 13 89 F R F D Y L P T E 13
114 A E F R Q P A V L 13 135 N D F L G S L E L 13 137 F L G S L E L
Q L 13 163 A R N G A G P R C 13 171 C N L F R C R R L 13 178 R L R
G W W P V V 13 250 K R P V G K G R K 13 295 L V L L L L V L L 13
300 L V L L T V F L L 13 301 V L L T V F L L L 13 HLA-B*4402
nonamers 114 A E F R Q P A V L 28 79 G E G N F N W R F 21 191 A E D
V E R E A Q 17 238 A E F E L L T V E 17 116 F R Q P A V L V L 16
166 G A G P R C N L F 16 248 A E K R P V G K G 16 25 S Y E L R V V
I W 15 51 S S D I Y V K S W 15 69 E T D V H F N S L 15 81 G N F N W
R F V F 15 135 N D F L G S L E L 15 214 K G R P E D L E F 15 263 L
E K P S R P K T 15 275 W F V N P L K T F 15 295 L V L L L L V L L
15 301 V L L T V F L L L 15 304 T V F L L L V F Y 15 18 I K P R Q P
I S Y 14 20 P R Q P I S Y E L 14 26 Y E L R V V I W N 14 42 D E N P
L T G E M 14 174 F R C R R L R G W 14 246 E E A E K R P V G 14 277
V N P L K T F V F 14 278 N P L K T F V F F 14 283 F V F F I W R R Y
14 284 V F F I W R R Y W 14 289 R R Y W R T L V L 14 290 R Y W R T
L V L L 14 291 Y W R T L V L L L 14 292 W R T L V L L L L 14 294 T
L V L L L L V L 14 300 L V L L T V F L L 14 HLA-B*5101 nonamers 314
I P G Q I S Q V I 25 10 V P A P P P V D I 23 94 L P T E R E V S V
23 24 I S Y E L R V V I 22 237 E A E F E L L T V 22 22 Q P I S Y E
L R V 21 118 Q P A V L V L Q V 21 113 E A E F R Q P A V 18 133 S A
N D F L G S L 18 180 R G W W P V V K L 18 297 L L L L V L L T V 18
306 F L L L V F Y T I 18 154 G P E L C S V Q L 17 261 E P L E K P S
R P 17 278 N P L K T F V F F 17 310 V F Y T I P G Q I 17 12 A P P P
V D I K P 16 92 D Y L P T E R E V 16 109 F A L E E A E F R 16 119 P
A V L V L Q V W 16 6 F P Q D V P A P P 15 46 L T G E M S S D I 15
80 E G N F N W R F V 15 202 Q A G K K K R K Q 15 228 N V Y I L T G
K V 15 251 R P V G K G R K Q 15 31 V I W N T E D V V 14 44 N P L T
G E M S S 14 145 L P D M V R G A R 14 165 N G A G P R C N L 14 190
E A E D V E R E A 14 200 E A Q A G K K K R 14 223 T D M G G N V Y I
14 255 K G R K Q P E P L 14 268 R P K T S F N W F 14 289 R R Y W R
T L V L 14 301 V L L T V F L L L 14 11 P A P P P V D I K 13 13 P P
P V D I K P R 13 23 P I S Y E L R V V 13 32 I W N T E D V V L 13
116 F R Q P A V L V L 13 124 L Q V W D Y D R I 13 141 L E L Q L P D
M V 13 162 L A R N G A G P R 13 183 W P V V K L K E A 13 186 V K L
K E A E D V 13 216 R P E D L E F T D 13 224 D M G G N V Y I L 13
247 E A E K R P V G K 13 279 P L K T F V F F I 13 300 L V L L T V F
L L 13 14 P P V D I K P R Q 12 16 V D I K P R Q P I 12 53 D I Y V K
S W V K 12 107 G P F A L E E A E 12 151 G A R G P E L C S 12 153 R
G P E L C S V Q 12 168 G P R C N L F R C 12 178 R L R G W W P V V
12 197 E A Q E A Q A G K 12 287 I W R R Y W R T L 12 291 Y W R T L
V L L L 12 293 R T L V L L L L V 12 294 T L V L L L L V L 12 295 L
V L L L L V L L 12 302 L L T V F L L L V 12 313 T I P G Q I S Q V
12 part 2: MHC Class I nonamer analysis of 158P3D2v.2a (aa1-236).
Pos 1 2 3 4 5 6 7 8 9 score HLA-A*0201 nonamers 117 K L L V R V Y V
V 28 11 N L I S M V G E I 26 165 P I F G E I L E L 25 91 L I Y P E
S E A V 24 158 Y I P K Q L N P I 23 162 Q L N P I F G E I 23 39 T L
K I Y N R S L 22 169 E I L E L S I S L 22 198 L I G E T H I D L 22
32 S P K K A V A T L 19 90 F L I Y P E S E A 19 114 R P I K L L V R
V 19 119 L V R V Y V V K A 19 177 L P A E T E L T V 19 179 A E T E
L T V A V 19 191 D L V G S D D L I 19 220 L A S Q Y E V W V 19 19 I
Q D Q G E A E V 18 175 I S L P A E T E L 18 176 S L P A E T E L T
18 184 T V A V F E H D L 18 5 G D S D G V N L I 17 23 G E A E V K G
T V 17 137 G K A D P Y V V V 17 14 S M V G E I Q D Q 16 29 G T V S
P K K A V 16 34 KK A V A T L K I 16 92 I Y P E S E A V L 16 98 A V
L F S E P Q I 16 116 I K L L V R V Y V 16 197 D L I G E T H I D 16
228 E A E F E L L T V 16 105 Q I S R G I P Q N 15 129 N L A P A D P
N G 15 170 I L E L S I S L P 15 215 R A N C G L A S Q 15 218 C G L
A S Q Y E V 15 8 D G V N L I S M V 14 46 S L E E E F N H F 14 52 N
H F E D W L N V 14 74 G E E E G S G H L 14 110 I P Q N R P I K L 14
118 L L V R V Y V V K 14 138 K A D P Y V V V S 14 185 V A V F E H D
L V 14 190 H D L V G S D D L 14 HLA-A1 nonamers 203 H I D L E N R F
Y 26 85 K F K G S F L I Y 25 6 D S D G V N L I S 24 56 D W L N V F
P L Y 22 101 F S E P Q I S R G 21 180 E T E L T V A V F 18 115 P I
K L L V R V Y 17 138 K A D P Y V V V S 17 167 F G E I L E L S I 17
216 A N C G L A S Q Y 17 1 M D D P G D S D G 16 46 S L E E E F N I
H F 16 95 E S E A V L F S E 16 134 D P N G K A D P Y 16 35 K A V A
T L K I Y 15 132 P A D P N G K A D 15 150 E R Q D T K E R Y 15 69 G
Q D G G G E E E 14 75 E E E G S G H L V 14 93 Y P E S E A V L F 14
148 G R E R Q D T K E 14 194 G S D D L I G E T 14 205 D L E N R F Y
S H 14 16 V G E I Q D Q G E 13 154 T K E R Y I P K Q 13 170 I L E L
S I S L P 13 189 E H D L V G S D D 13 195 S D D L I G E T H 13
HLA-A26 nonamers 180 E T E L T V A V F 31 169 E I L E L S I S L 28
165 P I F G E I L E L 27 115 P I K L L V R V Y 26 46 S L E E E F N
H F 25 26 E V K G T V S P K 24 85 K F K G S F L I Y 24 153 D T K E
R Y I P K 23 82 L V G K F K G S F 22 53 H F E D W L N V F 21 56 D W
L N V F P L Y 21 172 E L S I S L P A E 21 201 E T H I D L E N R 21
203 H I D L E N R F Y 21 50 E F N H F E D W L 20 59 N V F P L Y R G
Q 20 198 L I G E T H I D L 20 18 E I Q D Q G E A E 19 134 D P N G K
A D P Y 19 182 E L T V A V F E H 19 184 T V A V F E H D L 19 205 D
L E N R F Y S H 19 39 T L K I Y N R S L 18 55 E D W L N V F P L 18
150 E R Q D T K E R Y 18 197 D L I G E T H I D 18 158 Y I P K Q L N
P I 17 191 D L V G S D D L I 17 225 E V W V Q Q G P Q 17 11 N L I S
M V G E I 16 38 A T L K I Y N R S 16 78 G S G H L V G K F 16 81 H L
V G K F K G S 16 105 Q I S R G I P Q N 16 119 L V R V Y V V K A 16
32 S P K K A V A T L 15 162 Q L N P I F G E I 15 183 L T V A V F E
H D 15 202 T H I D L E N R F 15 216 A N C G L A S Q Y 15 219 G L A
S Q Y E V W 15 HLA-A3 nonamers 118 L L V R V Y V V K 32 26 E V K G
T V S P K 26 121 R V Y V V K A T N 26 147 A G R E R Q D T K 23 20 Q
D Q G E A E V K 21 30 T V S P K K A V A 21 41 K I Y N R S L E E 21
117 K L L V R V Y V V 21 186 A V F E H D L V G 21 62 P L Y R G Q G
G Q 20 115 P I K L L V R V Y 20 216 A N C G L A S Q Y 20 109 G I P
Q N R P I K 19 205 D L E N R F Y S H 19 33 P K K A V A T L K 18 57
W L N V F P L Y R 18 98 A V L F S E P Q I 18 105 Q I S R G I P Q N
18 82 L V G K F K G S F 17 119 L V R V Y V V K A 17 124 V V K A T N
L A P 17 143 V V V S A G R E R 17 174 S I S L P A E T E 17 9 G V N
L I S M V G 16 46 S L E E E F N H F 16 77 E G S G H L V G K 16 79 S
G H L V G K F K 16 90 F L I Y P E S E A 16 91 L I Y P E S E A V 16
162 Q L N P I F G E I 16 170 I L E L S I S L P 16 HLA-B*0702
nonamers 32 S P K K A V A T L 23 131 A P A D P N G K A 22 110 I P Q
N R P I K L 21 114 R P I K L L V R V 20 177 L P A E T E L T V 19 93
Y P E S E A V L F 18 159 I P K Q L N P I F 18 4 P G D S D G V N L
14 165 P I F G E I L E L 14 55 E D W L N V F P L 13 92 I Y P E S E
A V L 13 111 P Q N R P I K L L 13 134 D P N G K A D P Y 13 137 G K
A D P Y V V V 13 155 K E R Y I P K Q L 13 175 I S L P A E T E L 13
3 D P G D S D G V N 12 83 V G K F K G S F L 12 103 E P Q I S R G I
P 12 140 D P Y V V V S A G 12 179 A E T E L T V A V 12 228 V Q Q G
P Q E P F 12 30 T V S P K K A V A 11 34 K K A V A T L K I 11 50 E F
N H F E D W L 11 61 F P L Y R G Q G G 11 112 Q N R P I K L L V 11
119 L V R V Y V V K A 11 122 V Y V V K A T N L 11 139 A D P Y V V V
S A 11 163 L N P I F G E I L 11 169 E I L E L S I S L 11 184 T V A
V F E H D L 11 198 L I G E T H I D L 11 HLA-B*08 nonamers 83 V G K
F K G S F L 31 32 S P K K A V A T L 29 39 T L K I Y N R S L 27 110
I P Q N R P I K L 25 159 I P K Q L N P I F 24 122 V Y V V K A T N L
22 153 D T K E R Y I P K 20 81 H L V G K F K G S 18 155 K E R Y I P
K Q L 18 169 E I L E L S I S L 18 24 E A E V K G T V S 17 37 V A T
L K I Y N R 17 46 S L E E E F N H F 16 61 F P L Y R G Q G G 16 115
P I K L L V R V Y 16 117 K L L V R V Y V V i6 134 D P N G K A D P Y
16 147 A G R E R Q D T K 16 151 R Q D T K E R Y I 16 165 P I F G E
I L E L 16 198 L I G E T H I D L 16 HLA-B*1510 nonamers 202 T H I D
L E N R F 20 212 S HH R A N C G L 20 92 I Y P E S E A V L 15 175 I
S L P A E T E L 15 74 G E E E G S G H L 14 80 G H L V G K F K G 14
32 S P K K A V A T L 13 39 T L K I Y N R S L 13 55 E D W L N V F P
L 13 110 I P Q N R P I K L 13 165 P I F G E I L E L 13 184 T V A V
F E H D L 13 4 P G D S D G V N L 12 111 P Q N R P I K L L 12 169 E
I L E L S I S L 12 190 H D L V G S D D L 12 50 E F N H F E D W L 11
52 N H F E D W L N V 11 122 V Y V V K A T N L 11 155 K E R Y I P K
Q L 11 180 E T E L T V A V F 11 189 E H D L V G S D D 11 198 L I G
E T H I D L 11 213 H H R A N C G L A 11 53 H F E D W L N V F 10 83
V G K F K G S F L 10 93 Y P E S E A V L F 10 159 I P K Q L N P I F
10 163 L N P I F G E I L 10 HLA-B*2705 nonamers 113 N R P I K L L V
R 24 150 E R Q D T K E R Y 21 165 P I F G E I L E L 21 37 V A T L K
I Y N R 18 45 R S L E E E F N H 18 148 G R E R Q D T K E 18 74 G E
E E G S G H L 17 78 G S G H L V G K F 17 84 G K F K G S F L I 17
122 V Y V V K A T N L 17 149 R E R Q D T K E R 17 169 E I L E L S I
S L 17 175 I S L P A E T E L 17 100 L F S E P Q I S R 16 106 I S R
G I P Q N R 16 107 S R G I P Q N R P 16 109 G I P Q N R P I K 16
159 I P K Q L N P I F 16 202 T H I D L E N R F 16 208 N R F Y S H H
R A 16 20 Q D Q G E A E V K 15 27 V K G T V S P K K 15 32 S P K K A
V A T L 15 92 I Y P E S E A V L 15 147 A G R E R Q D T K 15 190 H D
L V G S D D L 15 216 A N C G L A S Q Y 15 228 V Q Q G P Q E P F 15
26 E V K G T V S P K 14 33 P K K A V A T L K 14 64 Y R G Q G G Q D
G 14 73 G G E E E G S G H 14 77 E G S G H L V G K 14 82 L V G K F K
G S F 14 85 K F K G S F L I Y 14 108 R G I P Q N R P I 14 110 I P Q
N R P I K L 14 111 P Q N R P I K L L 14 118 L L V R V Y V V K 14
141 P Y V V V S A G R 14 155 K E R Y I P K Q L 14 156 E R Y I P K Q
L N 14 180 E T E L T V A V F 14 4 R G D S D G V N L 13 5 G D S D G
V N L I 13 46 S L E E E F N H F 13 53 H F E D W L N V F 13 93 Y P E
S E A V L F 13 114 R P I K L L V R V 13 120 V R V Y V V K A T 13
157 R Y I P K Q L N P 13 201 E T H I D L E N R 13 35 K A V A T L K
I Y 12 39 T L K I Y N R S L 12 43 Y N R S L E E E F 12 44 N R S L E
E E F N 12 55 E D W L N V F P L 12 56 D W L N V F P L Y 12 79 S G H
L V G K F K 12 83 V G K F K G S F L 12 98 A V L F S E P Q I 12 115
P I K L L V R V Y 12 134 D P N G K A D P Y 12 143 V V V S A G R E R
12 153 D T K E R Y I P K 12 196 D D L I G E T H I 12 198 L I G E T
H I D L 12 HLA-B*2709 nonamers 114 R P I K L L V R V 15 4 P G D S D
G V N L 14 108 R G I P Q N R P I 14 117 K L L V R V Y V V 14 155 K
E R Y I P K Q L 14 175 I S L P A E T E L 14 29 G T V S P K K A V 13
52 N H F E D W L N V 13 74 G E E E G S G H L 13 84 G K F K G S F L
I 13 98 A V L F S E P Q I 13 122 V Y V V K A T N L 13 148 G R E R Q
D T K E 13 165 P I F G E I L E L 13 208 N R F Y S H H R A 13 5 G D
S D G V N L I 12 78 G S G I I L V G
K F 12 116 I K L L V R V Y V 12 120 V R V Y V V K A T 12 137 G K A
D P Y V V V 12 151 R Q D T K E R Y I 12 156 E R Y I P K Q L N 12
169 E I L E L S I S L 12 190 H D L V G S D D L 12 11 N L I S M V G
E I 11 23 G E A E V K G T V 11 32 S P K K A V A T L 11 34 K K A V A
T L K I 11 55 E D W L N V F P L 11 91 L I Y P E S E A V 11 92 I Y P
E S E A V L 11 93 Y P E S E A V L F 11 107 S R G I P Q N R P 11 110
I P Q N R P I K L 11 112 Q N R P I K L L V 11 113 N R P I K L L V R
11 150 E R Q D T K E R Y 11 179 A E T E L T V A V 11 214 H R A N C
G L A S 11 218 C G L A S Q Y E V 11 39 T L K I Y N R S L 10 44 N R
S L E E E F N 10 50 E F N H F E D W L 10 64 Y R G Q G G Q D G 10 83
V G K F K G S F L 10 111 P Q N R P I K L L 10 136 N G K A D P Y V V
10 159 I P K Q L N P I F 10 162 Q L N P I F G E I 10 163 L N P I F
G E I L 10 184 T V A V F E H D L 10 196 D D L I G E T H I 10 198 L
I G E T H I D L 10 202 T H I D L E N R F 10 212 S H H R A N C G L
10 2 D D P G D S D G V 9 8 D G V N L I S M V 9 19 I Q D Q G E A E V
9 43 Y N R S L E E E F 9 102 S E P Q I S R G I 9 135 P N G K A D P
Y V 9 167 F G E I L E L S I 9 177 L P A E T E L T V 9 180 E T E L T
V A V F 9 185 V A V F E H D L V 9 191 D L V G S D D L I 9 220 L A S
Q Y E V W V 9 7 S D G V N L I S M 8 46 S L E E E F N H F 8 53 H F E
D W L N V F 8 75 E E E G S G H L V 8 82 L V G K F K G S F 8 157 R Y
I P K Q L N P 8 158 Y I P K Q L N P I 8 228 V Q Q G P Q E P F 8 45
R S L E E E F N H 7 88 G S F L I Y P E S 7 209 R F Y S H H R A N 7
HLA-B*5101 nonamers 177 L P A E T E L T V 26 110 I P Q N R P I K L
22 114 R P I K L L V R V 22 140 D P Y V V V S A G 22 220 L A S Q Y
E V W V 22 32 S P K K A V A T L 21 136 N G K A D P Y V V 20 3 D P G
D S D G V N 19 8 D G V N L I S M V 19 185 V A V F E H D L V 19 108
R G I P Q N R P I 18 167 F G E I L E L S I 18 196 D D L I G E T H I
18 218 C G L A S Q Y E V 18 134 D P N G K A D P Y 17 138 K A D P Y
V V V S 17 130 L A P A D P N G K 16 158 Y I P K Q L N P I 16 178 P
A E T E L T V A 16 191 D L V G S D D L I 16 24 E A E V K G T V S 15
92 I Y P E S E A V L 15 116 I K L L V R V Y V 15 117 K L L V R V Y
V V 15 2 D D P G D S D G V 14 5 G D S D G V N L I 14 11 N L I S M V
G E I 14 23 G E A E V K G T V 14 35 K A V A T L K I Y 14 83 V G K P
K G S F L 14 91 L I Y P E S E A V 14 93 Y P E S E A V L F 14 131 A
P A D P N G K A 14 4 P G D S D G V N L 13 34 K K A V A T L K I 13
37 V A T L K I Y N R 13 52 N H F E D W L N V 13 61 F P L Y R G Q G
G 13 98 A V L F S E P Q I 13 137 G K A D P Y V V V 13 151 R Q D T K
E R Y I 13 159 I P K Q L N P I F 13 part 3: MHC Class I nonamer
analysis of 158P3D2 v.3 (aa 95-111, PTEREVSVRRRSGPFAL). Pos 1 2 3 4
5 6 7 8 9 score HLA-A1 nonamers 95 P T E R E V S V R 18 HLA-A26
nonamers 101 S V R R R S G P F 20 99 E V S V R R R S G 17 94 P T E
R E V S V R 15 HLA-A3 nonamers 101 S V R R R S G P F 24 99 E V S V
R R R S G 15 HLA-B*0702 nonamers 103 R R R S G P F A L 14 102 V R R
R S G P F A 11 HLA-B*08 nonamers 101 S V R R R S G P F 22 103 R R R
S G P F A L 17 HLA-B*1510 nonamers 103 R R R S G P F A L 13 96 E R
E V S V R R R 8 HLA-B*2705 nonamers 103 R R R S G P F A L 26 96 E R
E V S V R R R 24 95 T E R E V S V R R 16 94 P T E R E V S V R 14
HLA-B*2709 nonamers 103 R R R S G P F A L 25 part 4: MHC Class I
nonamer analysis of 158P3D2 v.4 (aa 94-110, LPTEREVSIWRRSGPFA). Pos
1 2 3 4 5 6 7 8 9 score HLA-A*0201 nonamers 94 L P T E R E V S I 15
HLA-A1 nonamers 95 P T E R E V S I W 17 HLA-A26 nonamers 101 S I W
R R S G P F 20 99 E V S I W R R S G 17 95 P T E R E V S I W 15
HLA-A3 nonamers 101 S I W R R S G P F 19 HLA-B*0702 nonamers 94 L P
T E R E V S I 18 102 I W R R S G P F A 12 HLA-B*08 nonamers 94 L P
T E R E V S I 23 101 S I W R R S G P F 20 HLA-B*2705 nonamers 4 E R
E V S I W R R 27 3 T E R E V S I W R 14 HLA-B*5101 nonamers 94 L P
T E R E V S I 25 part 5: MHC Class I nonamer analysis of 158P3D2
v.5a (aa 122-178, LVLQVWDYTASLPMTSLDPWSCSYQTWCV GPGAPSSALC
SWPAMGPGRG AICFAAAA) Pos 1 2 3 4 5 6 7 8 9 score HLA-A*0201
nonamers 125 Q V W D Y T A S L 23 164 A M G P G R G A I 23 123 V L
Q V W D Y T A 18 130 T A S L P M T S L 17 137 S L D P W S C S Y 16
170 G A I C F A A A A 15 157 S A L C S W P A M 14 158 A L C S W P A
M G 14 132 S L P M T S L D P 13 142 S C S Y Q T W C V 13 151 G P G
A P S S A L 13 121 L V L Q V W D Y T 12 129 Y T A S L P M T S 11
146 Q T W C V G P G A 11 149 G V G P G A P S S 11 HLA-A1 nonamers
16 S L D P W S C S Y 32 HLA-A26 noamers 16 S L D P W S C S Y 22 4 Q
V W D Y T A S L 21 8 Y T A S L P M T S 14 28 C V G P G A P S S 14 7
D Y T A S L P M T 13 9 T A S L P M T S L 13 25 Q T W C V G P G A 12
1 L V L Q V W D Y T 11 14 M T S L D P W S C 11 36 S A L C S W P A M
11 37 A L C S W P A M G 11 HLA-A3 nonamers 16 S L D P W S C S Y 22
28 G V G P G A P S S 20 4 Q V W D Y T A S L 18 37 A L C S W P A M G
18 2 V L Q V W D Y T A 14 11 S L P M T S L D P 14 1 L V L Q V W D Y
T 13 166 G P G R G A I C F 12 131 A S L P M T S L D 11 164 A M G P
G R G A I 11 HLA-B*0702 nonamers 151 G P G A P S S A L 26 166 G P G
R G A I C F 17 130 T A S L P M T S L 16 139 D P W S C S Y Q T 16
154 A P S S A L C S W 14 163 P A M G P G R G A 13 HLA-B*08 nonamers
151 G P G A P S S A L 18 130 T A S L P M T S L 15 166 G P G R G A I
C F 13 125 Q V W D Y T A S L 10 HLA-B*1510 nonamers 130 T A S L P M
T S L 14 151 G P G A P S S A L 13 125 Q V W D Y T A S L 11 157 S A
L C S W P A M 8 166 G P G R G A I C F 8 HLA-B*2705 nonamers 166 G P
G R G A I C F 17 151 G P G A P S S A L 16 130 T A S L P M T S L 15
168 G R G A I C F A A 14 125 Q V W D Y T A S L 12 127 W D Y T A S L
P M 12 157 S A L C S W P A M 12 137 S L D P W S C S Y 11 161 S W P
A M G P G R 11 164 A M G P G R G A I 10 HLA-B*2709 nonamers 168 G R
G A I C F A A 14 151 G P G A P S S A L 13 146 G P G R G A I C F 12
127 W D Y T A S L P M 11 157 S A L C S W P A M 11 125 Q V W D Y T A
S L 10 130 T A S L P M T S L 10 161 A M G P G R G A I 10 142 S C S
Y Q T W C V 8 HLA-B*4402 nonamers 164 A M G P G R G A I 17 137 S L
D P W S C S Y 15 154 A P S S A L C S W 15 133 L P M T S L D P W 13
166 G P G R G A I C F 13 125 Q V W D Y T A S L 12 130 T A S L P M T
S L 12 140 P W S C S Y Q T W 12 151 G P G A P S S A L 12 131 A S L
P M T S L D 9 HLA-B*5101 nonamers 130 T A S L P M T S L 18 151 G P
G A P S S A L 17 133 L P M T S L D P W 15 139 D P W S C S Y Q T 15
153 G A P S S A L C S 14 157 S A L C S W P A M 13 162 W P A M G P G
R G 12 163 P A M G P G R G A 12 166 G P G R G A I C F 12 154 A P S
S A L C S W 11 170 G A I C F A A A A 11 150 V G P G A P S S A 10
164 A M G P G R G A I 10 125 Q V W D Y T A S L 9 165 M G P G R G A
I C 9
[0778]
26TABLE XIXB MHC Class I Analysis of 158P3D2 (decamers) part 1: MHC
Class I decamer analysis of 158P3D2 v.1 (aa 1-328) Listed are
scores which correlate with the ligation strength to a defined HLA
type for a sequence of amino acids. The algorithms used are based
on the book "MHC Ligands and Peptide Motifs" by H. G. Rammensee, J.
Bachmann and S. Stevanovic. The probability of being processed and
presented is given in order to predict T-cell epitopes. Pos 1 2 3 4
5 6 7 8 9 0 score HLA-A*0201 decamers 296 V L L L L V L L T V 30
301 V L L T V F L L L V 28 93 Y L P T E R E V S V 26 294 T L V L L
L L V L L 26 298 L L L V L L T V F L 26 299 L L V L L T V F L L 26
312 Y T I P G Q I S Q V 24 151 G A R G P E L C S V 23 31 V I W N T
E D V V L 22 236 V E A E F E L L T V 22 286 F I W R R Y W R T L 22
140 S L E L Q L P D M V 21 293 R T L V L L L L V L 21 132 I S A N D
F L G S L 20 179 L R G W W P V V K L 20 123 V L Q V W D Y D R I 19
148 M V R G A R G P E L 19 300 L V L L T V F L L L 19 223 T D M G G
N V Y I L 18 297 L L L L V L L T V F 18 313 T I P G Q I S Q V I 18
317 Q I S Q V I F R P L 18 30 V V I W N T E D V V 17 230 Y I L T G
K V E A E 17 231 I L T G K V E A E F 17 289 R R Y W R T L V L L 17
291 Y W R T L V L L L L 17 295 L V L L L L V L L T 17 308 L L V F Y
T I P G Q 17 4 D I F P Q D V P A P 16 22 QP I S Y E L R V V 16 45 P
L T G E M S S D I 16 54 I Y V K S W V K G L 16 187 K L K E A E D V
E R 16 271 T S F N W F V N P L 16 290 R Y W R T L V L L L 16 302 L
L T V F L L L V F 16 39 V L D D E N P L T G 15 114 A E F R Q P A V
L V 15 117 R Q P A V L V L Q V 15 137 F L G S L E L Q L P 15 143 L
Q L P D M V R G A 15 222 F T D M G G N V Y I 15 244 T V E E A E K R
P V 15 HLA-A*0203 decamers 194 V E R E A Q E A Q A 18 3 I D I F P Q
D V P A 10 101 S V W R R S G P F A 10 105 R S G P F A L E E A 10
111 L E E A E F R Q P A 10 125 Q V W D Y D R I S A 10 143 L Q L P D
M V R G A 10 154 G P E L C S V Q L A 10 158 C S V Q L A R N G A 10
182 W W P V V K L K E A 10 189 K E A E D V E R E A 10 192 E D V E R
E A Q E A 10 229 V Y I L T G K V E A 10 239 E F E L L T V E E A 10
4 D I F P Q D Y P A P 9 102 V W R R S G P F A L 9 106 S G P F A L E
E A E 9 112 E E A E F R Q P A V 9 126 V W D Y D R I S A N 9 144 Q L
P D M V R G A R 9 155 P E L C S V Q L A R 9 159 S V Q L A R N G A G
9 183 W P V V K L K E A E 9 190 E A E D V E R E A Q 9 193 D V E R E
A Q E A Q 9 195 E R E A Q E A Q A G 9 230 Y I L T G K V E A E 9 240
F E L L T V E E A E 9 HLA-A1 decamers 17 D I K P R Q P I S Y 23 46
L T G E M S S D I Y 21 303 L T V F L L L V F Y 21 69 E T D V H F N
S L T 19 39 V L D D E N P L T G 18 222 F T D M G G N V Y I 18 235 K
V E A E F E L L T 18 25 S Y E L R V V I W N 17 120 A V L V L Q V W
D Y 17 134 A N D F L G S L E L 17 221 E F T D M G G N V Y 17 34 N T
E D V V L D D E 16 51 S S D I Y V K S W V 16 84 N W R F V F R F D Y
16 95 P T E R E V S V W R 16 110 A L E E A E F R Q P 15 282 T F V F
F I W R R Y 15 47 T G E M S S D I Y V 14 140 S L E L Q L P D M V 14
198 A Q E A Q A G K K K 14 259 Q P E P L E K P S R 14 262 P L E K P
S R P K T 14 154 G P E L C S V Q L A 13 216 R P E D L E F T D M 13
245 V E E A E K R P V G 13 247 E A E K R P V G K G 13 293 R T L V L
L L L V L 13 78 T G E G N F N W R F 12 181 G W W P V V K L K E 12
312 Y T I P G Q I S Q V 12 2 W I D I F P Q D V P 11 15 P V D I K P
R Q P I 11 62 G L E H D K Q E T D 11 64 E H D K Q E I D V H 11 111
L E E A E F R Q P A 11 113 E A E F R Q P A V L 11 126 V W D Y D R I
S A N 11 145 L P D M V R G A R G 11 166 G A G P R C N L F R 11 188
L K E A E D V E R E 11 190 E A E D V E R E A Q 11 191 A E D V E R E
A Q E 11 217 P E D L E F T D M G 11 219 D L E F T D M G G N 11 237
E A E F E L L T V E 11 239 E F E L L T V E E A 11 300 L Y L L T V F
L L L 11 HLA-A26 decamers 17 D I K P R Q P I S Y 30 4 D I F P Q D V
P A P 26 115 E F R Q P A V L V L 25 303 L T V F L L L V F Y 25 37 D
V V L D D E N P L 24 120 A V L V L Q V W D Y 24 136 D F L G S L E L
Q L 23 221 E F T D M G G N V Y 23 317 Q I S Q V I F R P L 23 46 L T
G E M S S D I Y 22 148 M V R G A R G P E L 22 231 I L T G K V E A E
F 22 276 F V N P L KT F V F 22 293 R T L V L L L L V L 22 297 L L L
L V L L T V F 22 300 L V L L T V F L L L 22 302 L L T V F L L L V F
22 71 D V H F N S L T G E 21 82 N F N W R F V F R F 21 156 E L C S
V Q L A R N 21 294 T L V L L L L V L L 21 142 E L Q L P D M V R G
20 299 L L V L L T V F L L 20 312 Y T I P G Q I S Q V 20 31 V I W N
T E D V V L 19 219 D L E F T D M G G N 19 264 E K P S R P K T S F
19 9 D V P A P P P V D I 18 53 D I Y V K S W V K G 18 69 E T D V H
F N S L T 18 99 E V S V W R R S G P 18 286 F I W R R Y W R T L 18
128 D Y D R I S A N D F 17 193 D V E R E A Q E A Q 17 239 E F E L L
T V E E A 17 281 K T F V F F I W R R 17 282 T F V F F I W R R Y 17
298 L L L V L L T V F L 17 77 L T G E G N F N W R 16 80 E G N F N W
R F V F 16 87 F V F R P D Y L P T 16 241 E L L T V E E A E K 16 267
S R P K T S F N W F 16 270 K T S F N W F V N P 16 274 N W F V N P L
K T F 16 277 V N P L K T F V F F 16 304 T V F L L L V F Y T 16 34 N
T E D V V L D D E 15 41 D D E N P L T G E M 15 110 A L E E A E F R
Q P 15 113 E A E F R Q P A V L 15 131 R I S A N D F L G S 15 139 G
S L E L Q L P D M 15 230 Y I L T G K V E A E 15 HLA-A3 decamers 178
R L R G W W P V V K 38 241 E L L T V E E A E K 25 161 Q L A R N G A
G P R 24 187 K L K E A E D V E R 23 2281 N V Y I L T G K V E 22 297
L L L L V L L T V F 22 17 D I K P R Q P I S Y 21 120 A V L V L Q V
W D Y 21 231 I L T G K V E A E F 21 276 F V N P L K T F V F 21 302
L L T V F L L L V F 21 144 Q L P D M V R G A R 20 296 V L L L L V L
L T V 20 39 V L D D E N P L T G 19 HLA-B*0702 decamers 19 K P R Q P
I S Y E L 23 314 I P G Q I S Q V I F 20 216 R P E D L E F T D M 19
278 N P L K T F V P F I 19 107 G P F A L E E A E F 18 268 R P K T S
F N W F V 18 22 Q P I S Y E L R V V 17 115 E F R Q P A V L V L 17
154 G P E L C S V Q L A 17 288 W R R Y W R T L V L 16 134 A N D F L
G S L E L 15 148 M V R G A R G P E L 15 265 K P S R P K T S F N 15
12 A P P P V D I K P R 14 136 D F L G S L E L Q L 14 164 R N G A G
P R C N L 14 179 L R G W W P V V K L 14 211 R R R K G R P E D L 14
223 T D M G G N V Y I L 14 251 R P V G K G R K Q P 14 290 R Y W R T
L V L L L 14 291 Y W R T L V L L L L 14 293 R T L V L L L L V L 14
298 L L L V L L T V F L 14 317 Q I S Q V I F R P L 14 10 V P A P P
P V D I K 13 31 V I W N T E D V V L 13 54 I Y V K S W V K G L 13
102 V W R R S G P F A L 13 113 E A E F R Q P A V L 13 129 Y D R I S
A N D F L 13 153 R G P E L C S V Q L 13 168 G P R C N L F R C R 13
289 R R Y W R T L V L L 13 300 L V L L T V F L L L 13 6 F P Q D V P
A P P P 12 23 P I S Y E L R V V I 12 94 L P T E R E V S V W 12 118
Q P A V L V L Q V W 12 132 I S A N D F L G S L 12 145 L P D M V R G
A R G 12 254 G K G R K Q P E P L 12 259 Q P E P L E K P S R 12 261
E P L E K P S R P K 12 271 T S F N W F V N P L 12 294 T L V L L L L
V L L 12 HLA-B*4402 decamers 68 Q E T D V H F N S L 23 114 A E F R
Q P A V L V 19 238 A E F E L L T V E E 17 263 L E K P S R P K T S
17 274 N W F V N P L K T F 17 248 A E K R P V G K G R 16 17 D I K P
R Q P I S Y 15 26 Y E L R V V I W N T 15 48 G E M S S D I Y V K 15
50 M S S D I Y V K S W 15 115 E F R Q P A V L V L 15 120 A V L V L
Q V W D Y 15 134 A N D F L G S L E L 15 191 A E D V E R E A Q E 15
221 E F T D M G G N V Y 15 271 T S F N W F V N P L 15 276 F V N P L
K T F V F 15 300 L V L L T V F L L L 15 80 E G N F N W R F V F 14
102 V W R R S G P F A L 14 112 E E A E F R Q P A V 14 113 E A E F R
Q P A V L 14 128 D Y D R I S A N D F 14 136 D F L G S L E L Q L 14
155 P E L C S V Q L A R 14 165 N G A G P R C N L F 14 240 F E L L T
V E E A E 14 246 E E A E K R P V G K 14 267 S R P K T S F N W F 14
277 V N P L K T F V F F 14 283 F V F F I W R R Y W 14 290 R Y W R T
L V L L L 14 291 Y W R T L V L L L L 14 293 R T L V L L L L V L 14
294 T L V L L L L V L L 14 297 L L L L V L L T V F 14 31 V I W N T
E D V V L 13 42 D E N P L T G E M S 13 54 I Y V K S W V K G L 13 85
W R F V F R F D Y L 13 153 R G P E L C S V Q L 13 179 L R G W W P V
V K L 13 199 Q E A Q A G K K K R 13 217 P E D L E F T D M G 13 220
L E F T D M G G N V 13 223 T D M G G N V Y I L 13 236 V E A E F E L
L T V 13 260 P E P L E K P S R P 13 264 E K P S R P K T S F 13 266
P S R P K T S F N W 13 286 F I W R R Y W R T L 13 288 W R R Y W R T
L V L 13 289 R R Y W R T L V L L 13 298 L L L V L L T V F L 13 299
L L V L L T V F L L 13 302 L L T V F L L L V F 13 317 Q I S Q V I F
R P L 13 12 A P P P V D I K P R 12 23 P I S Y E L R V V I 12 24 I S
Y E L R V V I W 12 37 D V V L D D E N P L 12 74 F N S L T G E G N F
12 76 S L T G E G N F N W 12 79 G E G N F N W R F V 12 82 N F N W R
F V F R F 12 84 N W R F V F R F D Y 12 94 L P T E R E V S V W 12 98
R E V S V W R R S G 12 107 G P F A L E E A E F 12 118 Q P A V L V L
Q V W 12 132 I S A N D F L G S L 12 141 L E L Q L P D M V R 12 170
R C N L F R C R R L 12 173 L F R C R R L R G W 12 174 F R C R R L R
G W W 12 189 K E A E D V E R E A 12 213 R K G R P E D L E F 12 234
G K V E A E F E L L 12 245 V E E A E K R P V G 12 254 G K G R K Q P
E P L 12 279 P L K T F V F F I W 12 303 L T V F L L L V F Y 12 305
V F L L L V F Y T I 12 309 L V F Y T I P G Q I 12 9 D V P A P P P V
D I 11 19 K P R Q P I S Y E L 11 35 T E D V V L D D E N 11 63 L E H
D K Q E T D V 11 65 H D K Q E T D V H F 11 78 T G E G N F N W R F
11 96 T E R E V S V W R R 11 111 L E E A E F R Q P A 11 148 M V R G
A R G P E L 11 164 R N G A G P R C N L 11 194 V E R E A Q E A Q A
11 211 R R R K G R P E D L 11 231 I L T G K V E A E F 11 278 N P L
K T F V F F I 11 282 T F V F F I W R R Y 11 313 T I P G Q I S Q V I
11 314 I P G Q I S Q V I F 11 part 2: MHC Class I decamer analysis
of 158P3D2 v.2a (aa 1-236). HLA-A*0201 decamers 91 L I Y P E S E A
V L 25 176 S L P A E T E L T V 25 219 G L A S Q Y E V W V 25 118 L
L V R V Y V V K A 24 90 F L I Y P E S E A V 23 162 Q L N P I F G E
I L 23 197 D L I G E T H I D L 23 174 S I S L P A E T E L 22 109 G
I P Q N E P I K L 21 18 E I Q D Q E A E V 20 38 A T L K I Y N R S L
20 31 V S P K K A V A T L 19 116 I K L L V R V Y V V 19 138 K A D P
Y V V V S A 19 164 N P I F G E I L E L 18 10 V N L I S M V G E I 17
157 R Y I P K Q L N P I 17 183 L T V A V F E H D L 17 7 S D G V N L
I S M V 16 41 KI Y N R S L E E E 16 57 W L N V F P L Y R G 16 82 L
V G K F K G S F L 16 113 N R P I K L L V R V 16 115 P I K L L V R V
Y V 16 129 N L A P A D P N G K 16 170 I L E L S I S L P A 16 184 T
V A V F E H D L V 16 186 A V F E H D L V G S 16 198 L I G E T H I D
L E 16 54 F E D W L N V F P L 15 110 I P Q N R P I K L L 15 117 K L
L V R V Y V V K 15 121 R V Y V V K A T N L 15 166 I F G E I L E L S
I 15 168 G E I L E L S I S L 15 172 E L S I S L P A E T 15 46 S L E
E E F N H F E 14 81 H L V G K F K G S F 14 99 V L F S E P Q I S R
14 119 L V R V Y V V K A T 14 124 V V K A T N L A P A 14 177 L P A
E T E L T V A 14 1 M D D P G D S D G V 13 30 T V S P K K A V A T 13
36 A V A T L K I Y N R 13 74 G E E E G S G H L V 13 134 D P N G K A
D P Y V 13 161 K Q L N P I F G E I 13 169 E I L E L S I S L P 13
178 P A E T E L T V A V 13 211 Y S H H R A N C G L 13 217 N C G L A
S Q Y E V 13 3 D P G D S D G V N L 12 11 N L I S M V G E I Q 12 14
S M V G E I Q D Q G 12 73 G G E E E G S G H L 12 130 L A P A D P N
G K A 12 165 P I F G E I L E L S 12 191 D L V G S D D L I G 12 193
V G S D D L I G E T 12 HLA-A*0203 decamers 29 G T V S P K K A V A
18 124 V V K A T N L A P A 18 16 V G E I Q D Q G E A 10 27 V K G T
V S P K K A 10 89 S F L I Y P E S E A 10 118 L L V R V Y V V K A 10
122 V Y V V K A T N L A 10 130 L A P A D P N G K A 10 138 K A D P Y
V V V S A 10 170 I L E L S I S L P A 10 177 L P A E T E L T V A 10
207 E N R F Y S H H R A 10 212 S H H R A N C G L A 10 17 G E I Q D
Q G E A E 9 28 K G T V S P K K A V 9 30 T V S P K K A V A T 9 90 F
L I Y P E S E A V 9
119 L V R V Y V V K A T 9 123 Y V V K A T N L A P 9 125 V K A T N L
A P A D 9 131 A P A D P N G K A D 9 139 A D P Y V V V S A G 9 171 L
E L S I S L P A E 9 178 P A E T E L T V A V 9 208 N R F Y S H H R A
N 9 213 H H R A N C G L A S 9 HLA-A1 decamers 84 G K F K G S F L I
Y 24 55 E D W L N V F P L Y 20 6 D S D G V N L I S M 19 93 Y P E S
E A V L F S 19 101 F S E P Q I S R G I 19 75 E E E G S G H L V G 18
95 E S E A V L F S E P 17 114 R P I K L L V R V Y 17 170 I L E L S
I S L P A 17 133 A D P N G K A D P Y 16 180 E T E L T V A V F E 16
199 I G E T H I D L E N 16 202 T H I D L E N R F Y 16 34 K K A V A
T L K I Y 15 138 K A D P Y V V V S A 15 149 R E R Q D T K E R Y 15
194 G S D D L I G E T H 15 215 R A N C G L A S Q Y 15 1 M D D P G D
S D G V 14 46 S L E E E F N H F E 14 132 P A D P N G K A D P 14 4 P
G D S D G V N L I 13 48 E E E F N H F E D W 13 74 G E E E G S G H L
V 13 54 F E D W L N V F P L 12 187 V F E H D L V G S D 12 195 S D D
L I G E T H I 12 HLA-A26 decamers 201 E T H I D L E N R F 27 197 D
L I G E T H I D L 26 153 D T K E R Y I P K Q 25 77 E G S G H L V G
K F 23 158 Y I P K Q L N P I F 23 169 E I L E L S I S L P 23 6 D S
D G V N L I S M 22 26 E V K G T V S P K K 21 55 E D W L N V F P L Y
21 81 HL V G K F K G S F 21 91 L I Y P E S E A V L 21 109 G I P Q N
R P I K L 21 227 W V Q Q G P Q E P F 21 38 A T L K I Y N R S L 20
82 L V G K F K G S F L 20 186 A V F E H D L V G S 20 205 D L E N R
F Y S H H 20 18 E I Q D Q G E A E V 19 121 R V Y V V K A T N L 19
174 S I S L P A E T E L 19 182 E L T V A V F E H D 19 52 N H F E D
W L N V F 18 162 Q L N P I F G E I L 18 165 P I F G E I L E L S 18
183 L T V A V F E H D L 18 225 E V W V Q Q G P Q E 18 3 D P G D S D
G V N L 17 45 R S L E E E F N H P 17 84 G K F K G S F L I Y 17 114
R P I K L L V R V Y 17 179 A E T E L T V A V F 17 180 E T E L T V A
V F E 17 9 G V N L I S M V G E 16 36 A V A T L K I Y N R 16 49 E E
F N H F E D W L 16 124 V V K A T N L A P A 16 172 E L S I S L P A E
T 16 191 D L V G S D D L I G 16 192 L V G S D D L I G E 16 198 L I
G E T H I D L E 16 31 V S P K K A V A T L 15 34 K K A V A T L K I Y
15 41 K I Y N R S L E E E 15 59 N V F P L Y R G Q G 15 119 L V R V
Y V V K A T 15 164 N P I F G E I L E L 15 189 E H D L V G S D D L
15 15 M V G E I Q D Q G E 14 30 T V S P K K A V A T 14 92 I Y P E S
E A V L F 14 100 L F S E P Q I S R G 14 118 L L V R V Y V V K A 14
21 D Q G E A E V K G T 13 54 F E D W L N V F P L 13 57 W L N V F P
L Y R G 13 76 E E G S G H L V G K 13 85 K F K G S F L I Y P 13 117
K L L V R V Y V V K 13 202 T H I D L E N R F Y 13 HLA-A3 decamers
117 K L L V R V Y V V K 33 129 N L A P A D P N G K 25 62 P L Y R G
Q G Q D 24 26 E V K G T V S P K K 23 91 L I Y P E S E A V L 22 121
R V Y V V K A T N L 22 30 T V S P K K A V A T 21 108 R G I P Q N R
P I K 21 176 S L P A E T E L T V 20 19 I Q D Q G E A E V K 19 81 H
L V G K F K G S F 19 112 Q N R P I K L L V R 19 215 R A N C G L A S
Q Y 19 36 A V A T L K I Y N R 18 59 N V F P L Y R G Q G 18 105 Q I
S R G I P Q N R 18 146 S A G R E R Q D T K 18 162 Q L N P I F G E I
L 18 186 A V F E H D L V G S 18 205 D L E N R F Y S H H 18 114 R P
I K L L V R V Y 17 118 L L V R V Y V K A 17 142 Y V V V S A G R E R
17 11 N L I S M V G E I Q 16 25 A E V K G T V S P K 16 32 S P K K A
V A T L K 16 39 T L K I Y N R S L E 16 41 K I Y N R S L E E E 16 82
L V G K F K G S F L 16 98 A V L F S E P Q I S 16 99 V L F S E P Q I
S R 16 124 V V K A T N L A P A 16 170 I L E L S I S L P A 16 219 G
L A S Q Y E V W V 16 225 E V W V Q Q Q P Q E 16 HLA-B*0702 decamers
3 D P G D S D G V N L 23 164 N P I F G E I L E L 22 110 I P Q N R P
I K L L 21 134 D P N G K A D P Y V 19 177 L P A E T E L T V A 19 93
Y P E S E A V L F S 14 1114 R P I K L L V R V Y 14 131 A P A D P N
G K A D 14 31 V S P K K A V A T L 13 38 A T L K I Y N R S L 13 54 F
E D W L N V F P L 13 82 L V G K F K G S F L 13 91 L I Y P E S E A V
L 13 174 S I S L P A E T E L 13 30 T V S P K K A V A T 12 32 S P K
K A V A T L K 12 77 E G S G H L V G K F 12 103 E P Q I S R G I P Q
12 121 R V Y V V K A T N L 12 138 K A D P Y V V V S A 12 159 I P K
Q L N P I F G 12 189 E H D L V G S D D L 12 197 D L I G E T H I D L
12 49 E E F N H F E D W L 11 140 D P Y V V V S A G R 11 162 Q L N P
I F G E I L 11 179 A E T E L T V A V F 11 183 L T V A V F E H D L
11 HLA-B*4402 decamers 49 E E F N H F E D W L 25 168 G E I L E L S
I S L 25 179 A E T E L T V A V F 25 48 E E E F N H F E D W 23 54 F
E D W L N V F P L 22 149 R E R Q D T K E R Y 20 164 N P I F G E I L
E L 18 110 I P Q N R P I K L L 17 52 N H F E D W L N V F 16 77 E G
S G H L V G K F 16 114 R P I K L L V R V Y 16 133 A D P N G K A D P
Y 16 157 R Y I P K Q L N P I 16 17 G E I Q D Q G E A E 15 38 A T L
K I Y N R S L 15 55 E D W L N V F P L Y 15 75 E E E G S G H L V G
15 154 T K E R Y I P K Q L 15 197 D L I G E T H I D L 15 202 T H I
D L E N R F Y 15 25 A E V K G T V S P K 14 34 K K A V A T L K I Y
14 76 E E G S G H L V G K 14 84 G K F K G S F L I Y 14 91 L I Y P E
S E A V L 14 92 I Y P E S E A V L F 14 109 G I P Q N R P I K L 14
171 L E L S I S L P A E 14 189 E H D L V G S D D L 14 31 V S P K K
A V A T L 13 45 R S L E E E F N H F 13 102 S E P Q I S R G I P 13
162 Q L N P I F G E I L 13 174 S I S L P A E T E L 13 181 T E L T V
A V F E H 13 201 E T H I D L E N R F 13 3 D P G D S D G V N L 12 4
P G D S D G V N L I 12 74 G E E E G S G H L V 12 94 P E S E A V L F
S E 12 97 E A V L F S E P Q I 12 101 F S E P Q I S R G I 12 150 E R
Q D T K E R Y I 12 155 K E R Y I P K Q L N 12 161 K Q L N P I F G E
I 12 206 L E N R F Y S H H R 12 215 R A N C G L A S Q Y 12 218 C G
L A S Q Y E V W 12 part 3: MHC Class I decamer analysis of 158P3D2
v.3 , (aa 94-103-112, LPTEREVSVRRRSGPFALE). HLA-A*0203 decamers 101
S V R R R S G P F A 10 102 V R R R S G P F A L 9 HLA-A1 decamers 95
P T E R E V S V R R 16 97 E R E V S V R R R S 11 HLA-A26 decamers
99 E V S V R R R S G P 18 HLA-A3 decamers 101 S V R R R S G P F A
20 99 E V S V R R R S G P 14 HLA-B*0702 decamers 102 V R R R S G P
F A L 13 94 L P T E R E V S V R 12 HLA-B*4402 decamers 102 V R R R
S G P F A L 14 96 T E R E V S V R R R 12 98 R E V S V R R R S G 12
100 V S V R R R S G P F 11 part 4: MHC Class I decamer analysis of
158P3D2 v.4 (aa 93-102-111, YLPTEREVSIWRRSGPFAL). Pos 1 2 3 4 5 6 7
8 9 0 score HLA-A*0201 decamers 101 S I W R R S G P F A 16
HLA-A*0203 decamers 101 S I W R R S G P F A 10 1021 I W R R S G P F
A L 9 HLA-A1 decamers 95 P T E R E V S I W R 20 97 E R E V S I W R
R S 10 HLA-A26 decamers 99 E V S I W R R S G P 18 HLA-B*0702
decamers 102 I W R R S G P F A L 14 HLA-B*4402 decamers 102 I W R R
S G P F A L 14 95 T E R E V S I W R R 13 100 V S I W R R S G P F 13
98 R E V S I W R R S G 12 94 L P T E R E V S I W 11 part 5: MHC
Class I decamer analysis of 158P3D2 via (aa 121-178,
VLVLQVWDYTSLPMTSLDPWSCSYQTWCVGPGA PSSALCSWPAMGPGRGAICFAAAA). Pos 1
2 3 4 5 6 7 8 9 0 score HLA-A*0201 decamers 9 Y T A S L P M T S L
20 4 L Q V W D Y T A S L 16 12 S L P M T S L D P W 16 1 V L V L Q W
D Y T 15 2 L V L Q V W D Y T A 15 17 S L D P W S C S Y Q 14 30 V G
P G A P S S A L 14 44 A M G P G R G A I C 14 38 A L C S W P A M G P
13 43 P A M G P G R G A I 13 3 V L Q V W D Y T A S 12 29 C V G P G
A P S S A 12 33 G A P S S A L C S W 11 7 W D Y T A S L P M T 10 21
W S C S Y Q T W C V 10 36 S S A L C S W P A M 10 37 S A L C S W P A
M G 10 HLA-A*0203 decamers 48 G R G A I C F A A A 27 49 R G A I C F
A A A A 27 47 P G R G A I C F A A 19 HLA-A1 decamers 16 T S L D P W
S C S Y 19 6 V W D Y T A S L P M 17 17 S L D P W S C S Y Q 17 11 A
S L P M T S L D P 15 32 P G A P S S A L C S 10 40 C S W P A M G P G
R 9 HLA-A26 decamers 9 Y T A S L P MT S L 25 29 C V G P G A P S S A
14 3 V L Q V W D Y T A S 13 12 S L P M T S L D P W 13 30 V G P G A
P S S A L 13 45 M G P G R G A I C F 13 5 Q V W D Y T A S L P 12 16
T S L D P W S C S Y 12 17 S L D P W S C S Y Q 12 19 D P W S C S Y Q
T W 12 HLA-A3 decamers 5 Q V W D Y T A S L P 19 29 C V G P G A P S
S A 19 17 S L D P W S C S Y Q 17 38 A L C S W P A M G P 17 2 L V L
Q V W D Y T A 16 49 R G A I C F A A A A 13 3 V L Q V W D Y T A S 12
44 A M G P G R G A I C 12 1 V L V L Q V W D Y T 11 11 A S L P M T S
L D P 11 12 S L P M T S L D P W 11 16 T S L D P W S C S Y 11 32 P G
A P S S A L C S 10 40 C S W P A M G P G R 10 45 M G P G R G A I C F
9 HLA-B*0702 decamers 46 G P G R G A I C F A 18 42 W P A M G P G R
G A 17 34 A P S S A L C S W P 14 30 V G P G A P S S A L 13 31 G P G
A P S S A L C 13 4 L Q V W D Y T A S L 12 9 Y T A S L P M T S L 12
13 L P M T S L D P W S 12 43 P A M G P G R G A I 11 47 P G R G A I
C F A A 11 48 G R G A I C F A A A 11 6 V W D Y T A S L P M 10 19 D
P W S C S Y Q T W 10 35 P S S A L C S W P A 10 49 R G A I C F A A A
A 10 36 S S A L C S W P A M 9 HLA-B*4402 decamers 30 V G P G A P S
S A L 14 45 M G P G R G A I C F 14 12 S L P M T S L D P W 13 43 P A
M G P G R G A I 13 16 T S L D P W S C S Y 12 33 G A P S S A L C S W
12 4 L Q V W D Y T A S L 11 19 D P W S C S Y Q T W 11 9 Y T A S L P
M T S L 10 11 A S L P M T S L D P 8
[0779]
27TABLE XIXC MHC Class II Analysis of 158P3D2 part 1: MHC Class 111
5-mer analysis of 1 58P3D2 v.1 (aa 1-328). Listed are scores which
correlate with the ligation strength to a defined HLA type for a
sequence of amino acids. The algorithms used are based on the book
"MHC Ligands and Peptide Motifs" by H.G.Ranimensee, J. Bachmann and
S. Stevanovic. The probability of being processed and presented is
given in order to predict T-cell epitopes. Pos 1 2 3 4 5 6 7 8 9 0
1 2 3 4 5 score HLA-DRB1*0101 15-mers 126 V W D Y D R I S A N D F L
G S 32 308 L L V F Y T I P G Q I S Q V I 32 274 N W F V N P L K T F
V F F I W 31 296 V L L L L V L L T V F L L L V 30 71 D V H F N S L
T G E G N F N W 29 138 L G S L E L Q L P D M V R G A 29 226 G G N V
Y I L T G K V E A E F 28 289 R R Y W R T L V L L L L V L L 28 311 F
Y T I P G Q I S Q V I F R P 28 100 V S V W R R S G P F A L E E A 27
183 W P V V K L K E A E D V E R E 27 237 E A E F E L L T V E E A E
K R 27 303 L T V F L L L V F Y T I P G Q 27 27 E L R V V I W N T E
D V V L D 26 146 P D M V R G A R G P E L C S V 26 173 L F R C R R L
R G W W P V V K 26 219 D L E F T D M G G N V Y I L T 26 292 W R T L
V L L L L Y L L T V F 26 297 L L L L V L L T V F L L L V F 26 40 L
D D E N P L T G E M S S D I 25 135 N D F L G S L E L Q L P D M V 25
180 R G W W P V V K L K E A E D V 25 294 T L V L L L L V L L T V F
L L 25 3 I D I F P Q D V P A P P P V D 24 52 S D I Y V K S W V K G
L E H D 24 88 V F R F D Y L P T E R E V S V 24 99 E V S V W R R S G
P F A L E E 24 132 I S A N D F L G S L E L Q L P 24 295 L V L L L L
V L L T V F L L L 24 304 T V F L L L V F Y T I P G Q I 24 43 E N P
L T G E M S S D I Y V K 23 2 W I D I F P Q D V P A P P P V 22 4 D I
F P Q D V P A P P P V D I 22 7 P Q D V P A P P P V D I K P R 22 12
A P P P V D I K P R Q P I S Y 22 112 E E A E F R Q P A V L V L Q V
22 151 G A R G P E L C S V Q L A R N 22 225 M G G N V Y I L T G K V
E A E 22 299 L L V L L T V F L L L V F Y T 22 307 L L L V F Y T I P
G Q I S Q V 22 285 F F I W R R Y W R T L V L L L 21 84 N W R F V F
R F D Y L P T E R 20 106 S G P F A L E E A E F R Q P A 20 113 E A E
F R Q P A V L V L Q V W 20 144 Q L P D M V R G A R G P E L C 20 227
G N V Y I L T G K V E A E F E 20 273 F N W F V N P L K T F V F F I
20 13 P P P V D I K P R Q P I S Y E 19 57 K S W V K G L E H D K Q E
T D 19 80 E G N F N W R F V F R F D Y L 19 82 N F N W R F V F R F D
Y L P T 19 90 R F D Y L P T E R E V S V W R 19 156 E L C S V Q L A
R N G A G P R 19 182 W W P V V K L K E A E D V E R 19 240 F E L L T
V E E A E K R P V G 19 272 S F N W F V N P L K T F V F F 19 35 T E
D V V L D D E N P L T G E 18 97 E R E V S V W R R S G P F A L 18
108 P F A L E E A E F R Q P A V L 18 129 Y D R I S A N D F L G S L
E L 18 134 A N D F L G S L E L Q L P D M 18 158 C S V Q L A R N G A
G P R C N 18 190 E A E D V E R E A Q E A Q A G 18 209 K Q R R R K G
R P E D L E F T 18 214 K G R P E D L E F T D M G G N 18 242 L L T V
E E A E K R P V G K G 18 288 W R R Y W R T L V L L L L V L 18 29 R
V V I W N T E D V V L D D E 17 34 N T E D V V L D D E N P L T G 17
47 T G E M S S D I Y V K S W V K 17 105 R S G P F A L E E A E F R Q
P 17 159 S V Q L A R N G A G P R C N L 17 179 L R G W W P V V K L K
E A E D 17 229 V Y I L T G K V E A E F E L L 17 230 Y I L T G K V E
A E F E L L T 17 284 V F F I W R R Y W R T L V L L 17 293 R T L V L
L L L V L L T V F L 17 298 L L L V L L T V F L L L V F Y 17 300 L V
L L T V F L L L V F Y T I 17 302 L L T V F L L L V F Y T I P G 17
17 D I K P R Q P I S Y E L R V V 16 21 R Q P I S Y E L R V V I W N
T 16 25 S Y E L R V V I W N T E D V V 16 28 L R V V I W N T E D V V
L D D 16 91 F D Y L P T E R E V S V W R R 16 118 Q P A V L V L Q V
W D Y D R I 16 119 P A V L V L Q V W D Y D R I S 16 121 V L V L Q V
W D Y D R I S A N 16 123 V L Q V W D Y D R I S A N D F 16 137 F L G
S L E L Q L P D M V R G 16 176 C R R L R G W W P V V K L K E 16 196
R E A Q E A Q A G K K K R K Q 16 218 E D L E F T D M G G N V Y I L
16 261 E P L E K P S R P K T S F N W 16 281 K T F V F F I W R R Y W
R T L 16 286 F I W R R Y W R T L V L L L L 16 290 R Y W R T L V L L
L L V L L T 16 291 Y W R T L V L L L L V L L T V 16 HLA-DRB1*0301
(DR17) 15-mers 35 T E D V V L D D E N P L T G E 37 36 E D V V L D D
E N P L T G E M 30 60 V K G L E H D K Q E T D V H F 27 134 A N D F
L G S L E L Q L P D M 26 229 V Y I L T G K V E A E F E L L 26 47 T
G E M S S D I Y V K S W V K 23 292 W R T L V L L L L V L L T V F 23
298 L L L V L L T V F L L L V F Y 23 295 L V L L L L V L L T V F L
L L 22 296 V L L L L V L L T V F L L L V 22 297 L L L L V L L T V F
L L L V F 21 300 L V L L T V F L L L V F Y T I 21 15 P V D I K P R
Q P I S Y E L R 20 29 R V V I W N T E D V V L D D E 20 118 Q P A V
L V L Q V W D Y D R I 20 239 E F E L L T V E E A E K R P V 20 274 N
W F V N P L K T F V F F I W 20 3 I D I F P Q D V P A P P P V D 19
53 D I Y V K S W V K G L E H D K 19 74 F N S L T G E G N F N W R F
V 19 86 R F V F R F D Y L P T E R E V 19 146 P D M V R G A R G P E
L C S V 19 182 W W P V V K L K E A E D V E R 19 219 D L E F T D M G
G N V Y I L T 19 13 P P P V D I K P R Q P I S Y E 18 21 R Q P I S Y
E L R V V I W N T 18 113 E A E F R Q P A V L V L Q V W 18 130 D R I
S A N D F L G S L E L Q 18 157 L C S V Q L A R N G A G P R C 18 213
R K G R P E D L E F T D M G G 18 233 T G K V E A E F E L L T V E E
18 242 L L T V E E A E K R P V G K G 18 260 P E P L E K P S R P K T
S F N 18 284 V F F I W R R Y W R T L V L L 18 HLA-DRB1*0401
(DR4Dw4) 15-mers 270 K T S F N W F V N P L K T F V 28 21 R Q P I S
Y E L R V V I W N T 26 36 E D V V L D D E N P L T G E M 26 43 E N P
L T G E M S S D I Y V K 26 57 K S W V K G L E H D K Q E T D 26 191
A E D V E R E A Q E A Q A G K 26 296 V L L L L V L L T V F L L L V
26 71 D V H F N S L T G E G N F N W 22 82 N F N W R F V F R F D Y L
P T 22 88 V F R F D Y L P T E R E V S V 22 90 R F D Y L P T E R E V
S V W R 22 124 L Q V W D Y D R I S A N D F L 22 180 R G W W P V V K
L K E A E D V 22 237 E A E F E L L T V E E A E K R 22 285 F F I W R
R Y W R T L V L L L 22 289 R R Y W R T L V L L L L V L L 22 303 L T
V F L L L V F Y T I P G Q 22 308 L L V F Y T I P G Q I S Q V I 22
309 L V F Y T I P G Q I S Q V I F 22 27 E L R V V I W N T E D V V L
D 20 35 T E D V V L D D E N P L T G E 20 47 T G E M S S D I Y V K S
W V K 20 60 V K G L E H D K Q E T D V H F 20 74 F N S L T G E G N F
N W R F V 20 85 W R F V F R F D Y L P T E R E 20 91 F D Y L P T E R
E V S V W R R 20 123 V L Q V W D Y D R I S A N D F 20 146 P D M V R
G A R G P E L C S V 20 154 G P E L C S V Q L A R N G A G 20 157 L C
S V Q L A R N G A G P R C 20 233 T G K V E A E F E L L T V E E 20
239 E F E L L T V E E A E K R P V 20 242 L L T V E E A E K R P V G
K G 20 260 P E P L E K P S R P K T S F N 20 274 N W F V N P L K T F
V F F I W 20 281 K T F V F F I W R R Y W R T L 20 292 W R T L V L L
L L V L L T V F 20 293 R T L V L L L L V L L T V F L 20 294 T L V L
L L L V L L T V F L L 20 297 L L L L V L L T V F L L L V F 20 299 L
L V L L T V F L L L V F Y T 20 302 L L T V F L L L V F Y T I P G 20
305 V F L L L V F Y T I P G Q I S 20 311 F Y T I P G Q I S Q V I F
R P 20 50 M S S D I Y V K S W V K G L E 18 65 H D K Q E T D V H F N
S L T G 18 109 F A L E E A E F R Q P A V L V 18 110 A L E E A E F R
Q P A V L V L 18 132 I S A N D F L G S L E L Q L P 18 151 G A R G P
E L C S V Q L A R N 18 156 E L C S V Q L A R N G A G P R 18 188 L K
E A E D V E R E A Q E A Q 18 194 V E R E A Q E A Q A G K K K R 18
225 M G G N V Y I L T G K V E A E 18 3 I D I F P Q D V P A P P P V
D 16 30 V V I W N T E D V V L D D E N 16 52 S D I Y V K S W V K G L
E H D 16 56 V K S W V K G L E H D K Q E T 16 86 R F V F R F D Y L P
T E R E V 16 100 V S V W R R S G P F A L E E A 16 106 S G P F A L E
E A E F R Q P A 16 113 E A E F R Q P A V L V L Q V W 16 126 V W D Y
D R I S A N D F L G S 16 134 A N D F L G S L E L Q L P D M 16 179 L
R G W W P V V K L K E A E D 16 227 G N V Y I L T G K V E A E F E 16
273 F N W F V N P L K T F V F F I 16 280 L K T F V F F I W R R Y W
R T 16 282 T F V F F I W R R Y W R T L V 16 288 W R R Y W R T L V L
L L L V L 16 13 P P P V D I K P R Q P I S Y E 15 7 P Q D V P A P P
P V D I K P R 14 25 S Y E L R V V I W N T E D V V 14 28 L R V V I W
N T E D V V L D D 14 29 R V V I W N T E D V V L D D E 14 37 D V V L
D D E N P L T G E M S 14 97 E R E V S V W R R S G P F A L 14 108 P
F A L E E A E F R Q P A V L 14 118 Q P A V L V L Q V W D Y D R I 14
120 A V L V L Q V W D Y D R I S A 14 121 V L V L Q V W D Y D R I S
A N 14 129 Y D R I S A N D F L G S L E L 14 135 N D F L G S L E L Q
L P D M V 14 138 L G S L E L Q L P D M V R G A 14 142 E L Q L P D M
V R G A R G P E 14 145 L P D M V R G A R G P E L C S 14 170 R C N L
F R C R R L R G W W P 14 176 C R R L R G W W P V V K L K E 14 182 W
W P V V K L K E A E D V E R 14 185 V V K L K E A E D V E R E A Q 14
217 P E D L E F T D M G G N V Y I 14 222 F T D M G G N V Y I L T G
K V 14 226 G G N V Y I L T G K V E A E F 14 240 F E L L T V E E A E
K R P V G 14 277 V N P L K T F V F F I W R R Y 14 295 L V L L L L V
L L T V F L L L 14 298 L L L V L L T V F L L L V F Y 14 300 L V L L
T V F L L L V F Y T I 14 304 T V F L L L V F Y T I P G Q I 14 306 F
L L L V F Y T I P G Q I S Q 14 307 L L L V F Y T I P G Q I S Q V 14
HLA-DRB1*1101 15-mers 179 L R G W W P V V K L K E A E D 27 90 R F D
Y L P T E R E V S V W R 25 82 N F N W R F V F R F D Y L P T 24 227
G N V Y I L T G K V E A E F E 24 170 R C N L F R C R R L R G W W P
23 180 R G W W P V V K L K E A E D V 23 308 L L V F Y T I P G Q I S
Q V I 23 142 E L Q L P D M V R G A R G P E 22 237 E A E F E L L T V
E E A E K R 22 281 K T F V F F I W R R Y W R T L 22 57 K S W V K G
L E H D K Q E T D 21 96 T E R E V S V W R R S G P F A 21 97 E R E V
S V W R R S G P F A L 21 123 V L Q V W D Y D R I S A N D F 20 156 E
L G S V Q L A R N G A G P R 20 135 N D F L G S L E L Q L P D M V 19
219 D L E F T D M G G N V Y I L T 19 282 T F V F F I W R R Y W R T
L V 19 285 F F I W R R Y W R T L V L L L 19 289 R R Y W R T L V L L
L L V L L 19 304 T V F L L L V F Y T I P G Q I 19 3 I D I F P Q D V
P A P P P V D 18 88 V F R F D Y L P T E R E V S V 18 273 F N W F V
N P L K T F V F F I 18 303 L T V F L L L V F Y T I P G Q 18 53 D I
Y V K S W V K G L E H D K 17 84 N W R F V F R F D Y L P T E R 17 65
H D K Q E T D V H F N S L T G 16 71 D V H F N S L T G E G N F N W
16 126 V W D Y D R I S A N D F L G S 16 167 A G P R C N L F R C R R
L R G 16 204 G K K K R K Q R R R K G R P E 16 206 K K R K Q R R R K
G R P E D L 16 247 E A E K R P V G K G R K Q P E 16 21 R Q P I S Y
E L R V V I W N T 15 242 L L T V E E A E K R P V G K G 15 243 L T V
E E A E K R P V G K G R 15 260 P E P L E K P S R P K T S F N 15 13
P P P V D I K P R Q P I S Y E 14 51 S S D I Y V K S W V K G L E H
14 109 F A L E E A E F R Q P A V L V 14 140 S L E L Q L P D M V R G
A R G 14 143 L Q L P D M V R G A R G P E L 14 145 L P D M V R G A R
G P E L C S 14 154 G P E L C S V Q L A R N G A G 14 188 L K E A E D
V E R E A Q E A Q 14 249 E K R P V G K G R K Q P E P L 14 250 K R P
V G K G R K Q P E P L E 14 257 R K Q P E P L E K P S R P K T 14 294
T L V L L L L V L L T V F L L 14 2 W I D I F P Q D V P A P P P V 13
12 A P P P V D I K P R Q P I S Y 13 25 S Y E L R V V I W N T E D V
V 13 34 N T E D V V L D D E N P L T G 13 47 T G E M S S D I Y V K S
W V K 13 108 P F A L E E A E F R Q P A V L 13 118 Q P A V L V L Q V
W D Y D R I 13 226 G G N V Y I L T G K V E A E F 13 270 K T S F N W
F V N P L K T F V 13 274 N W F V N P L K T F V F F I W 13 280 L K T
F V F F I W R R Y W R T 13 292 W R T L V L L L L V L L T V F 13 293
R T L V L L L L V L L T V F L 13 295 L V L L L L V L L T V F L L L
13 296 V L L L L V L L T V F L L L V 13 297 L L L L V L L T V F L L
L V F 13 299 L L V L L T V F L L L V F Y T 13 302 L L T V F L L L V
F Y T I P G 13 305 V F L L L V F Y T I P G Q I S 13 part 2: MHC
Class 1115-met analysis of 158P3D2 v.2a (aa 1-236) Pos 1 2 3 4 5 6
7 8 9 0 1 2 3 4 5 score HLA-DRB1*0101 15-mers 119 L V R V Y V V K A
T N L A P A 31 160 P K Q L N P I F G E I L E L S 31 61 F P L Y R G
Q G G Q D G G G E 27 90 F L I Y P E S E A V L F S E P 27 80 G H L V
G K F K G S F L I Y P 26 113 N R P I K L L V R V Y V V K A 26 120 V
R V Y V V K A T N L A P A D 25 139 A D P Y V V V S A G R E R Q D 25
96 S E A V L F S E P Q I S R G I 24 116 I K L L V R V Y V V K A T N
L 24 156 E R Y I P K Q L N P I F G E I 24 164 N P I F G E I L E L S
I S L P 24 167 F G E I L E L S I S L P A E T 24 16 V G E I Q D Q G
E A E V K G T 23 21 D Q G E A E V K G T V S P K K 23 88 G S F L I Y
P E S E A V L F S 23 99 V L F S E P Q I S R G I P Q N 23 107 S R G
I P Q N R P I K L L V R 23 124 V V K A T N L A P A D P N G K 23 168
G E I L E L S I S L P A E T E 23 9 G V N L I S M V G E I Q D Q G 22
25 A E V K G T V S P K K A V A T 22 31 V S P K K A V A T L K I Y N
R 22 54 F E D W L N V F P L Y R G Q G 22 187 V F E H D L V G S D D
L I G E 22 217 N C G L A S Q Y E V W V Q Q G 22 58 L N V F P L Y R
G Q G G Q D G 21 87 K G S F L I Y P E S E A V L F 21 165 P I F G E
I L E L S I S L P A 20 221 A S Q Y E V W V Q Q G P Q E P 20 40 L K
I Y N R S L E E E F N H F 19
83 V G K F K G S F L I Y P E S E 19 121 R V Y V V K A T N L A P A D
P 19 7 S D G V N L I S M V G E I Q D 18 51 F N H F E D W L N V F P
L Y R 18 140 D P Y V V V S A G R E R Q D T 18 155 K E R Y I P K Q L
N P I F G E 18 172 E L S I S L P A E T E L T V A 18 184 T V A V F E
H D L V G S D D L 18 208 N R F Y S H H R A N C G L A S 18 211 Y S H
H R A N C G L A S Q Y E 18 14 S M V G E I Q D Q G E A E V K 17 36 A
V A T L K I Y N R S L E E E 17 76 E E G S G H L V G K F K G S F 17
79 S G H L V G K F K G S F L I Y 17 81 H L V G K F K G S F L I Y P
E 17 85 K F K G S F L I Y P E S E A V 17 122 V Y V V K A T N L A P
A D P N 17 170 I L E L S I S L P A E T E L T 17 177 L P A E T E L T
V A V F E H D 17 182 E L T V A V F E H D L V G S D 17 193 V G S D D
L I G E T H I D L E 17 201 E T H I D L E N R F Y S H H R 17 1 M D D
P G D S D G V N L I S M 16 10 V N L I S M V G E I Q D Q G E 16 13 I
S M V G E I Q D Q G E A E V 16 24 E A E V K G T V S P K K A V A 16
28 K G T V S P K K A V A T L K I 16 34 K K A V A T L K I Y N R S L
E 16 52 N H F E D W L N V F P L Y R G 16 57 W L N V F P L Y R G Q G
G Q D 16 60 V F P L Y R G Q G G Q D G G G 16 64 Y R G Q G G Q D G G
G E E E G 16 72 G G G E E E G S G H L V G K F 16 89 S F L I Y P E S
E A V L F S E 16 104 P Q I S R G I P Q N R P I K L 16 127 A T N L A
P A D P N G K A D P 16 133 A D P N G K A D P Y V V V S A 16 163 L N
P I F G E I L E L S I S L 16 171 L E L S I S L P A E T E L T V 16
174 S I S L P A E T E L T V A V F 16 186 A V F E H D L V G S D D L
I G 16 192 L V G S D D L I G E T H I D L 16 195 S D D L I G E T H I
D L E N R 16 6 D S D G V N L I S M V G E I Q 15 22 Q G E A E V K G
T V S P K K A 15 71 D G G G E E E G S G H L V G K 15 114 R P I K L
L V R V Y V V K A T 15 152 Q D T K E R Y I P K Q L N P I 15 157 R Y
I P K Q L N P I F G E I L 15 166 I F G E I L E L S I S L P A E 15
181 T E L T V A V F E H D L V G S 15 HLA-DRB1*0301 (DR17) 15-mers
89 S F L I Y P E S E A V L F S E 27 199 I G E T H I D L E N R F Y S
H 26 79 S G H L V G K F K G S F L I Y 25 147 A G R E R Q D T K E R
Y I P K 25 156 E R Y I P K Q L N P I F G E I 25 172 E L S I S L P A
E T E L T V A 25 190 H D L V G S D D L I G E T H I 21 50 E F N H F
E D W L N V F P L Y 20 119 L V R V Y V V K A T N L A P A 20 80 G H
L V G K F K G S F L I Y P 19 107 S R G I P Q N R P I K L L V R 19
113 N R P I K L L V R V Y V V K A 19 121 R V Y V V K A T N L A P A
D P 19 141 P Y V V V S A G R E R Q D T K 19 160 P K Q L N P I F G E
I L E L S 19 185 V A V F E H D L V G S D D L I 19 195 S D D L I G E
T H I D L E N R 19 10 V N L I S M V G E I Q D Q G E 18 16 V G E I Q
D Q G E A E V K G T 18 37 V A T L K I Y N R S L E E E F 18 97 E A V
L F S E P Q I S R G I P 18 128 T N L A P A D P N G K A D P Y 18 217
N C G L A S Q Y E V W V Q Q G 18 12 L I S M V G E I Q D Q G E A E
17 44 N R S L E E E F N H F E D W L 17 57 W L N V F P L Y R G Q G G
Q D 17 87 K G S F L I Y P E S E A V L F 17 164 N P I F G E I L E L
S I S L P 17 174 S I S L P A E T E L T V A V F 17 201 E T H I D L E
N R F Y S H H R 17 207 E N R F Y S H H R A N C G L A 17 36 A V A T
L K I Y N R S L E E E 16 51 F N H F E D W L N V F P L Y R 16 142 Y
V V V S A G R E R Q D T K E 16 200 G E T H I D L E N R F Y S H H 16
40 L K I Y N R S L E E E F N H F 15 167 F G E I L E L S I S L P A E
T 15 181 T E L T V A V F E H D L V G S 15 2 D D P G D S D G V N L I
S M V 14 28 K G T V S P K K A V A T L K I 14 47 L E E E F N H F E D
W L N V F 14 96 S E A V L F S E P Q I S R G I 14 209 R F Y S H H R
A N C G L A S Q 14 7 S D G V N L I S M V G E I Q D 13 43 Y N R S L
E E E F N H F E D W 13 88 G S F L I Y P E S E A V L F S 13 115 P I
K L L V R V Y V V K A T N 13 116 I K L L V R V Y V V K A T N L 13
134 D P N G K A D P Y V V V S A G 13 HLA-DRB1*0401 (DR4Dw4) 15-mers
113 N R P I K L L V R V Y V V K A 26 141 P Y V V V S A G R E R Q D
T K 26 182 E L T V A V F E H D L V G S D 26 195 S D D L I G E T H I
D L E N R 26 201 E T H I D L E N R F Y S H H R 26 48 E E E F N H F
E D W L N V F P 22 164 N P I F G E I L E L S I S L P 22 12 L I S M
V G E I Q D Q G E A E 20 24 E A E V K G T V S P K K A V A 20 44 N R
S L E E E F N H F E D W L 20 57 W L N V F P L Y R G Q G G Q D 20 80
G H L V G K F K G S F L I Y P 20 88 G S F L I Y P E S E A V L F S
20 89 S F L I Y P E S E A V L F S E 20 97 E A V L F S E P Q I S R G
I P 20 116 I K L L V R V Y V V K A T N L 20 119 L V R V Y V V K A T
N L A P A 20 121 R V Y V V K A T N L A P A D P 20 127 A T N L A P A
D P N G K A D P 20 160 P K Q L N P I F G E I L E L S 20 163 L N P I
F G E I L E L S I S L 20 168 G E I L E L S I S L P A E T E 20 174 S
I S L P A E T E L T V A V F 20 31 V S P K K A V A T L K I Y N R 18
36 A V A T L K I Y N R S L E E E 18 71 D G G G E E E G S G H L V G
K 18 94 P E S E A V L F S E P Q I S R 18 128 T N L A P A D P N G K
A D P Y 18 144 V V S A G R E R Q D T K E R Y 18 166 I F G E I L E L
S I S L P A E 18 173 L S I S L P A E T E L T V A V 18 176 S L P A E
T E L T V A V F E H 18 187 V F E H D L V G S D D L I G E 18 215 R A
N C G L A S Q Y E V W V Q 18 222 S Q Y E V W V Q Q G P Q E P F 18
120 V R V Y V V K A T N L A P A D 17 51 F N H F E D W L N V F P L Y
R 16 54 P E D W L N V F P L Y R G Q G 16 87 K G S F L I Y P E S E A
V L F 16 139 A D P Y V V V S A G R E R Q D 16 185 V A V F E H D L V
G S D D L I 16 207 E N R F Y S H H R A N C G L A 16 221 A S Q Y E V
W V Q Q G P Q E P 16 7 S D G V N L I S M V G E I Q D 14 9 G V N L I
S M V G E I Q D Q G 14 10 V N L I S M V G E I Q D Q G E 14 13 I S M
V G E I Q D Q G E A E V 14 16 V G E I Q D Q G E A E V K G T 14 34 K
K A V A T L K I Y N R S L E 14 37 V A T L K I Y N R S L E E E F 14
55 E D W L N V F P L Y R G Q G G 14 96 S E A V L F S E P Q I S R G
I 14 107 S R G I P Q N R P I K L L V R 14 117 K L L V R V Y V V K A
T N L A 14 122 V Y V V K A T N L A P A D P N 14 156 E R Y I P K Q L
N P I F G E I 14 167 F G E I L E L S I S L P A E T 14 170 I L E L S
I S L P A E T E L T 14 172 E L S I S L P A E T E L T V A 14 180 E T
E L T V A V F E H D L V G 14 184 T V A V F E H D L V G S D D L 14
190 H D L V G S D D L I G E T H I 14 217 N C G L A S Q Y E V W V Q
Q G 14 HLA-DRB1*1101 15-mers 57 W L N V F P L Y R G Q G G Q D 27
113 N R P I K L L V R V Y V V K A 22 77 E G S G H L V G K F K G S F
L 21 100 L F S E P Q I S R G I P Q N R 21 201 E T H I D L E N R F Y
S H H R 20 116 I K L L V R V Y V V K A T N L 19 139 A D P Y V V V S
A G R E R Q D 19 167 F G E I L E L S I S L P A E T 19 221 A S Q Y E
V W V Q Q G P Q E P 19 98 A V L F S E P Q I S R G I P Q 18 120 V R
V Y V V K A T N L A P A D 18 207 E N R F Y S H H R A N C G L A 18
51 F N H F E D W L N V F P L Y R 16 54 F E D W L N V F P L Y R G Q
G 16 58 L N V F P L Y R G Q G G Q D G 16 61 F P L Y R G Q G G Q D G
G G E 16 83 V G K F K G S F L I Y P E S E 16 87 K G S F L I Y P E S
E A V L F 16 118 L L V R V Y V V K A T N L A P 16 141 P Y V V V S A
G R E R Q D T K 16 164 N P I F G E I L E L S I S L P 16 182 E L T V
A V F E H D L V G S D 16 205 D L E N R F Y S H H R A N C G 16 208 N
R F Y S H H R A N C G L A S 16 9 G V N L I S M V G E I Q D Q G 15
27 V K G T V S P K K A V A T L K 15 37 V A T L K I Y N R S L E E E
F 15 55 E D W L N V F P L Y R G Q G G 15 73 G G E E E G S G H L V G
K F K 15 149 R E R Q D T K E R Y I P K Q L 15 153 D T K E R Y I P K
Q L N P I F 15 79 S G H L V G K F K G S F L I Y 14 104 P Q I S R G
I P Q N R P I K L 14 124 V V K A T N L A P A D P N G K 14 130 L A P
A D P N G K A D P Y V V 14 137 G K A D P Y V V V S A G R E R 14 163
L N P I F G E I L E L S I S L 14 195 S D D L I G E T H I D L E N R
14 6 D S D G V N L I S M V G E I Q 13 21 D Q G E A E V K G T V S P
K K 13 25 A E V K G T V S P K K A V A T 13 96 S E A V L F S E P Q I
S R G I 13 119 L V R V Y V V K A T N L A P A 13 160 P K Q L N P I F
G E I L E L S 13 165 P I F G E I L E L S I S L P A 13 184 T V A V F
E H D L V G S D D L 13 189 E H D L V G S D D L I G E T H 13 part 3:
MHC Class 1115-met analysis of 158P3D2 v.3 (aa 89-103-117,
FRFDYLPTEREVSVRRRSGPFALEEAEFR) Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5
score HLA-DRB1*0101 15-mers 11 E V S V R R R S G P F A L E E 26 2 R
F D Y L P T E R E V S V R R 19 9 E R E V S V R R R S G P F A L 18
12 V S V R R R S G P F A L E E A 17 3 F D Y L P T E R E V S V R R R
16 10 R E V S V R R R S G P F A L E 15 HLA-DRB1*0301 (DR17) 15-mers
3 F D Y L P T E R E V S V R R R 17 9 E R E V S V R R R S G P F A L
16 11 E V S V R R R S G P F A L E E 12 12 V S V R R R S G P F A L E
E A 11 2 R F D Y L P T E R E V S V R R 10 10 R E V S V R R R S G P
F A L E 9 8 T E R E V S V R R R S G P F A 8 HLA-DRB1*0401 (DR4Dw4)
15-mers 2 R F D Y L P T E R E V S V R R 22 3 F D Y L P T E R E V S
V R R R 20 5 Y L P T E R E V S V R R R S G 12 7 P T E R E V S V R R
R S G P F 12 8 T E R E V S V R R R S G P F A 12 15 R R R S G P F A
L E E A E F R 12 HLA-DRB1*1101 15-mers 2 R F D Y L P T E R E V S V
R R 25 8 T E R E V S V R R R S G P F A 21 9 E R E V S V R R R S G P
F A L 21 7 P T E R E V S V R R R S G P F 20 11 E V S V R R R S G P
F A L E E 12 part 4: MHC Class II 15-mer analysis of 158P3D2 v.4
(aa 88-102-116, VFRFDYLPTEREVSIWRRSGPFALEEAEF) Pos 1 2 3 4 5 6 7 8
9 0 1 2 3 4 5 score HLA-DRB1*0101 15-mers 13 V S I W R R S G P F A
L E E A 27 1 V F R F D Y L P T E R E V S I 24 12 E V S I W R R S G
P F A L E E 24 10 E R E V S I W R R S G P F A L 21 3 R F D Y L P T
E R E V S I W R 19 4 F D Y L P T E R E V S I W R R 16 11 R E V S I
W R R S G P F A L E 14 HLA-DRB1*0301 (DR17) 15-mers 4 F D Y L P T E
R E V S I W R R 17 10 E R E V S I W R R S G P F A L 16 12 E V S I W
R R S G P F A L E E 11 13 V S I W R R S G P F A L E E A 11 3 R F D
Y L P T E R E V S I W R 10 1 V F R F D Y L P T E R E V S I 9 11 R E
V S I W R R S G P F A L E 9 9 T E R E V S I W R R S G P F A 8
HLA-DRB1*0401 (DR4Dw4) 15-mers 1 V F R F D Y L P T E R E V S I 22 3
R F D Y L P T E R E V S I W R 22 4 F D Y L P T E R E V S I W R R 20
13 V S I W R R S G P F A L E E A 16 10 E R E V S I W R R S G P F A
L 14 6 Y L P T E R E V S I W R R S G 12 9 T E R E V S I W R R S G P
F A 12 HLA-DRB1*1101 15-mers 3 R F D Y L P T E R E V S I W R 25 9 T
E R E V S I W R R S G P F A 21 10 E R E V S I W R R S G P F A L 20
1 V F R F D Y L P T E R E V S I 18 12 E V S I W R R S G P P A L E E
12 part 5: MHC Class II 15-mer analysis of 158P3D2 v.5a (aa
116-178). Pos 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 score HLA-DRB1*0101
15-mers 30 Y Q T W C V G P G A P S S A L 26 41 S S A L C S W P A M
G P G R G 26 11 V W D Y T A S L P M T S L D P 25 8 V L Q V W D Y T
A S L P M T S 24 9 L Q V W D Y T A S L P M T S L 24 17 S L P M T S
L D P W S C S Y Q 23 44 L C S W P A M G P G R G A I C 23 5 A V L V
L Q V W D Y T A S L P 22 32 T W C V G P G A P S S A L C S 22 38 G A
P S S A L C S W P A M G P 22 29 S Y Q T W C V G P G A P S S A 21 14
Y T A S L P M T S L D P W S C 20 45 C S W P A M G P G R G A I C F
20 28 C S Y Q T W C V G P G A P S S 19 31 Q T W C V G P G A P S S A
L C 18 6 V L V L Q V W D Y T A S L P M 17 12 W D Y T A S L P M T S
L D P W 17 48 P A M G P G R G A I C F A A A 17 3 Q P A V L V L Q V
W D Y T A S 16 4 P A V L V L Q V W D Y T A S L 16 35 V G P G A P S
S A L C S W P A 16 47 W P A M G P G R G A I C F A A 16 7 L V L Q V
W D Y T A S L P M T 15 24 D P W S C S Y Q T W C V G P G 14 33 W C V
G P G A P S S A L C S W 14 HLA-DRB1*0301 (DR17) 15-mers 3 Q P A V L
V L Q V W D Y T A S 20 7 L V L Q V W D Y T A S L P M T 20 4 P A V L
V L Q V W D Y T A S L 12 5 A V L V L Q V W D Y T A S L P 12 15 T A
S L P M T S L D P W S C S 12 17 S L P M T S L D P W S C S Y Q 12 18
L P M T S L D P W S C S Y Q T 12 20 M T S L D P W S C S Y Q T W C
12 41 S S A L C S W P A M G P G R G 12 47 W P A M G P G R G A I C F
A A 12 6 V L V L Q V W D Y T A S L P M 11 8 V L Q V W D Y T A S L P
M T S 11 32 T W C V G P G A P S S A L C S 11 2 R Q P A V L V L Q V
W D Y T A 10 12 W D Y T A S L P M T S L D P W 10 19 P M T S L D P W
S C S Y Q T W 10 33 W C V G P G A P S S A L C S W 10 HLA-DRB1*0401
(DR4Dw4) 15-mers 9 L Q V W D Y T A S L P M T S L 22 5 A V L V L Q V
W D Y T A S L P 20 7 L V L Q V W D Y T A S L P M T 18 33 W C V G P
G A P S S A L C S W 18 38 G A P S S A L C S W P A M G P 18 11 V W D
Y T A S L P M T S L D P 16 23 L D P W S C S Y Q T W C V G P 16 30 Y
Q T W C V G P G A P S S A L 16 3 Q P A V L V L Q V W D Y T A S 14 4
P A V L V L Q V W D Y T A S L 14 6 V L V L Q V W D Y T A S L P M 14
17 S L P M T S L D P W S C S Y Q 14 20 M T S L D P W S C S Y Q T W
C 14 32 T W C V G P G A P S S A L C S 14 2 R Q P A V L V L Q V W D
Y T A 12 10 Q V W D Y T A S L P M T S L D 12 12 W D Y T A S L P M T
S L D P W 12 18 L P M T S L D P W S C S Y Q T 12 21 T S L D P W S C
S Y Q T W C V 12 24 D P W S C S Y Q T W C V G P G 12 46 S W P A M G
P G R G A I C F A 12 HLA-DRB1*1101 15-mers 44 L C S W P A M G P G R
G A I C 24 5 A V L V L Q V W D Y T A S L P 18 11 V W D Y T A S L P
M T S L D P 18 30 Y Q T W C V G P G A P S S A L 17 27 S C S Y Q T W
C V G P G A P S 16 17 S L P M T S L D P W S C S Y Q 14 3 Q P A V L
V L Q V W D Y T A S 13 6 V L V L Q V W D Y T A S L P M 13 8 V L Q V
W D Y T A S L P M T S 13 14 Y T A S L P M T S L D P W S C 12
29 S Y Q T W C V G P G A P S S A 12 32 T W C V G P G A P S S A L C
S 12 38 G A P S S A L C S W P A M G P 12 41 S S A L C S W P A M G P
G R G 12
[0780]
28TABLE XX Frequently Occurring Motifs avrg. % Name identity
Description Potential Function zf-C2H2 34% Zinc finger, C2H2 type
Nucleic acid-binding protein functions as transcription factor,
nuclear location probable cytochrome b N 68% Cytochrome b(N-
membrane bound oxidase, generate terminal)/b6/petB superoxide ig
19% Immunoglobulin domain domains are one hundred amino acids long
and include a conserved intradomain disulfide bond. WD40 18% WD
domain, G-beta repeat tandem repeats of about 40 residues, each
containing a Trp-Asp motif. Function in signal transduction and
protein interaction PDZ 23% PDZ domain may function in targeting
signaling molecules to sub-membranous sites LRR 28% Leucine Rich
Repeat short sequence motifs involved in protein- protein
interactions pkinase 23% Protein kinase domain conserved catalytic
core common to both serine/threonine and tyrosine protein kinases
containing an ATP binding site and a catalytic site PH 16% PH
domain pleckstrin homology involved in intracellular signaling or
as constituents of the cytoskeleton EGF 34% EGF-like domain 30-40
amino-acid long found in the extracellular domain of membrane-bound
proteins or in secreted proteins rvt 49% Reverse transcriptase
(RNA-dependent DNA polymerase) ank 25% Ank repeat Cytoplasmic
protein, associates integral membrane proteins to the cytoskeleton
oxidored q1 32% NADH Membrane associated. Involved in proton
Ubiquinone/plastoquinone translocation across the membrane (complex
I), various chains ethand 24% EF hand calcium-binding domain,
consists of a 12 residue loop flanked on both sides by a 12 residue
alpha-helical domain rvp 79% Retroviral aspartyl protease Aspartyl
or acid proteases, centered on a catalytic aspartyl residue
Collagen 42% Collagen triple helix repeat extracellular structural
proteins involved in (20 copies) formation of connective tissue.
The sequence consists of the G-X-Y and the polypeptide chains forms
a triple helix. fn3 20% Fibronectin type III domain Located in the
extracellular ligand-binding region of receptors and is about 200
amino acid residues long with two pairs of cysteines involved in
disulfide bonds 7tm 1 19% 7 transmembrane receptor seven
hydrophobic transmembrane regions, (rhodopsin family) with the
N-terminus located extracellularly while the C-terminus is
cytoplasmic. Signal through G proteins
[0781]
29TABLE XXI Motifs and Post-translational Modifications of 158P3D2
Protein kinase C phosphorylation site 96-98 TeR 233-235 TgK Casein
kinase II phosphorylation site. 96-99 TerE 133-136 SanD 244-247
TveE Amidation site. 203-206 aGKK 255-258 kGRK Aminoacyl-transfer
RNA synthetases class-II signature .1 89-113
FRfDylpterevsvwrRsgpFaleE C2-domain. 13-142
[0782]
30TABLE XXII Properties of 158P3D2 Bioinformatic Variant 1 Program
URL Outcome ORF ORF finder Protein length 328 aa Transmembrane
region TM Pred http://www.ch.embnet.org/ 1 TM helix 295-312 HMMTop
http://www.enzim.hu/hmmtop/ N terminus extracellular, 1TM helix aa
295-314 Sosui http://www.genome.ad.jp/SOSui/ 1 TM helix 291-313
TMHMM http://www.cbs.dtu.dk/services/TMHMM N terminus
extracellular, 1 TM helix 292-314 Signal Peptide Signal P
http://www.cbs.dtu.dk/services/SignalP/ none pI pI/MW tool
http://www.expasy.ch/tools/ 8.64 Molecular weight pI/MW tool
http://www.expasy.ch/tools/ 38.4 kDa Localization PSORT
http://psort.nibb.ac.jp/ 85% endoplasmic reticulum, 64% peroxisome,
44% plasma membrane, 35% nucleus PSORT II http://psort.nibb.ac.jp/
33.3% vesicles of secretory system, 22.2% cytoplasmic Motifs Pfam
http://www.sanger.ac.uk/Pfam/ 7TM chemoreceptor Prints
http://www.biochem.ucl.ac.uk/ No significant motif Blocks
http://www.blocks.fhcrc.org/ C2 domain Bioinformatic Variant 2A
Program URL Outcome ORF ORF finder Protein length 236 aa
Transmembrane region TM Pred http://www.ch.embnet.org/ no TM HMMTop
http://www.enzim.hu/hmmtop/ no TM, extracellular Sosui
http://www.genome.ad.jp/SOSui/ no TM, soluble protein TMHMM
http://www.cbs.dtu.dk/services/TMHMM no TM Signal Peptide Signal P
http://www.cbs.dtu.dk/services/SignalP/ none pI pI/MW tool
http://www.expasy.ch/tools/ 4.7 Molecular weight pI/MW tool
http://www.expasy.ch/tools/ 26.1 kDa Localization PSORT
http://psort.nibb.ac.jp/ 65% cytoplasm, 10% mitochondrial matrix
space, 10% lysosome PSORT II http://psort.nibb.ac.jp/ 60.9%
cytoplasm, 21.7% nuclear Motifs Pfam http://www.sanger.ac.uk/Pfam/
C2 domain, glutamine synthetase Prints
http://www.biochem.ucl.ac.uk/ no significant motif Blocks
http://www.blocks.fhcrc.org/ C2 domain Bioinformatic Variant 2B
Program URL Outcome ORF ORF finder Protein length 181 aa
Transmembrane region TM Pred http://www.ch.embnet.org/ 1TM helix at
aa 148-165 HMMTop http://www.enzim.hu/hmmtop/ N terminus
intracellular 1TM helix at aa 148-167 Sosui
http://www.genome.ad.jp/SOSui/ 1TM helix at aa 144-166 TMHMM
http://www.cbs.dtu.dk/services/TMHMM N terminus intracellular 1TM
helix at aa 148-167 Signal Peptide Signal P
http://www.cbs.dtu.dk/services/SignalP/ none pI pI/MW tool
http://www.expasy.ch/tools/ 10.37 Molecular weight pI/MW tool
http://www.expasy.ch/tools/ 21.19 kDa Localization PSORT
http://psort.nibb.ac.jp/ 85% endoplasmic reticulum, 58% peroxisome,
44% plasma membrane PSORT II http://psort.nibb.ac.jp/ 33.3%
vesicles of secretory system, 22.2% plasma membrane Motifs Pfam
http://www.sanger.ac.uk/Pfam/ 7TM chemoreceptor Prints http://www
biochem.ucl.ac.uk/ No significant motif Blocks
http://www.blocks.fhcrc.org/ no significant motif Bioinformatic
Variant 5A Program URL Outcome ORF ORF finder Protein length 178 aa
Transmembrane region TM Pred http://www.ch.embnet.org/ N terminus
extracellular, 1 TM helix 145-165 HMMTop
http://www.enzim.hu/hmmtop/ N terminus extracellular, no TM Sosui
http://www.genome.ad.jp/SOSu- i/ no TM, soluble protein TMHMM
http://www.cbs.dtu.dk/services/TMH- MM N terminus extracellular, no
TM Signal Peptide Signal P http://www.cbs.dtu.dk/services/SignalP/
none pI pI/MW tool http://www.expasy.ch/tools/ 4.49 Molecular
weight pI/MW tool http://www.expasy.ch/tools/ 20.16 kDa
Localization PSORT http://psort.nibb.ac.jp/ 64% peroxisome, 45%
cyto- plasmic, 15.3% lysosome PSORT II http://psort.nibb.ac.jp/
52.2% cytoplasmic, 34.8% nuclear Motifs Pfam
http://www.sanger.ac.uk/Pfam/ none Prints http://www.biochem.ucl.-
ac.uk/ none Blocks http://www.blocks.fhcrc.org/ none
[0783]
31 Exon Number Start End TABLE XXIIIA Exon compositions of 158P3D2
var1 Exon 1 1 836 Exon 2 837 922 Exon 3 923 1021 Exon 4 1022 1263
Exon 5 1264 1547 Exon 6 1548 1648 Exon 7 1649 1961 TABLE XXIIIB
Exon compositions of 158P3D2 var2 Exon 1 1 95 Exon 2 96 138 Exon 3
139 239 Exon 4 240 377 Exon 5 378 494 Exon 6 495 623 Exon 7 624
1835 Exon 8 1836 1921 Exon 9 1922 2020 Exon 10 2021 2222 Exon 11
2223 2506 Exon 12 2507 2607 Exon 13 2608 2918
[0784]
32TABLE XXIIIB Exon compositions of 158P3D2 var2 Exon Number Start
End Exon 1 1 95 Exon 2 96 138 Exon 3 139 239 Exon 4 240 377 Exon 5
378 494 Exon 6 495 623 Exon 7 624 1835 Exon 8 1836 1921 Exon 9 1922
2020 Exon 10 2021 2222 Exon 11 2223 2506 Exon 12 2507 2607 Exon 13
2608 2918
[0785]
33TABLE XXIV Nucleotide sequence of transcript variant 158P3D2
atcaaggccc tgggctggag gaagacatcc cagatccaga ggagctcgac tgggggtcca
60 agtactatgc gtcgctgcag gagctccagg ggcagcacaa ctttgatgaa
gatgaaatgg 120 atgatcctgg agattcagat ggggtcaacc tcatttctat
ggttggggag atccaagacc 180 agggtgaggc tgaagtcaaa ggcactgtgt
ccccaaaaaa agcagttgcc accctgaaga 240 tctacaacag gtccctggag
gaagaattta accactttga agactggctg aatgtgtttc 300 ctctgtaccg
agggcaaggg ggccaggatg gaggtggaga agaggaagga tctggacacc 360
ttgtgggcaa gttcaagggc tccttcctca tttaccctga atcagaggca gtgttgttct
420 ctgagcccca gatctctcgg gggatcccac agaaccggcc catcaagctc
ctggtcagag 480 tgtatgttgt aaaggctacc aacctggctc ctgcagaccc
caatggcaaa gcagaccctt 540 acgtggtggt gagcgctggc cgggagcggc
aggacaccaa ggaacgctac atccccaagc 600 agctcaaccc catctttgga
gagatcctgg agctaagcat ctctctccca gctgagacgg 660 agctgacggt
cgccgtattt gaacatgacc tcgtgggttc tgacgacctc atcggggaga 720
cccacattga tctggaaaac cgattctata gccaccacag agcaaactgt gggctggcct
780 cccagtatga agtgtgggtc cagcagggcc cacaggagcc attctgagtt
tctggccaaa 840 cacattcaag ctcacattcc cttttgtgtc tccagatcct
atgatttcat ggaaggggac 900 cctcccaccc accgccactg ccaaccaaga
catagctcag tggtcaagac ttgggcttgg 960 gagtcgggat cctgtaacga
atgtcacttg accgctttct ttttttatga aacagtctcg 1020 ctctgtctcc
caggttggag tgcagtggca cgatctcggc tgactgcaac ctccacctcc 1080
tgggttcaag cgattctcct gcctcagcct ccccagtagc tgggattaca ggcgtgggcc
1140 cccatgtcca gctaattttt atattttcgc tctgtctccc aggttggagt
gcagtggcac 1200 gatctcggct gactgcaacc tccacctcct gggttcaagc
gattctcctg cctcagcctc 1260 cccagtagct gggattacag gcgtgggccc
ccatgtccag ctaattttta tatttttagt 1320 agagacaggg tttcaccatg
ttgtccaggc tggtcttgaa cccctgacct caagtgatcc 1380 acccacctct
gcctcccaaa gtgctgggat tacaggtgtg agccaccatg ccaggccctc 1440
ttaacctctt caagtctgtt ttctcatctg caaaacagag gtaataagat cagtatcttc
1500 ttaatggaag cacctgggct acattttttt cattcattgt tatcataaat
gaggactaac 1560 ctgtctcccg ttgggagttt tgaacctaga cctcatgtct
tcatgacgtc atcactgccc 1620 caggcccagc tgtgtcccta caccagcccc
agctgacgca tcttcttttt ctgcctgtag 1680 agatggttac aatgcctggc
gtgatgcatt ctggccttcg cagatcctgg cggggctgtg 1740 ccaacgctgt
ggcctccctg cccctgaata ccgagccggt gctgtcaagg tgggcagcaa 1800
agtcttcctg acaccaccgg agaccctgcc cccagggatc caagcctcgg cagccaatca
1860 tctttcctca agatgtgcct gctccacccc cagttgacat caagcctcgg
cagccaatca 1920 gctatgagct cagagttgtc atctggaaca cggaggatgt
ggttctggat gacgagaatc 1980 cactcaccgg agagatgtcg agtgacatct
atgtgaagag ctgggtgaag gggttggagc 2040 atgacaagca ggagacagac
gttcacttca actccctgac tggggagggg aacttcaatt 2100 ggcgctttgt
gttccgcttt gactacctgc ccacggagcg ggaggtgagc gtctggcgca 2160
ggtctggacc ctttgccctg gaggaggcgg agttccggca gcctgcagtg ctggtcctgc
2220 aggatccctg gagttgcagc taccagacat ggtgcgtggg gcccggggcc
ccgagctctg 2280 ctctgtgcag ctggcccgca atggggccgg gccgaggtgc
aatctgtttc gctgccgccg 2340 cctgaggggc tggtggccgg tagtgaagct
gaaggaggca gaggacgtgg agcgggaggc 2400 gcaggaggct caggctggca
agaagaagcg aaagcagagg aggaggaagg gccggccaga 2460 cccgctgaag
acctttgtct tcttcatctg gcgccggtac tggcgcaccc tggtgctgct 2700
gctactggtg ctgctcaccg tcttcctcct cctggtcttc tacaccatcc ctggccagat
2760 cagccaggtc atcttccgtc ccctccacaa gtgactctcg ctgaccttgg
acactcaccc 2820 agggtgccaa cccttcaatg cctgctcctg gaagtctttc
ttacccatgt gagctacccc 2880 agagtctagt gcttcctctg aataaaccta
tcacagcc 2918
[0786]
34TABLE XXV Nucleotide sequence alignment of 158P3D2 var1 and
158P3D2 var2 Score = 2348 bits (1221), Expect = 0.0Identities =
1223/1224 (99%) Strand = Plus/Plus Query: 1
tttttttatgaaacagtctcgctctgtctcccaggttg- gagtgcagtggcacgatctcgg 60
.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..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline..vertline..vertline. Sbjct: 1000
tttttttatgaaacagtctcgctctgtctcccaggttggagtgcagtggcacgatctcgg 1059
Query: 61 ctgactgcaacctccacctcctgggttcaagcgattctcctgcctcagcctcccc-
agtag 120 .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..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline. Sbjct: 1060
ctgactgcaacctccacctcctgggtt- caagcgattctcctgcctcagcctccccagtag 1119
Query: 121
ctgggattacaggcgtgggcccccatgtccagctaatttttatattttcgctctgtctcc 180
.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..-
vertline. Sbjct: 1120
ctgggattacaggcgtgggcccccatgtccagctaatttttatat- tttcgctctgtctcc 1179
Query: 181 caggttggagtgcagtggcacgatct-
cggctgactgcaacctccacctcctgggttcaag 240 .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..vertline..vertline..ver-
tline..vertline..vertline..vertline..vertline..vertline. Sbjct:
1180 caggttggagtgcagtggcacgatctcggctgactgcaacctccacctcctgggttcaag
1239 Query: 241 cgattctcctgcctcagcctccccagtagctgggattacaggcgtgggc-
ccccatgtcca 300 .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..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline. Sbjct: 1240
cgattctcctgcctcagcctccccagtagctgggattacaggcgtgggcccccatgtcca 1299
Query: 301 gctaatttttatatttttagtagagacagggtttcaccatgttgtccaggctgg-
tcttga 360 .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..vertline. Sbjct: 1300 gctaatttttatatttttagtagag-
acagggtttcaccatgttgtccaggctggtcttga 1359 Query: 361
acccctgacctcaagtgatccacccacctctgcctcccaaagtgctgggattacaggtgt 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..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline. Sbjct: 1360 acccctgacctcaagtgatccacccacctctgcctcccaaagtg-
ctgggattacaggtgt 1419 Query: 421 gagccaccatgccaggccctcttaa-
cctcttcaagtctgttttctcatctgcaaaacaga 480 .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..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline. Sbjct:
1420 gagccaccatgccaggccctcttaacctcttcaagtctgttttctcatctgcaaaacaga
1479 Query: 481 ggtaataagatcagtatcttcttaatggaagcacctggactacattttt-
ttcattcattg 540 .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..vertlin-
e..vertline..vertline..vertline..vertline..vertline..vertline..vertline..v-
ertline..vertline..vertline..vertline..vertline..vertline..vertline..vertl-
ine..vertline..vertline. Sbjct: 1480 ggtaataagatcagtatcttcttaatgga-
agcacctgggctacatttttttcattcattg 1539 Query: 541
ttatcataaatgaggactaacctgtctcccgttgggagttttgaacctagacctcatgtc 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..vertline..vertline..vertli-
ne..vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline. Sbjct: 1540 ttatcataaatgaggactaacctgtctcccgttgggagttttga-
acctagacctcatgtc 1599 Query: 601 ttcatgacgtcatcactgccccagg-
cccagctgtgtccctacaccagccccagctgacgc 660 .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..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline. Sbjct:
1600 ttcatgacgtcatcactgccccaggcccagctgtgtccctacaccagccccagctgacgc
1659 Query: 661 atcttctttttctgcctgtagagatggttacaatgcctggcgtgatgca-
ttctggccttc 720 .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..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline. Sbjct: 1660
atcttctttttctgcctgtagagatggttacaatgcctggcgtgatgcattctggccttc 1719
Query: 721 gcagatcctggcggggctgtgccaacgctgtggcctccctgcccctgaataccg-
agccgg 780 .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..vertline. Sbjct: 1720 gcagatcctggcggggctgtgccaa-
cgctgtggcctccctgcccctgaataccgagccgg 1779 Query: 781
tgctgtcaaggtgggcagcaaagtcttcctgacaccaccggagaccctgcccccagggat 840
.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..-
vertline. Sbjct: 1780 tgctgtcaaggtgggcagcaaagtcttcctgacaccaccggaga-
ccctgcccccagggat 1839 Query: 841 ctcttcacatgtggattgacatctt-
tcctcaagatgtgcctgctccacccccagttgaca 900 .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..vertline..vertline..vertline..vertline-
..vertline..vertline..vertline..vertline..vertline..vertline..vertline..ve-
rtline..vertline..vertline..vertline..vertline..vertline. Sbjct:
1840 ctcttcacatgtggattgacatctttcctcaagatgtgcctgctccacccccagttgaca
1899 Query: 901 tcaagcctcggcagccaatcagctatgagctcagagttgtcatctggaa-
cacggaggatg 960 .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..ve-
rtline..vertline..vertline..vertline..vertline..vertline..vertline..vertli-
ne..vertline..vertline..vertline. Sbjct: 1900
tcaagcctcggcagccaatcagctatgagctcagagttgtcatctggaacacggaggatg 1959
Query: 961 tggttctggatgacgagaatccactcaccggagagatgtcgagtgacatctatg-
tgaaga 1020 .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: 1960 tggttctggatgacgagaatccac-
tcaccggagagatgtcgagtgacatctatgtgaaga 2019 Query: 1021
gctgggtgaaggggttggagcatgacaagcaggagacagacgttcacttcaactccctga 1080
.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.-
.vertline. Sbjct: 2020 gctgggtgaaggggttggagcatgacaagcaggagacagacgt-
tcacttcaactccctga 2079 Query: 1081 ctggggaggggaacttcaattgg-
cgctttgtgttccgctttgactacctgcccacggagc 1140
.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..-
vertline. Sbjct: 2080 ctggggaggggaacttcaattggcgctttgtgttccgctttgac-
tacctgcccacggagc 2139 Query: 1141 gggaggtgagcgtctggcgcaggt-
ctggaccctttgccctggaggaggcggagttccggc 1200 .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..vertline..vertline..vertline. 1111
1I1III1I1I11111111111111 III111111111 111111111 1111 III Sbjct:
2140 gggaggtgagcgtctggcgcaggtctggaccctttgccctggaggaggcggagttccggc
2199 Query: 1201 agcctgcagtgctggtcctgcagg 1224
.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. Sbjct: 2200 agcctgcagtgctggtcctgcagg 2223 Score
= 1340 bits (697), Expect = 0.0ldentities = 697/697 (100%) Strand =
Plus/Plus Query: 1263
ggatccctggagttgcagctaccagacatggtgcgtggggcccggggccccgagctctgc 1322
.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..vertline..vertline..vertline..vertline-
..vertline. Sbjct: 2222 ggatccctggagttgcagctaccagacatggtgcgtggggcc-
cggggccccgagctctgc 2281 Query: 1323
tctgtgcagctggcccgcaatggggccgggccgaggtgcaatctgtttcgctgccgccgc 1382
.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.-
.vertline. Sbjct: 2282 tctgtgcagctggcccgcaatggggccgggccgaggtgcaatc-
tgtttcgctgccgccgc 2341 Query: 1383 ctgaggggctggtggccggtagt-
gaagctgaaggaggcagaggacgtggagcgggaggcg 1442
.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..-
vertline. Sbjct: 2342 ctgaggggctggtggccggtagtgaagctgaaggaggcagagga-
cgtggagcgggaggcg 2401 Query: 1443 caggaggctcaggctggcaagaag-
aagcgaaagcagaggaggaggaagggccggccagaa 1502 .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..vertline..vertline..vertline. Sbjct:
2402 caggaggctcaggctggcaagaagaagcgaaagcagaggaggaggaagggccggccagaa
2461 Query: 1503 gacctggagttcacagacatgggtggcaatgtgtacatcctcacgggc-
aaggtggaggca 1562 .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..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline. Sbjct: 2462
gacctggagttcacagacatgggtggcaatgtgtacatcctcacgggcaaggtggaggca 2521
Query: 1563 gagtttgagctgctgactgtggaggaggccgagaaacggccagtggggaaggg-
gcggaag 1622 .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.-
.vertline..vertline..vertline. Sbjct: 2522 gagtttgagctgctgactgtgga-
ggaggccgagaaacggccagtggggaaggggcggaag 2581 Query: 1623
cagccagagcctctggagaaacccagccgccccaaaacttccttcaactggtttgtgaac 1682
.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.-
.vertline. Sbjct: 2582 cagccagagcctctggagaaacccagccgccccaaaacttcct-
tcaactggtttgtgaac 2641 Query: 1683 ccgctgaagacctttgtcttctt-
catctggcgccggtactggcgcaccctggtgctgctg 1742
.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..-
vertline. Sbjct: 2642 ccgctgaagacctttgtcttcttcatctggcgccggtactggcg-
caccctggtgctgctg 2701 Query: 1743 ctactggtgctgctcaccgtcttc-
ctcctcctggtcttctacaccatccctggccagatc 1802 .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..vertline..vertline..vertline. Sbjct:
2702 ctactggtgctgctcaccgtcttcctcctcctggtcttctacaccatccctggccagatc
2761 Query: 1803 agccaggtcatcttccgtcccctccacaagtgactctcgctgaccttg-
gacactcaccca 1862 .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..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline. Sbjct: 2762
agccaggtcatcttccgtcccctccacaagtgactctcgctgaccttggacactcaccca 2821
Query: 1863 gggtgccaacccttcaatgcctgctcctggaagtctttcttacccatgtgagc-
tacccca 1922 .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.-
.vertline..vertline..vertline. Sbjct: 2822 gggtgccaacccttcaatgcctg-
ctcctggaagtctttcttacccatgtgagctacccca 2881 Query: 1923
gagtctagtgcttcctctgaataaacctatcacagcc 1959
.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-
. Sbjct: 2882 gagtctagtgcttcctctgaataaacctatcacagcc 2918
[0787]
35TABLE XXVI Peptide sequences of protein coded by 158P3D2 var2
>158P3D2 var2a MDDPGDSDGV NLISMVGEIQ DQGEAEVKGT VSPKKAVATL
KIYNRSLEEE FNHFEDWLNV 60 FPLYRGQGGQ DGGGEEEGSG HLVGKFKGSF
LIYPESEAVL FSEPQISRGI PQNRPIKLLV 120 RVYVVKATNL APADPNGKAD
PYVVVSAGRE RQDTKERYIP KQLNPIFGEI LELSISLPAE 180 TELTVAVFEH
DLVGSDDLIG ETHIDLENRF YSHHRANCGL ASQYEVWVQQ GPQEPF 236 >158P3D2
var2b MVRGARGPEL CSVQLARNGA GPRCNLFRCR RLRGWWPVVK LKEAEDVERE
AQEAQAGKKK 60 RKQRRRKGRP EDLEFTDMGG NVYILTGKVE AEFELLTVEE
AEKRPVGKGR KQPEPLEKPS 120 RPKTSFNWFV NPLKTFVFFI WRRYWRTLVL
LLLVLLTVFL LLVFYTIPGQ ISQVIFRPLH 180 K 181
[0788]
36TABLE XXVII Amino acid sequence alignment of 158P3D2 var1 and
158P3D2 var2 Score = 372 bits (956), Expect = e - 103Identities =
181/181 (100%), Positives = 181/181 (100%) Query: 148
MVRGARGPELCSVQLARNGAGPRCNLFRCRRLRGWWPVVK- LKEAEDVEREAQEAQAGKKK 207
MVRGARGPELCSVQLARNGAGPRCNLFRCR- RLRGWWPVVKLKEAEDVEREAQEAQAGKKK
Sbjct: 1
MVRGARGPELCSVQLARNGAGPRCNLFRCRRLRGWWPVVKLKEAEDVEREAQEAQAGKKK 60
Query: 208 RKQRRRKGRPEDLEFTDMGGNVUILTGKVEAEFELLTVEEAEKRPVGKGRKQPEP-
LEKPS 267 RKQRRRKGRPEDLEFTDMGGNVYILTGKVEAEFELLTVEEAEKRPV-
GKGRKQPEPLEKPS Sbjct: 61 RKQRRRKGRPEDLEFTDMGGNVYILTGKVEAE-
FELLTVEEAEKRPVGKGRKQPEPLEKPS 120 Query: 268
RPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLLVLLTVFLLLVFYTIPGQISQVIFRPLH 327
RPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLLVLLTVFLLLVFYTIPGQISQVIFRPLH Sbjct:
121 RPKTSFNWFVNPLKTFVFFIWRRYWRTLVLLLLVLLTVFLLLVFYTIPG- QISQVIFRPLH
180 Query: 328 K 328 K Sbjct: 181 K 181 Note: Protein variant
158P3D2 var2a does not share common sequence with protein 158P3D2
var1.
[0789]
Sequence CWU 0
0
* * * * *
References