# Patent

## Title
Nucleic acids and corresponding proteins entitled 282P1G3 useful in treatment and detection of cancer

## Abstract

A novel gene 282P1G3 and its encoded protein, and variants thereof, are described wherein 282P1G3 exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, 282P1G3 provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The 282P1G3 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 282P1G3 can be used in active or passive immunization.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a non-provisional utility patent application that is a continuation of United States non-provisional utility application U.S. Ser. No. 10/435,751, filed 9 May 2003, now U.S. Pat. No. 7,115,727, and this application claims priority from U.S. provisional patent application U.S. Ser. No. 60/404,306, filed 16 Aug. 2002, and this application claims priority from U.S. provisional patent application U.S. Ser. No. 60/423,290, filed 1 Nov. 2002. The contents of the applications listed in this paragraph are fully incorporated by reference herein.


STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH
Not applicable.
FIELD OF THE INVENTION
The invention described herein relates to genes and their encoded proteins, termed 282P1G3, expressed in certain cancers, and to diagnostic and therapeutic methods and compositions useful in the management of cancers that express 282P1G3.
SUBMISSION ON COMPACT DISC
The content of the following submission on compact discs is incorporated herein by reference in its entirety: A computer readable form (CRF) of the Sequence Listing on compact disc (file name: 511582008401, date recorded: Jul. 18, 2006, size: 1,013,760 bytes); a duplicate compact disc copy of the Sequence Listing (COPY 1) (file name: 511582008401, date recorded: Jul. 18, 2006, size: 1,013,760 bytes); and a duplicate compact disc copy of the Sequence Listing (COPY 2) (file name: 511582008401, date recorded: Jul. 18, 2006, size: 1,013,760 bytes).
BACKGROUND OF THE INVENTION
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.
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.
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.
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.
Progress in identifying additional specific markers for prostate cancer has been improved by the generation of prostate cancer xenografts that can recapitulate different stages of the disease in mice. The LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer xenografts that have survived passage in severe combined immune deficient (SCID) mice and have exhibited the capacity to mimic the transition from androgen dependence to androgen independence (Klein et al., 1997, Nat. Med. 3:402). More recently identified prostate cancer markers include PCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res 1996 Sep. 2 (9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad Sci USA. 1999 Dec. 7; 96(25): 14523-8) and prostate stem cell antigen (PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).
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.
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.
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.
Of all new cases of cancer in the United States, bladder cancer represents approximately 5 percent in men (fifth most common neoplasm) and 3 percent in women (eighth most common neoplasm). The incidence is increasing slowly, concurrent with an increasing older population. In 1998, there was an estimated 54,500 cases, including 39,500 in men and 15,000 in women. The age-adjusted incidence in the United States is 32 per 100,000 for men and eight per 100,000 in women. The historic male/female ratio of 3:1 may be decreasing related to smoking patterns in women. There were an estimated 11,000 deaths from bladder cancer in 1998 (7,800 in men and 3,900 in women). Bladder cancer incidence and mortality strongly increase with age and will be an increasing problem as the population becomes more elderly.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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
The present invention relates to a gene, designated 282P1G3, that has now been found to be over-expressed in the cancer(s) listed in Table 1. Northern blot expression analysis of 282P1G3 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 282P1G3 are provided. The tissue-related profile of 282P1G3 in normal adult tissues, combined with the over-expression observed in the tissues listed in Table 1, shows that 282P1G3 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 1.
The invention provides polynucleotides corresponding or complementary to all or part of the 282P1G3 genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding 282P1G3-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100 or more than 100 contiguous amino acids of a 282P1G3-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 282P1G3 genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the 282P1G3 genes, mRNAs, or to 282P1G3-encoding polynucleotides. Also provided are means for isolating cDNAs and the genes encoding 282P1G3. Recombinant DNA molecules containing 282P1G3 polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 282P1G3 gene products are also provided. The invention further provides antibodies that bind to 282P1G3 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.
The invention further provides methods for detecting the presence and status of 282P1G3 polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 282P1G3. A typical embodiment of this invention provides methods for monitoring 282P1G3 gene products in a tissue or hematology sample having or suspected of having some form of growth dysregulation such as cancer.
The invention further provides various immunogenic or therapeutic compositions and strategies for treating cancers that express 282P1G3 such as cancers of tissues listed in Table I, including therapies aimed at inhibiting the transcription, translation, processing or function of 282P1G3 as well as cancer vaccines. In one aspect, the invention provides compositions, and methods comprising them, for treating a cancer that expresses 282P1G3 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 282P1G3. Preferably, the carrier is a uniquely human carrier. In another aspect of the invention, the agent is a moiety that is immunoreactive with 282P1G3 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.
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 282P1G3 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 282P1G3 as described above. The one or more than one nucleic acid molecule may also be, or encodes, a molecule that inhibits production of 282P1G3. Non-limiting examples of such molecules include, but are not limited to, those complementary to a nucleotide sequence essential for production of 282P1G3 (e.g. antisense sequences or molecules that form a triple helix with a nucleotide double helix essential for 282P1G3 production) or a ribozyme effective to lyse 282P1G3 mRNA.
Note that to determine the starting position of any peptide set forth in Tables VIII-XXI and XXII to XLIX (collectively HLA Peptide Tables) respective to its parental protein, e.g., variant 1, variant 2, etc., reference is made to three factors: the particular variant, the length of the peptide in an HLA Peptide Table, and the Search Peptides in Table VII. Generally, a unique Search Peptide is used to obtain HLA peptides of a particular for a particular variant. The position of each Search Peptide relative to its respective parent molecule is listed in Table VII. Accordingly, if a Search Peptide begins at position “X”, one must add the value “X-1” to each position in Tables VIII-XXI and XXII to XLIX to obtain the actual position of the HLA peptides in their parental molecule. For example, if a particular Search Peptide begins at position 150 of its parental molecule, one must add 150-1, i.e., 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.
One embodiment of the invention comprises an HLA peptide, that occurs at least twice in Tables VIII-XXI and XXII to XLIX collectively, or an oligonucleotide that encodes the HLA peptide. Another embodiment of the invention comprises an HLA peptide that occurs at least once in Tables VIII-XXI and at least once in tables XXII to XLIX, or an oligonucleotide that encodes the HLA peptide.
Another embodiment of the invention is antibody epitopes, which comprise a peptide regions, or an oligonucleotide encoding the peptide region, that has one two, three, four, or five of the following characteristics:
i) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Hydrophilicity profile of FIG. 5;
ii) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or less than 0.5, 0.4, 0.3, 0.2, 0.1, or having a value equal to 0.0, in the Hydropathicity profile of FIG. 6;
iii) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Percent Accessible Residues profile of FIG. 7;
iv) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Average Flexibility profile of FIG. 8; or
v) a peptide region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Beta-turn profile of FIG. 9.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. The 282P1G3 SSH sequence of 321 nucleotides.
FIG. 2. A) The cDNA and amino acid sequence of 282P1G3 variant 1 (also called “282P1G3 v.1” or “282P1G3 variant 1”) is shown in FIG. 2A. The start methionine is underlined. The open reading frame extends from nucleic acid 272-3946 including the stop codon.
B) The cDNA and amino acid sequence of 282P1G3 variant 2 (also called “282P1G3 v.2”) is shown in FIG. 2B. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 272-3787 including the stop codon.
C) The cDNA and amino acid sequence of 282P1G3 variant 3 (also called “282P1G3 v.3”) is shown in FIG. 2C. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 272-2953 including the stop codon.
D) The cDNA and amino acid sequence of 282P1G3 variant 4 (also called “282P1G3 v.4”) is shown in FIG. 2D. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 272-3625 including the stop codon.
E) The cDNA and amino acid sequence of 282P1G3 variant 5 (also called “282P1G3 v.5”) is shown in FIG. 2E. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 272-3898 including the stop codon.
F) The cDNA and amino acid sequence of 282P1G3 variant 6 (also called “282P1G3 v.6”) is shown in FIG. 2F. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 272-3823 including the stop codon.
G) The cDNA and amino acid sequence of 282P1G3 variant 7 (also called “282P1G3 v.7”) is shown in FIG. 2G. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 272-3982 including the stop codon.
H) The cDNA and amino acid sequence of 282P1G3 variant 8 (also called “282P1G3 v.8”) is shown in FIG. 2H. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 272-3859 including the stop codon.
I) The cDNA and amino acid sequence of 282P1G3 variant 28 (also called “282P1G3 v.28”) is shown in FIG. 21. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 192-3866 including the stop codon.
J) The cDNA and amino acid sequence of 282P1G3 variant 14 (also called “282P1G3 v.14”) is shown in FIG. 2J. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 272-3946 including the stop codon.
K) SNP variants of 282P1G3 v.1. 282P1G3 v.9 through v.25. The 282P1G3 v.9 through v.23 proteins have 1224 amino acids. Variants 282P1G3 v.9 through v.25 are variants with single nucleotide difference from 282P1G3 v.1. 282P1G3 v.9, v.10, v.11, v.24 and v.25 proteins differ from 282P1G3v.1 by one amino acid. 282P1G3v.12 through v.23, v.26 and v.27 code for the same protein as v.1. Though these SNP variants are shown separately, they can also occur in any combinations and in any of the transcript variants listed above in FIGS. 2A through 2I.
FIG. 3. A) The amino acid sequence of 282P1G3 v.1 is shown in FIG. 3A; it has 1224 amino acids.
B) The amino acid sequence of 282P1G3 v.2 is shown in FIG. 3B; it has 1171 amino acids.
C) The amino acid sequence of 282P1G3 v.3 is shown in FIG. 3C; it has 893 amino acids.
D) The amino acid sequence of 282P1G3 v.4 is shown in FIG. 3D; it has 1117 amino acids.
E) The amino acid sequence of 282P1G3 v.5 is shown in FIG. 3E; it has 1208 amino acids.
F) The amino acid sequence of 282P1G3 v.6 is shown in FIG. 3F; it has 1183 amino acids.
G) The amino acid sequence of 282P1G3 v.7 is shown in FIG. 3G; it has 1236 amino acids.
H) The amino acid sequence of 282P1G3 v.8 is shown in FIG. 3H; it has 1195 amino acids.
I) The amino acid sequence of 282P1G3 v.9 is shown in FIG. 31; it has 1224 amino acids.
J) The amino acid sequence of 282P1G3 v.10 is shown in FIG. 3J; it has 1224 amino acids.
K) The amino acid sequence of 282P1G3 v.11 is shown in FIG. 3k; it has 1224 amino acids.
L) The amino acid sequence of 282P1G3 v.24 is shown in FIG. 3L; it has 1224 amino acids.
M) The amino acid sequence of 282P1G3 v.25 is shown in FIG. 3M; it has 1224 amino acids.
As used herein, a reference to 282P1G3 includes all variants thereof, including those shown in FIGS. 2, 3, 10, and 11, unless the context clearly indicates otherwise.
FIG. 4. FIG. 4A: Alignment of 282P1G3 with human close homolog of L1 (gi 27894376). FIG. 4B: Alignment of 282P1G3 with mouse close homolog of L1 (gi6680936).
FIG. 5. FIGS. 5(a)-(c): Hydrophilicity amino acid profile of 282P1G3v.1, v.3, and v.7 determined by computer algorithm sequence analysis using the method of Hopp and Woods (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828) accessed on the Protscale website located on the World Wide Web at (expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
FIG. 6. FIGS. 6(a)-(c): Hydropathicity amino acid profile of 282P1G3v.1, v.3, and v.7 determined by computer algorithm sequence analysis using the method of Kyte and Doolittle (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
FIG. 7. FIGS. 7(a)-(c): Percent accessible residues amino acid profile of 282P1G3v.1, v.3, and v.7 determined by computer algorithm sequence analysis using the method of Janin (Janin J., 1979 Nature 277:491-492) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
FIG. 8. FIGS. 8(a)-(c): Average flexibility amino acid profile of 282P1G3v.1, v.3, and v.7 determined by computer algorithm sequence analysis using the method of Bhaskaran and Ponnuswamy (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
FIG. 9. FIGS. 9(a)-(c): Beta-turn amino acid profile of 282P1G3v.1, v.3, and v.7 determined by computer algorithm sequence analysis using the method of Deleage and Roux (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294) accessed on the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
FIG. 10. Schematic alignment of SNP variants of 282P1G03 v.1. Variants 282P1G03 v.9 through v.27 are variants with single nucleotide difference from v.1. Variant v.14 inserted a ‘T’ between 4635 and 4636 of v.1. Though these SNP variants are shown separately, they can also occur in any combinations and in any transcript variants as shown in FIG. 12, e.g. v.2, that contains the bases. Numbers correspond to those of 282P1G03 v.1. Black box shows the same sequence as 282P1G03 v.1. SNPs are indicated above the box.
FIG. 11. Schematic alignment of protein variants of 282P1G03. Protein variants are named to correspond to nucleotide variants. Variants v.2 through v.8 were translated from splice variants. Variants v.7 and v.8 had an insertion of 12 amino acids. Variants v.9 through v.11, v.24, and v.25 were translated from SNP variants. Nucleotide variants 282P1G03 v.12 through v.23 coded for the same protein as v.1. Single amino acid differences among the proteins translated from SNP variants were indicated above the boxes. Black boxes represent the same sequence as 282P1G03 v.1. Numbers underneath the box correspond to positions in 282P1G03 v.1.
FIG. 12. Structures of transcript variants of 282P1G03. Variant 282P1G03 v.2 through v.8 and v.28 are transcript variants of 282P1G03 v.1. Variant 282P1G03 v.3 deleted exons 22 through 27, 3′ portion of exon 21 and 5′ portion of exon 28 of variant 282P1G03 v.1. Variants v.2, v.4, v.5 and v.6 spliced out exon 25, exons 21-22, exon 8, and exon 6, respectively, in v.1. Variant 282P1G03 v.7 extended 36 bp at the 5′ end of exon 11 of variant 282P1G03v.1. In addition to such an extension of 36 bp to exon 11 of v.1, variant 282P1G03 v.8 deleted exon 6 of variant 282P1G03 v.1. The 11th potential exon had two forms: the longer form was 36 bp longer than the shorter form. The 21st and 28th potential exons could also have a long and a short form, as seen in v. 3. Poly A tails are not shown here. Numbers in “( )” underneath the boxes correspond to those of 282P1G03 v. 1. Lengths of introns and exons are not proportional.
FIG. 13. Secondary structure and transmembrane domains prediction for 282P1G3B protein variants.
The secondary structure of 282P1G3B protein variants 1 through 8 (FIGS. 13A (SEQ ID NO: 199), 13B (SEQ ID NO: 200), 13C (SEQ ID NO: 201), 13D (SEQ ID NO: 202), 13E (SEQ ID NO: 203), 13F (SEQ ID NO: 204), 13G (SEQ ID NO: 205), and 13H (SEQ ID NO: 206) respectively) were predicted using the HNN—Hierarchical Neural Network method (NPS @: Network Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]:147-150 Combet C., Blanchet C., Geourjon C. and Deléage G accessed from the ExPasy molecular biology server located on the World Wide Web at (.expasy.ch/tools/). This method predicts the presence and location of alpha helices, extended strands, and random coils from the primary protein sequence. The percent of the protein in a given secondary structure is also listed.
FIGS. 13I, 13K, 13M, 13O, 13Q, 13S, 13U, and 13W: Show schematic representations of the probability of existence of transmembrane regions and orientation of 282P1G3B variants 1 through 9, respectively, based on the TMpred algorithm of Hofmann and Stoffel which utilizes TMBASE (K. Hofmann, W. Stoffel. TMBASE—A database of membrane spanning protein segments Biol. Chem. Hoppe-Seyler 374:166, 1993). FIGS. 13J, 13L, 13N, 13P, 13R, 13T, 13V, and 13X: Show schematic representations of the probability of the existence of transmembrane regions and the extracellular and intracellular orientation of 282P1G3B variants 1 through 9, respectively, based on the TMHMM algorithm of Sonnhammer, von Heijne, and Krogh (Erik L. L. Sonnhammer, Gunnar von Heijne, and Anders Krogh: A hidden Markov model for predicting transmembrane helices in protein sequences. In Proc. of Sixth Int. Conf. on Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, Calif.: AAAI Press, 1998). The TMpred and TMHMM algorithms are accessed from the ExPasy molecular biology server located on the World Wide Web at (.expasy.ch/tools/).
FIG. 14. 282P1G3 Expression by RT-PCR. First strand cDNA was prepared from (A) vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), normal pancreas, ovary cancer pool, and pancreas cancer pool; (B) normal stomach, normal brain, normal heart, normal liver, normal skeletal muscle, normal testis, normal prostate, normal bladder, normal kidney, normal colon, normal lung, normal pancreas, and a pool of cancer specimens from pancreas cancer patients, ovary cancer patients, and cancer metastasis specimens. Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primers to 282P1G3, was performed at 26 and 30 cycles of amplification. (A) Expression of 282P1G3 was detected in ovary cancer pool, pancreas cancer pool vital pool 1, but not in vital pool 2 nor in normal pancreas. (B) Samples were run on an agarose gel, and PCR products were quantitated using the Alphalmager software. Results show strong expression in pancreas cancer, ovary cancer, cancer metastasis, and normal brain compared to all other normal tissues tested.
FIG. 15. 282P1G3 expression in normal tissues. Two multiple tissue northern blots (Clontech) both with 2 ug of mRNA/lane were probed with the 282P1G3 sequence. Size standards in kilobases (kb) are indicated on the side. Results show expression of an approximately 9-10 kb 282P1G3 transcript in normal brain, but not in any other normal tissue tested.
FIG. 16. Expression of 282P1G3 in Pancreas Cancer Patient Specimens. RNA was extracted from pancreas cancer cell lines (CL), normal pancreas (N), and pancreas cancer patient tumor (T). Northern blots with 10 ug of total RNA were probed with the 282P1G3 DNA probe. Size standards in kilobases are on the side. Results show expression of 282P1G3 in pancreas cancer patient tumor specimen but not in the cell lines nor in the normal pancreas.
FIG. 17. Expression of 282P1G3 in Ovary Cancer Patient Specimens. RNA was extracted from ovary cancer cell lines (CL), normal ovary (N), and ovary cancer patient tumor (T). Northern blots with 10 ug of total RNA were probed with the 282P1G3 DNA probe. Size standards in kilobases are on the side. Results show expression of 282P1G3 in ovary cancer patient tumor specimen but not in the cell lines nor in the normal ovary.
FIG. 18. Expression of 282P1G3 in Lymphoma Cancer Patient Specimens. RNA was extracted from peripheral blood lymphocytes, cord blood isolated from normal individuals, and from lymphoma patient cancer specimens. Northern blots with 10 ug of total RNA were probed with the 282P1G3 sequence. Size standards in kilobases are on the side. Results show expression of 282P1G3 in lymphoma patient specimens but not in the normal blood cells tested.
FIG. 19. 282P1G3 Expression in 293T Cells Following Transfection of 282P1G3.pcDNA3.1/MycHis Construct. The complete ORF of 282P1G3 v.2 was cloned into the pcDNA3.1/MycHis construct to generate 282P1G3.pcDNA3.1/MycHis. 293T cells were transfected with either 282P1G3.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 282P1G3 from the 282P1G3.pcDNA3.1/MycHis construct in the lysates of transfected cells.
FIG. 20. 282P1G3 Expression in 293T Cells Following Transfection of 282P1G3.pcDNA3.1/MycHis Construct. The extracellular domain, amino acids 26-1043, of 282P1G3 v.2 was cloned into the pTag5 construct to generate 282P1G3.pTag5. 293T cells were transfected with 282P1G3.pTag5 construct. Forty hours later, supernatant as well as 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 and secretion of 282P1G3 from the 282P1G3.pTag5 transfected cells.



DETAILED DESCRIPTION OF THE INVENTION
Outline of Sections
I.) Definitions
II.) 282P1G3 Polynucleotides
II.A.) Uses of 282P1G3 Polynucleotides
II.A.1.) Monitoring of Genetic Abnormalities
II.A.2.) Antisense Embodiments
II.A.3.) Primers and Primer Pairs
II.A.4.) Isolation of 282P1G3-Encoding Nucleic Acid Molecules
II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems
III.) 282P1G3-related Proteins

    
    
        III.A.) Motif-bearing Protein Embodiments
        III.B.) Expression of 282P1G3-related Proteins
        III.C.) Modifications of 282P1G3-related Proteins
        III.D.) Uses of 282P1G3-related Proteins
    
    


IV.) 282P1G3 Antibodies
V.) 282P1G3 Cellular Immune Responses
VI.) 282P1G3 Transgenic Animals
VII.) Methods for the Detection of 282P1G3
VIII.) Methods for Monitoring the Status of 282P1G3-related Genes and Their Products
IX.) Identification of Molecules That Interact With 282P1G3
X.) Therapeutic Methods and Compositions

    
    
        X.A.) Anti-Cancer Vaccines
        X.B.) 282P1G3 as a Target for Antibody-Based Therapy
        X.C.) 282P1G3 as a Target for Cellular Immune Responses
        X.C.1. Minigene Vaccines
        X.C.2. Combinations of CTL Peptides with Helper Peptides
        X.C.3. Combinations of CTL Peptides with T Cell Priming Agents
        X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides
    
    


X.D.) Adoptive Immunotherapy
X.E.) Administration of Vaccines for Therapeutic or Prophylactic Purposes
XI.) Diagnostic and Prognostic Embodiments of 282P1G3.
XII.) Inhibition of 282P1G3 Protein Function

    
    
        XII.A.) Inhibition of 282P1G3 With Intracellular Antibodies
        XII.B.) Inhibition of 282P1G3 with Recombinant Proteins
        XII.C.) Inhibition of 282P1G3 Transcription or Translation
        XII.D.) General Considerations for Therapeutic Strategies
    
    


XIII.) Identification, Characterization and Use of Modulators of 282P1G3
XIV.) KITS/Articles of Manufacture
I.) Definitions:
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.
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.
“Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence 282P1G3 (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 282P1G3. 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.
The term “analog” refers to a molecule which is structurally similar or shares similar or corresponding attributes with another molecule (e.g. a 282P1G3-related protein). For example, an analog of a 282P1G3 protein can be specifically bound by an antibody or T cell that specifically binds to 282P1G3.
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-282P1G3 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.
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-282P1G3 antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-282P1G3 antibody compositions with polyepitopic specificity.
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.”
A “combinatorial library” is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical “building blocks” such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide (e.g., mutein) library, is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Numerous chemical compounds are synthesized through such combinatorial mixing of chemical building blocks (Gallop et al., J. Med. Chem. 37(9): 1233-1251 (1994)).
Preparation and screening of combinatorial libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Pept. Prot. Res. 37:487-493 (1991), Houghton et al., Nature, 354:84-88 (1991)), peptoids (PCT Publication No WO 91/19735), encoded peptides (PCT Publication WO 93/20242), random bio-oligomers (PCT Publication WO 92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913(1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568(1992)), nonpeptidal peptidomimetics with a Beta-D-Glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho, et al., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)). See, generally, Gordon et al., J. Med. Chem. 37:1385 (1994), nucleic acid libraries (see, e.g., Stratagene, Corp.), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology 14(3): 309-314 (1996), and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science 274:1520-1522 (1996), and U.S. Pat. No. 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Baum, C&EN, January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No. 5,288,514; and the like).
Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 NIPS, 390 NIPS, Advanced Chem Tech, Louisville Ky.; Symphony, Rainin, Woburn, Mass.; 433A, Applied Biosystems, Foster City, Calif.; 9050, Plus, Millipore, Bedford, NIA). A number of well-known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations such as the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate H, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.), which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art. In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J.; Asinex, Moscow, RU; Tripos, Inc., St. Louis, Mo.; ChemStar, Ltd, Moscow, RU; 3D Pharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md.; etc.).
The term “cytotoxic agent” refers to a substance that inhibits or prevents the expression activity of cells, function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Examples of cytotoxic agents include, but are not limited to auristatins, auromycins, maytansinoids, yttrium, bismuth, ricin, ricin A-chain, combrestatin, duocarmycins, dolostatins, doxorubicin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Sapaonaria officinalis inhibitor, and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32 and radioactive isotopes of Lu including Lu177. Antibodies may also be conjugated to an anti-cancer pro-drug activating enzyme capable of converting the pro-drug to its active form.
The “gene product” is sometimes referred to herein as a protein or mRNA. For example, a “gene product of the invention” is sometimes referred to herein as a “cancer amino acid sequence”, “cancer protein”, “protein of a cancer listed in Table I”, a “cancer mRNA”, “mRNA of a cancer listed in Table I”, etc. In one embodiment, the cancer protein is encoded by a nucleic acid of FIG. 2. The cancer protein can be a fragment, or alternatively, be the full-length protein to the fragment encoded by the nucleic acids of FIG. 2. In one embodiment, a cancer amino acid sequence is used to determine sequence identity or similarity. In another embodiment, the sequences are naturally occurring allelic variants of a protein encoded by a nucleic acid of FIG. 2. In another embodiment, the sequences are sequence variants as further described herein.
“High throughput screening” assays for the presence, absence, quantification, or other properties of particular nucleic acids or protein products are well known to those of skill in the art. Similarly, binding assays and reporter gene assays are similarly well known. Thus, e.g., U.S. Pat. No. 5,559,410 discloses high throughput screening methods for proteins; U.S. Pat. No. 5,585,639 discloses high throughput screening methods for nucleic acid binding (i.e., in arrays); while U.S. Pat. Nos. 5,576,220 and 5,541,061 disclose high throughput methods of screening for ligand/antibody binding.
In addition, high throughput screening systems are commercially available (see, e.g., Amersham Biosciences, Piscataway, N.J.; Zymark Corp., Hopkinton, Mass.; Air Technical Industries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.; Precision Systems, Inc., Natick, Mass.; etc.). These systems typically automate entire procedures, including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for various high throughput systems. Thus, e.g., Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.
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.
“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, 8TH ED., Lange Publishing, Los Altos, Calif. (1994).
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×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperatures for hybridization are above 37 degrees C. and temperatures for washing in 0.1×SSC/0.1% SDS are above 55 degrees C.
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 282P1G3 genes or that encode polypeptides other than 282P1G3 gene product or fragments thereof. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated 282P1G3 polynucleotide. A protein is said to be “isolated,” for example, when physical, mechanical or chemical methods are employed to remove the 282P1G3 proteins from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated 282P1G3 protein. Alternatively, an isolated protein can be prepared by chemical means.
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.
The terms “metastatic prostate cancer” and “metastatic disease” mean prostate cancers that have spread to regional lymph nodes or to distant sites, and are meant to include stage D disease under the AUA system and stage T×N×M+under the TNM system. As is the case with locally advanced prostate cancer, surgery is generally not indicated for patients with metastatic disease, and hormonal (androgen ablation) therapy is a preferred treatment modality. Patients with metastatic prostate cancer eventually develop an androgen-refractory state within 12 to 18 months of treatment initiation. Approximately half of these androgen-refractory patients die within 6 months after developing that status. The most common site for prostate cancer metastasis is bone. Prostate cancer bone metastases are often osteoblastic rather than osteolytic (i.e., resulting in net bone formation). Bone metastases are found most frequently in the spine, followed by the femur, pelvis, rib cage, skull and humerus. Other common sites for metastasis include lymph nodes, lung, liver and brain. Metastatic prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body radionuclide scans, skeletal radiography, and/or bone lesion biopsy.
The term “modulator” or “test compound” or “drug candidate” or grammatical equivalents as used herein describe any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., to be tested for the capacity to directly or indirectly alter the cancer phenotype or the expression of a cancer sequence, e.g., a nucleic acid or protein sequences, or effects of cancer sequences (e.g., signaling, gene expression, protein interaction, etc.) In one aspect, a modulator will neutralize the effect of a cancer protein of the invention. By “neutralize” is meant that an activity of a protein is inhibited or blocked, along with the consequent effect on the cell. In another aspect, a modulator will neutralize the effect of a gene, and its corresponding protein, of the invention by normalizing levels of said protein. In preferred embodiments, modulators alter expression profiles, or expression profile nucleic acids or proteins provided herein, or downstream effector pathways. In one embodiment, the modulator suppresses a cancer phenotype, e.g. to a normal tissue fingerprint. In another embodiment, a modulator induced a cancer phenotype. Generally, a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.
Modulators, drug candidates or test compounds encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 Daltons. Preferred small molecules are less than 2000, or less than 1500 or less than 1000 or less than 500 D. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Modulators also comprise biomolecules such as peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides. One class of modulators are peptides, for example of from about five to about 35 amino acids, with from about five to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. Preferably, the cancer modulatory protein is soluble, includes a non-transmembrane region, and/or, has an N-terminal Cys to aid in solubility. In one embodiment, the C-terminus of the fragment is kept as a free acid and the N-terminus is a free amine to aid in coupling, i.e., to cysteine. In one embodiment, a cancer protein of the invention is conjugated to an immunogenic agent as discussed herein. In one embodiment, the cancer protein is conjugated to BSA. The peptides of the invention, e.g., of preferred lengths, can be linked to each other or to other amino acids to create a longer peptide/protein. The modulatory peptides can be digests of naturally occurring proteins as is outlined above, random peptides, or ‘biased’ random peptides. In a preferred embodiment, peptide/protein-based modulators are antibodies, and fragments thereof, as defined herein.
Modulators of cancer can also be nucleic acids. Nucleic acid modulating agents can be naturally occurring nucleic acids, random nucleic acids, or “biased” random nucleic acids. For example, digests of prokaryotic or eukaryotic genomes can be used in an approach analogous to that outlined above for proteins.
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.
A “motif”, as in biological motif of a 282P1G3-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.
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.
“Pharmaceutically acceptable” refers to a non-toxic, inert, and/or composition that is physiologically compatible with humans or other mammals.
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).
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”.
An HLA “primary anchor residue” is an amino acid at a specific position along a peptide sequence which is understood to provide a contact point between the immunogenic peptide and the HLA molecule. One to three, usually two, primary anchor residues within a peptide of defined length generally defines a “motif” for an immunogenic peptide. These residues are understood to fit in close contact with peptide binding groove of an HLA molecule, with their side chains buried in specific pockets of the binding groove. In one embodiment, for example, the primary anchor residues for an HLA class I molecule are located at position 2 (from the amino terminal position) and at the carboxyl terminal position of a 8, 9, 10, 11, or 12 residue peptide epitope in accordance with the invention. Alternatively, in another embodiment, the primary anchor residues of a peptide binds an HLA class II molecule are spaced relative to each other, rather than to the termini of a peptide, where the peptide is generally of at least 9 amino acids in length. The primary anchor positions for each motif and supermotif are set forth in Table IV. For example, analog peptides can be created by altering the presence or absence of particular residues in the primary and/or secondary anchor positions shown in Table IV. Such analogs are used to modulate the binding affinity and/or population coverage of a peptide comprising a particular HLA motif or supermotif.
“Radioisotopes” include, but are not limited to the following (non-limiting exemplary uses are also set forth):










Examples of Medical Isotopes:








Isotope
Description of use





Actinium-225
See Thorium-229 (Th-229)


(AC-225)


Actinium-227
Parent of Radium-223 (Ra-223) which is an alpha emitter used to treat metastases in the skeleton resulting


(AC-227)
from cancer (i.e., breast and prostate cancers), and cancer radioimmunotherapy


Bismuth-212
See Thorium-228 (Th-228)


(Bi-212)


Bismuth-213
See Thorium-229 (Th-229)


(Bi-213)


Cadmium-109
Cancer detection


(Cd-109)


Cobalt-60
Radiation source for radiotherapy of cancer, for food irradiators, and for sterilization of medical supplies


(Co-60)


Copper-64
A positron emitter used for cancer therapy and SPECT imaging


(Cu-64)


Copper-67
Beta/gamma emitter used in cancer radioimmunotherapy and diagnostic studies (i.e., breast and colon


(Cu-67)
cancers, and lymphoma)


Dysprosium-166
Cancer radioimmunotherapy


(Dy-166)


Erbium-169
Rheumatoid arthritis treatment, particularly for the small joints associated with fingers and toes


(Er-169)


Europium-152
Radiation source for food irradiation and for sterilization of medical supplies


(Eu-152)


Europium-154
Radiation source for food irradiation and for sterilization of medical supplies


(Eu-154)


Gadolinium-153
Osteoporosis detection and nuclear medical quality assurance devices


(Gd-153)


Gold-198
Implant and intracavity therapy of ovarian, prostate, and brain cancers


(Au-198)


Holmium-166
Multiple myeloma treatment in targeted skeletal therapy, cancer radioimmunotherapy, bone marrow ablation,


(Ho-166)
and rheumatoid arthritis treatment


Iodine-125
Osteoporosis detection, diagnostic imaging, tracer drugs, brain cancer treatment, radiolabeling, tumor


(I-125)
imaging, mapping of receptors in the brain, interstitial radiation therapy, brachytherapy for treatment of



prostate cancer, determination of glomerular filtration rate (GFR), determination of plasma volume, detection



of deep vein thrombosis of the legs


Iodine-131
Thyroid function evaluation, thyroid disease detection, treatment of thyroid cancer as well as other non-


(I-131)
malignant thyroid diseases (i.e., Graves disease, goiters, and hyperthyroidism), treatment of leukemia,



lymphoma, and other forms of cancer (e.g., breast cancer) using radioimmunotherapy


Iridium-192
Brachytherapy, brain and spinal cord tumor treatment, treatment of blocked arteries (i.e., arteriosclerosis and


(Ir-192)
restenosis), and implants for breast and prostate tumors


Lutetium-177
Cancer radioimmunotherapy and treatment of blocked arteries (i.e., arteriosclerosis and restenosis)


(Lu-177)


Molybdenum-99
Parent of Technetium-99m (Tc-99m) which is used for imaging the brain, liver, lungs, heart, and other organs.


(Mo-99)
Currently, Tc-99m is the most widely used radioisotope used for diagnostic imaging of various cancers and



diseases involving the brain, heart, liver, lungs; also used in detection of deep vein thrombosis of the legs


Osmium-194
Cancer radioimmunotherapy


(Os-194)


Palladium-103
Prostate cancer treatment


(Pd-103)


Platinum-195m
Studies on biodistribution and metabolism of cisplatin, a chemotherapeutic drug


(Pt-195m)


Phosphorus-32
Polycythemia rubra vera (blood cell disease) and leukemia treatment, bone cancer diagnosis/treatment;


(P-32)
colon, pancreatic, and liver cancer treatment; radiolabeling nucleic acids for in vitro research, diagnosis of



superficial tumors, treatment of blocked arteries (i.e., arteriosclerosis and restenosis), and intracavity therapy


Phosphorus-33
Leukemia treatment, bone disease diagnosis/treatment, radiolabeling, and treatment of blocked arteries (i.e.,


(P-33)
arteriosclerosis and restenosis)


Radium-223
See Actinium-227 (Ac-227)


(Ra-223)


Rhenium-186
Bone cancer pain relief, rheumatoid arthritis treatment, and diagnosis and treatment of lymphoma and bone,


(Re-186)
breast, colon, and liver cancers using radioimmunotherapy


Rhenium-188
Cancer diagnosis and treatment using radioimmunotherapy, bone cancer pain relief, treatment of rheumatoid


(Re-188)
arthritis, and treatment of prostate cancer


Rhodium-105
Cancer radioimmunotherapy


(Rh-105)


Samarium-145
Ocular cancer treatment


(Sm-145)


Samarium-153
Cancer radioimmunotherapy and bone cancer pain relief


(Sm-153)


Scandium-47
Cancer radioimmunotherapy and bone cancer pain relief


(Sc-47)


Selenium-75
Radiotracer used in brain studies, imaging of adrenal cortex by gamma-scintigraphy, lateral locations of


(Se-75)
steroid secreting tumors, pancreatic scanning, detection of hyperactive parathyroid glands, measure rate of



bile acid loss from the endogenous pool


Strontium-85
Bone cancer detection and brain scans


(Sr-85)


Strontium-89
Bone cancer pain relief, multiple myeloma treatment, and osteoblastic therapy


(Sr-89)


Technetium-99m
See Molybdenum-99 (Mo-99)


(Tc-99m)


Thorium-228
Parent of Bismuth-212 (Bi-212) which is an alpha emitter used in cancer radioimmunotherapy


(Th-228)


Thorium-229
Parent of Actinium-225 (Ac-225) and grandparent of Bismuth-213 (Bi-213) which are alpha emitters used in


(Th-229)
cancer radioimmunotherapy


Thulium-170
Gamma source for blood irradiators, energy source for implanted medical devices


(Tm-170)


Tin-117m
Cancer immunotherapy and bone cancer pain relief


(Sn-117m)


Tungsten-188
Parent for Rhenium-188 (Re-188) which is used for cancer diagnostics/treatment, bone cancer pain relief,


(W-188)
rheumatoid arthritis treatment, and treatment of blocked arteries (i.e., arteriosclerosis and restenosis)


Xenon-127
Neuroimaging of brain disorders, high resolution SPECT studies, pulmonary function tests, and cerebral


(Xe-127)
blood flow studies


Ytterbium-175
Cancer radioimmunotherapy


(Yb-175)


Yttrium-90
Microseeds obtained from irradiating Yttrium-89 (Y-89) for liver cancer treatment


(Y-90)


Yttrium-91
A gamma-emitting label for Yttrium-90 (Y-90) which is used for cancer radioimmunotherapy (i.e., lymphoma,


(Y-91)
breast, colon, kidney, lung, ovarian, prostate, pancreatic, and inoperable liver cancers)









By “randomized” or grammatical equivalents as herein applied to nucleic acids and proteins is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. These random peptides (or nucleic acids, discussed herein) can incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.
In one embodiment, a library is “fully randomized,” with no sequence preferences or constants at any position. In another embodiment, the library is a “biased random” library. That is, some positions within the sequence either are held constant, or are selected from a limited number of possibilities. For example, the nucleotides or amino acid residues are randomized within a defined class, e.g., of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of nucleic acid binding domains, the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.
A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that has been subjected to molecular manipulation in vitro.
Non-limiting examples of small molecules include compounds that bind or interact with 282P1G3, ligands including hormones, neuropeptides, chemokines, odorants, phospholipids, and functional equivalents thereof that bind and preferably inhibit 282P1G3 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, 282P1G3 protein; are not found in naturally occurring metabolic pathways; and/or are more soluble in aqueous than non-aqueous solutions
“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).
“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° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° 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° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50° 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.
An HLA “supermotif” is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles. Overall phenotypic frequencies of HLA-supertypes in different ethnic populations are set forth in Table IV (F). The non-limiting constituents of various supetypes are as follows:
A2: A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*6802, A*6901, A*0207
A3: A3, A11, A31, A*3301, A*6801, A*0301, A*1101, A*3101
B7: B7, B*3501-03, B*51, B*5301, B*5401, B*5501, B*5502, B*5601, B*6701, B*7801, B*0702, B*5101, B*5602
B44: B*3701, B*4402, B*4403, B*60 (B*4001), B61 (B*4006)
A1: A*0102, A*2604, A*3601, A*4301, A*8001
A24: A*24, A*30, A*2403, A*2404, A*3002, A*3003
B27: B*1401-02, B*1503, B*1509, B*1510, B*1518, B*3801-02, B*3901, B*3902, B*3903-04, B*4801-02, B*7301, B*2701-08
B58: B*1516, B*1517, B*5701, B*5702, B58
B62: B*4601, B52, B*1501 (B62), B*1502 (B75), B*1513 (B77)
Calculated population coverage afforded by different HLA-supertype combinations are set forth in Table IV (G).
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.
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.
As used herein, an HLA or cellular immune response “vaccine” is a composition that contains or encodes one or more peptides of the invention. There are numerous embodiments of such vaccines, such as a cocktail of one or more individual peptides; one or more peptides of the invention comprised by a polyepitopic peptide; or nucleic acids that encode such individual peptides or polypeptides, e.g., a minigene that encodes a polyepitopic peptide. The “one or more peptides” can include any whole unit integer from 1-150 or more, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 or more peptides of the invention. The peptides or polypeptides can optionally be modified, such as by lipidation, addition of targeting or other sequences. HLA class I peptides of the invention can be admixed with, or linked to, HLA class II peptides, to facilitate activation of both cytotoxic T lymphocytes and helper T lymphocytes. HLA vaccines can also comprise peptide-pulsed antigen presenting cells, e.g., dendritic cells.
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 282P1G3 protein shown in FIG. 2 or FIG. 3. An analog is an example of a variant protein. Splice isoforms and single nucleotides polymorphisms (SNPs) are further examples of variants.
The “282P1G3-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 282P1G3 proteins or fragments thereof, as well as fusion proteins of a 282P1G3 protein and a heterologous polypeptide are also included. Such 282P1G3 proteins are collectively referred to as the 282P1G3-related proteins, the proteins of the invention, or 282P1G3. The term “282P1G3-related protein” refers to a polypeptide fragment or a 282P1G3 protein sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, or 576 or more amino acids.
II.) 282P1G3 Polynucleotides
One aspect of the invention provides polynucleotides corresponding or complementary to all or part of a 282P1G3 gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding a 282P1G3-related protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to a 282P1G3 gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides that hybridize to a 282P1G3 gene, mRNA, or to a 282P1G3 encoding polynucleotide (collectively, “282P1G3 polynucleotides”). In all instances when referred to in this section, T can also be U in FIG. 2.
Embodiments of a 282P1G3 polynucleotide include: a 282P1G3 polynucleotide having the sequence shown in FIG. 2, the nucleotide sequence of 282P1G3 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 282P1G3 nucleotides comprise, without limitation:
(I) a polynucleotide comprising, consisting essentially of, or consisting of a sequence as shown in FIG. 2, wherein T can also be U;
(II) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2A, from nucleotide residue number 272 through nucleotide residue number 3946, including the stop codon, wherein T can also be U;
(III) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2B, from nucleotide residue number 272 through nucleotide residue number 3787, including the stop codon, wherein T can also be U;
(IV) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2C, from nucleotide residue number 272 through nucleotide residue number 3953, including the a stop codon, wherein T can also be U;
(V) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2D, from nucleotide residue number 272 through nucleotide residue number 3625, including the stop codon, wherein T can also be U;
(VI) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2E, from nucleotide residue number 272 through nucleotide residue number 3898, including the stop codon, wherein T can also be U;
(VII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2F, from nucleotide residue number 272 through nucleotide residue number 3823, including the stop codon, wherein T can also be U;
(VIII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2G, from nucleotide residue number 272 through nucleotide residue number 3982, including the stop codon, wherein T can also be U;
(IX) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2H, from nucleotide residue number 272 through nucleotide residue number 3859, including the stop codon, wherein T can also be U;
(X) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 21, from nucleotide residue number 192 through nucleotide residue number 3866, including the stop codon, wherein T can also be U;
(XI) a polynucleotide that encodes a 282P1G3-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homologous to an entire amino acid sequence shown in FIG. 2A-J;
(XII) a polynucleotide that encodes a 282P1G3-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to an entire amino acid sequence shown in FIG. 2A-J;
(XIII) a polynucleotide that encodes at least one peptide set forth in Tables VIII-XXI and XXII-XLIX;
(XIV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIGS. 3A and 3I-3M in any whole number increment up to 1224 that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
(XV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIGS. 3A and 3I-3M in any whole number increment up to 1224 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
(XVI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIGS. 3A and 3I-3M in any whole number increment up to 1224 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
(XVII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIGS. 3A and 3I-3M in any whole number increment up to 1224 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
(XVIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIGS. 3A and 3I-3M in any whole number increment up to 1224 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;
(XIX) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3B in any whole number increment up to 1171 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
(XX) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3B in any whole number increment up to 1171 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
(XXI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3B in any whole number increment up to 1171 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
(XXII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3B in any whole number increment up to 1171 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
(XXIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3B in any whole number increment up to 1171 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9
(XXIV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3C in any whole number increment up to 893 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
(XXV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3C in any whole number increment up to 893 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
(XXVI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3C in any whole number increment up to 893 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
(XXVII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3C in any whole number increment up to 893 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
(XXVIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3C in any whole number increment up to 893 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9
(XXIX) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3D in any whole number increment up to 1117 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
(XXX) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3D in any whole number increment up to 1117 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
(XXXI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3D in any whole number increment up to 1117 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
(XXXII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3D in any whole number increment up to 1117 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
(XXXIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3D in any whole number increment up to 1117 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9
(XXXIV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3E in any whole number increment up to 1208 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
(XXXV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3E in any whole number increment up to 1208 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
(XXXVI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3E in any whole number increment up to 1208 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
(XXXVII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3E in any whole number increment up to 1208 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
(XXXVIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3E in any whole number increment up to 1208 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9
(XXXIX) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3F in any whole number increment up to 1183 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
(XL) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3F in any whole number increment up to 1183 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
(XLI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3F in any whole number increment up to 1183 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
(XLII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3F in any whole number increment up to 1183 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
(XLIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3F in any whole number increment up to 1183 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9
(XLIV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3G in any whole number increment up to 1236 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
(XLV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3G in any whole number increment up to 1236 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
(XLVI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3G in any whole number increment up to 1236 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
(XLVII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3G in any whole number increment up to 1236 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG.
(XLVIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3G in any whole number increment up to 1236 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9
(XLIX) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3E in any whole number increment up to 1208 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
(L) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3H in any whole number increment up to 1195 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
(LI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3H in any whole number increment up to 1195 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
(LII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3H in any whole number increment up to 1195 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid positon(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
(LIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3H in any whole number increment up to 1195 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9
(LIV) a polynucleotide that is fully complementary to a polynucleotide of any one of (I)-(LIII).
(LV) a peptide that is encoded by any of (I) to (LIV); and
(LVI) a composition comprising a polynucleotide of any of (I)-(LIV) or peptide of (LV) together with a pharmaceutical excipient and/or in a human unit dose form.
(LVII) a method of using a polynucleotide of any (I)-(LIV) or peptide of (LV) or a composition of (LVI) in a method to modulate a cell expressing 282P1G3,
(LVIII) a method of using a polynucleotide of any (I)-(LIV) or peptide of (LV) or a composition of (LVI) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 282P1G3
(LIX) a method of using a polynucleotide of any (I)-(LIV) or peptide of (LV) or a composition of (LVI) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 282P1G3, said cell from a cancer of a tissue listed in Table I;
(LX) a method of using a polynucleotide of any (I)-(LIV) or peptide of (LV) or a composition of (LVI) in a method to diagnose, prophylax, prognose, or treat a a cancer;
(LXI) a method of using a polynucleotide of any (I)-(LIV) or peptide of (LV) or a composition of (LVI) in a method to diagnose, prophylax, prognose, or treat a a cancer of a tissue listed in Table I; and,
(LXII) a method of using a polynucleotide of any (I)-(LIV) or peptide of (LV) or a composition of (LVI) in a method to identify or characterize a modulator of a cell expressing 282P1G3.
As used herein, a range is understood to disclose specifically all whole unit positions thereof.
Typical embodiments of the invention disclosed herein include 282P1G3 polynucleotides that encode specific portions of 282P1G3 mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example:
(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1210, 1220, and 1224 or more contiguous amino acids of 282P1G3 variant 1; the maximal lengths relevant for other variants are: variant 2, 1171 amino acids; variant 3, 893 amino acids, variant 4, 1117 amino acids, variant 5, 1208 amino acids, variant 6, 1183 amono acids, variant 7, 1236 amino acids, variant 8, 1195 amino acids, variant 9, 1224 amino acids, variant 10, 1224 amino acids, variant 11, 1224 amino acids, variant 24, 1224 amino acids, and variant 25, 1224 amino acids.
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 282P1G3 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the 282P1G3 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the 282P1G3 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 30 to about amino acid 40 of the 282P1G3 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 40 to about amino acid 50 of the 282P1G3 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 50 to about amino acid 60 of the 282P1G3 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 60 to about amino acid 70 of the 282P1G3 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 70 to about amino acid 80 of the 282P1G3 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 80 to about amino acid 90 of the 282P1G3 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 90 to about amino acid 100 of the 282P1G3 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 282P1G3 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.
Polynucleotides encoding relatively long portions of a 282P1G3 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 282P1G3 protein “or variant” shown in FIG. 2 or FIG. 3 can be generated by a variety of techniques well known in the art. These polynucleotide fragments can include any portion of the 282P1G3 sequence as shown in FIG. 2.
Additional illustrative embodiments of the invention disclosed herein include 282P1G3 polynucleotide fragments encoding one or more of the biological motifs contained within a 282P1G3 protein “or variant” sequence, including one or more of the motif-bearing subsequences of a 282P1G3 protein “or variant” set forth in Tables VIII-XXI and XXII-XLIX. In another embodiment, typical polynucleotide fragments of the invention encode one or more of the regions of 282P1G3 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 282P1G3 protein or variant N-glycosylation sites, cAMP and cGMP-dependent protein kinase phosphorylation sites, casein kinase 11 phosphorylation sites or N-myristoylation site and amidation sites.
Note that to determine the starting position of any peptide set forth in Tables VIII-XXI and Tables XXII to XLIX (collectively HLA Peptide Tables) respective to its parental protein, e.g., variant 1, variant 2, etc., reference is made to three factors: the particular variant, the length of the peptide in an HLA Peptide Table, and the Search Peptides listed in Table VII. Generally, a unique Search Peptide is used to obtain HLA peptides for a particular variant. The position of each Search Peptide relative to its respective parent molecule is listed in Table VII. Accordingly, if a Search Peptide begins at position “X”, one must add the value “X minus 1” to each position in Tables VIII-XXI and Tables XXII-IL to obtain the actual position of the HLA peptides in their parental molecule. For example if a particular Search Peptide begins at position 150 of its parental molecule, one must add 150−1, i.e., 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.
II.A.) Uses of 282P1G3 Polynucleotides
II.A.1.) Monitoring of Genetic Abnormalities
The polynucleotides of the preceding paragraphs have a number of different specific uses. The human 282P1G3 gene maps to the chromosomal location set forth in the Example entitled “Chromosomal Mapping of 282P1G3.” For example, because the 282P1G3 gene maps to this chromosome, polynucleotides that encode different regions of the 282P1G3 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 282P1G3 proteins provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the chromosomal region that encodes 282P1G3 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)).
Furthermore, as 282P1G3 was shown to be highly expressed in prostate and other cancers, 282P1G3 polynucleotides are used in methods assessing the status of 282P1G3 gene products in normal versus cancerous tissues. Typically, polynucleotides that encode specific regions of the 282P1G3 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 282P1G3 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.
II.A.2.) Antisense Embodiments
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 282P1G3. 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 282P1G3 polynucleotides and polynucleotide sequences disclosed herein.
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., 282P1G3. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The 282P1G3 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 282P1G3 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).
The 282P1G3 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 282P1G3 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 282P1G3 mRNA and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, 282P1G3 antisense oligonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to 282P1G3 mRNA. Optionally, 282P1G3 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 282P1G3. Alternatively, the antisense molecules are modified to employ ribozymes in the inhibition of 282P1G3 expression, see, e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).
II.A.3.) Primers and Primer Pairs
Further specific embodiments of these nucleotides of the invention include primers and primer pairs, which allow the specific amplification of polynucleotides of the invention or of any specific parts thereof, and probes that selectively or specifically hybridize to nucleic acid molecules of the invention or to any part thereof. Probes can be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme. Such probes and primers are used to detect the presence of a 282P1G3 polynucleotide in a sample and as a means for detecting a cell expressing a 282P1G3 protein.
Examples of such probes include polypeptides comprising all or part of the human 282P1G3 cDNA sequence shown in FIG. 2. Examples of primer pairs capable of specifically amplifying 282P1G3 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 282P1G3 mRNA.
The 282P1G3 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 282P1G3 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 282P1G3 polypeptides; as tools for modulating or inhibiting the expression of the 282P1G3 gene(s) and/or translation of the 282P1G3 transcript(s); and as therapeutic agents.
The present invention includes the use of any probe as described herein to identify and isolate a 282P1G3 or 282P1G3 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.
II.A.4.) Isolation of 282P1G3-Encoding Nucleic Acid Molecules
The 282P1G3 cDNA sequences described herein enable the isolation of other polynucleotides encoding 282P1G3 gene product(s), as well as the isolation of polynucleotides encoding 282P1G3 gene product homologs, alternatively spliced isoforms, allelic variants, and mutant forms of a 282P1G3 gene product as well as polynucleotides that encode analogs of 282P1G3-related proteins. Various molecular cloning methods that can be employed to isolate full length cDNAs encoding a 282P1G3 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 282P1G3 gene cDNAs can be identified by probing with a labeled 282P1G3 cDNA or a fragment thereof. For example, in one embodiment, a 282P1G3 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 282P1G3 gene. A 282P1G3 gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with 282P1G3 DNA probes or primers.
II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems
The invention also provides recombinant DNA or RNA molecules containing a 282P1G3 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).
The invention further provides a host-vector system comprising a recombinant DNA molecule containing a 282P1G3 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 282P1G3 or a fragment, analog or homolog thereof can be used to generate 282P1G3 proteins or fragments thereof using any number of host-vector systems routinely used and widely known in the art.
A wide range of host-vector systems suitable for the expression of 282P1G3 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 pSRαtkneo (Muller et al., 1991, MCB 11:1785). Using these expression vectors, 282P1G3 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 282P1G3 protein or fragment thereof. Such host-vector systems can be employed to study the functional properties of 282P1G3 and 282P1G3 mutations or analogs.
Recombinant human 282P1G3 protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a 282P1G3-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding 282P1G3 or fragment, analog or homolog thereof, a 282P1G3-related protein is expressed in the 293T cells, and the recombinant 282P1G3 protein is isolated using standard purification methods (e.g., affinity purification using anti-282P1G3 antibodies). In another embodiment, a 282P1G3 coding sequence is subcloned into the retroviral vector pSRαMSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293 and rat-1 in order to establish 282P1G3 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 282P1G3 coding sequence can be used for the generation of a secreted form of recombinant 282P1G3 protein.
As discussed herein, redundancy in the genetic code permits variation in 282P1G3 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.
Additional sequence modifications are known to enhance protein expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon/intron splice site signals, transposon-like repeats, and/or other such well-characterized sequences that are deleterious to gene expression. The GC content of the sequence is adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Where possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures. Other useful modifications include the addition of a translational initiation consensus sequence at the start of the open reading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080 (1989). Skilled artisans understand that the general rule that eukaryotic ribosomes initiate translation exclusively at the 5′ proximal AUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS 92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).
III.) 282P1G3-Related Proteins
Another aspect of the present invention provides 282P1G3-related proteins. Specific embodiments of 282P1G3 proteins comprise a polypeptide having all or part of the amino acid sequence of human 282P1G3 as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of 282P1G3 proteins comprise variant, homolog or analog polypeptides that have alterations in the amino acid sequence of 282P1G3 shown in FIG. 2 or FIG. 3.
Embodiments of a 282P1G3 polypeptide include: a 282P1G3 polypeptide having a sequence shown in FIG. 2, a peptide sequence of a 282P1G3 as shown in FIG. 2 wherein T is U; at least 10 contiguous nucleotides of a polypeptide having the sequence as shown in FIG. 2; or, at least 10 contiguous peptides of a polypeptide having the sequence as shown in FIG. 2 where T is U. For example, embodiments of 282P1G3 peptides comprise, without limitation:
(I) a protein comprising, consisting essentially of, or consisting of an amino acid sequence as shown in FIG. 2A-J or FIG. 3A-M;
(II) a 282P1G3-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homologous to an entire amino acid sequence shown in FIG. 2A-J or 3A-M;
(III) a 282P1G3-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to an entire amino acid sequence shown in FIG. 2A-J or 3A-M;
(IV) a protein that comprises at least one peptide set forth in Tables VIII to XLIX, optionally with a proviso that it is not an entire protein of FIG. 2;
(V) a protein that comprises at least one peptide set forth in Tables VIII-XXI, collectively, which peptide is also set forth in Tables XXII to XLIX, collectively, optionally with a proviso that it is not an entire protein of FIG. 2;
(VI) a protein that comprises at least two peptides selected from the peptides set forth in Tables VIII-XLIX, optionally with a proviso that it is not an entire protein of FIG. 2;
(VII) a protein that comprises at least two peptides selected from the peptides set forth in Tables VIII to XLIX collectively, with a proviso that the protein is not a contiguous sequence from an amino acid sequence of FIG. 2;
(VIII) a protein that comprises at least one peptide selected from the peptides set forth in Tables VIII-XXI; and at least one peptide selected from the peptides set forth in Tables XXII to XLIX, with a proviso that the protein is not a contiguous sequence from an amino acid sequence of FIG. 2;
(IX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3A, 3I-3M in any whole number increment up to 1224 respectively that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
(x) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3A, 3I-3M, in any whole number increment up to 1224 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
(XI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3A, 3I-3M, in any whole number increment up to 1224 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
(XII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3A, 3I-3M, in any whole number increment up to 1224 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
(XIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIG. 3A, 3I-3M in any whole number increment up to 1224 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;
(XIV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3B, in any whole number increment up to 1171 respectively that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
(XV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3B, in any whole number increment up to 1171 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
(XVI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3B, in any whole number increment up to 1171 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
(XVII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3B, in any whole number increment up to 1171 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
(XVIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIG. 3B in any whole number increment up to 1171 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;
(XIX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3C, in any whole number increment up to 893 respectively that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
(XX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3C, in any whole number increment up to 893 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
(XXI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3C, in any whole number increment up to 893 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
(XXII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3C, in any whole number increment up to 893 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
(XXIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIG. 3C in any whole number increment up to 893 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;
(XXIV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3D, in any whole number increment up to 1117 respectively that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
(XXV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3D, in any whole number increment up to 1117 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
(XXVI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3D, in any whole number increment up to 1117 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
(XXVII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3D, in any whole number increment up to 1117 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
(XXVIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIG. 3D in any whole number increment up to 1117 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;
(XXIX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3E, in any whole number increment up to 1208 respectively that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
(XXX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3E, in any whole number increment up to 1208 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
(XXXI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3E, in any whole number increment up to 1208 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
(XXXII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3E, in any whole number increment up to 1208 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
(XXXIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIG. 3E in any whole number increment up to 1208 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;
(XXXIV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3F, in any whole number increment up to 1183 respectively that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
(XXXV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3F, in any whole number increment up to 1183 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
(XXXVI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3F, in any whole number increment up to 1183 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
(XXXVII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3F, in any whole number increment up to 1183 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
(XXXVIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIG. 3F in any whole number increment up to 1183 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;
(XXXIX) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3G, in any whole number increment up to 1236 respectively that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
(XL) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3G, in any whole number increment up to 1236 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
(XLI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3G, in any whole number increment up to 1236 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
(XLII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3G, in any whole number increment up to 1236 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
(XLIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIG. 3G in any whole number increment up to 1236 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;
(XLIV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3H, in any whole number increment up to 1195 respectively that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
(XLV) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3H, in any whole number increment up to 1195 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
(XLVI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3H, in any whole number increment up to 1195 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
(XLVII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIG. 3H, in any whole number increment up to 1195 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
(XLVIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, amino acids of a protein of FIG. 3H in any whole number increment up to 1195 respectively that includes at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;
(XLIX) a peptide that occurs at least twice in Tables VIII-XXI and XXII to XLIX, collectively;
(L) a peptide that occurs at least three times in Tables VIII-XXI and XXII to XLIX, collectively;
(LI) a peptide that occurs at least four times in Tables VIII-XXI and XXII to XLIX, collectively;
(LII) a peptide that occurs at least five times in Tables VIII-XXI and XXII to XLIX, collectively;
(LIII) a peptide that occurs at least once in Tables VIII-XXI, and at least once in tables XXII to XLIX;
(LIV) a peptide that occurs at least once in Tables VIII-XXI, and at least twice in tables XXII to XLIX;
(LV) a peptide that occurs at least twice in Tables VIII-XXI, and at least once in tables XXII to XLIX;
(LVI) a peptide that occurs at least twice in Tables VIII-XXI, and at least twice in tables XXII to XLIX;
(LVII) a peptide which comprises one two, three, four, or five of the following characteristics, or an oligonucleotide encoding such peptide:

    
    
        i) a region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Hydrophilicity profile of FIG. 5;
        ii) a region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or less than 0.5, 0.4, 0.3, 0.2, 0.1, or having a value equal to 0.0, in the Hydropathicity profile of FIG. 6;
        iii) a region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Percent Accessible Residues profile of FIG. 7;
        iv) a region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Average Flexibility profile of FIG. 8; or,
        v) a region of at least 5 amino acids of a particular peptide of FIG. 3, in any whole number increment up to the full length of that protein in FIG. 3, that includes an amino acid position having a value equal to or greater than 0.5, 0.6, 0.7, 0.8, 0.9, or having a value equal to 1.0, in the Beta-turn profile of FIG. 9;
    
    


(LVIII) a composition comprising a peptide of (I)-(LVII) or an antibody or binding region thereof together with a pharmaceutical excipient and/or in a human unit dose form.
(LIX) a method of using a peptide of (I)-(LVII), or an antibody or binding region thereof or a composition of (LVIII) in a method to modulate a cell expressing 282P1G3,
(LX) a method of using a peptide of (I)-(LVII) or an antibody or binding region thereof or a composition of (LVIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 282P1G3
(LXI) a method of using a peptide of (1)-(LVII) or an antibody or binding region thereof or a composition (LVIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 282P1G3, said cell from a cancer of a tissue listed in Table I;
(LXII) a method of using a peptide of (I)-(LVII) or an antibody or binding region thereof or a composition of (LVIII) in a method to diagnose, prophylax, prognose, or treat a a cancer;
(LXIII) a method of using a peptide of (I)-(LVII) or an antibody or binding region thereof or a composition of (LVIII) in a method to diagnose, prophylax, prognose, or treat a cancer of a tissue listed in Table I; and,
(LXIV) a method of using a a peptide of (1)-(LVII) or an antibody or binding region thereof or a composition (LVIII) in a method to identify or characterize a modulator of a cell expressing 282P1G3.
As used herein, a range is understood to specifically disclose all whole unit positions thereof.
Typical embodiments of the invention disclosed herein include 282P1G3 polynucleotides that encode specific portions of 282P1G3 mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example:
(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1125, 1150, 1175, 1200, 1210, 1215, 1220, and 1224 or more contiguous amino acids of 282P1G3 variant 1; the maximal lengths relevant for other variants are: variant 2, 1171 amino acids; variant 3, 893 amino acids, variant 4, 1117 amino acids, variant 5, 1208 amino acids, variant 6, 1183 amino acids, variant 7, 1236 amino acids, variant 8, 1195 amino acids, variant 9, 1224 amino acids, variant 10, 1224 amino acids, variant 11, 1224 amino acids, variant 24, 1224 amino acids, and variant 25, 1224 amino acids.
In general, naturally occurring alelic variants of human 282P1G3 share a high degree of structural identity and homology (e.g., 90% or more homology). Typically, allelic variants of a 282P1G3 protein contain conservative amino acid substitutions within the 282P1G3 sequences described herein or contain a substitution of an amino acid from a corresponding position in a homologue of 282P1G3. One class of 282P1G3 allelic variants are proteins that share a high degree of homology with at least a small region of a particular 282P1G3 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.
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” 2nd ED. Lubert Stryer ed (Stanford University); Henikoff et al., PNAS 1992 Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19; 270(20):11882-6).
Embodiments of the invention disclosed herein include a wide variety of art-accepted variants or analogs of 282P1G3 proteins such as polypeptides having amino acid insertions, deletions and substitutions. 282P1G3 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 282P1G3 variant DNA.
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.
As defined herein, 282P1G3 variants, analogs or homologs, have the distinguishing attribute of having at least one epitope that is “cross reactive” with a 282P1G3 protein having an amino acid sequence of FIG. 3. As used in this sentence, “cross reactive” means that an antibody or T cell that specifically binds to a 282P1G3 variant also specifically binds to a 282P1G3 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 282P1G3 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.
Other classes of 282P1G3-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 282P1G3 protein variants or analogs comprises one or more of the 282P1G3 biological motifs described herein or presently known in the art. Thus, encompassed by the present invention are analogs of 282P1G3 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.
As discussed herein, embodiments of the claimed invention include polypeptides containing less than the full amino acid sequence of a 282P1G3 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 282P1G3 protein shown in FIG. 2 or FIG. 3.
Moreover, representative embodiments of the invention disclosed herein include polypeptides consisting of about amino acid 1 to about amino acid 10 of a 282P1G3 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 10 to about amino acid 20 of a 282P1G3 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 20 to about amino acid 30 of a 282P1G3 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 30 to about amino acid 40 of a 282P1G3 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 40 to about amino acid 50 of a 282P1G3 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 50 to about amino acid 60 of a 282P1G3 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 60 to about amino acid 70 of a 282P1G3 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 70 to about amino acid 80 of a 282P1G3 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 80 to about amino acid 90 of a 282P1G3 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 90 to about amino acid 100 of a 282P1G3 protein shown in FIG. 2 or FIG. 3, etc. throughout the entirety of a 282P1G3 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 282P1G3 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.
282P1G3-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 282P1G3-related protein. In one embodiment, nucleic acid molecules provide a means to generate defined fragments of a 282P1G3 protein (or variants, homologs or analogs thereof).
III.A.) Motif-Bearing Protein Embodiments
Additional illustrative embodiments of the invention disclosed herein include 282P1G3 polypeptides comprising the amino acid residues of one or more of the biological motifs contained within a 282P1G3 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., EpimabixTMand EpimerTM, Brown University, and BIMAS).
Motif bearing subsequences of all 282P1G3 variant proteins are set forth and identified in Tables VIII-XXI and XXII-XLIX.
Table V sets forth several frequently occurring motifs based on pfam searches on the INTERNET. The columns of Table V list (1) motif name abbreviation, (2) percent identity found amongst the different member of the motif family, (3) motif name or description and (4) most common function; location information is included if the motif is relevant for location.
Polypeptides comprising one or more of the 282P1G3 motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 282P1G3 motifs discussed above are associated with growth dysregulation and because 282P1G3 is overexpressed in certain cancers (See, e.g., Table I). Casein kinase 11, 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)).
In another embodiment, proteins of the invention comprise one or more of the immunoreactive epitopes identified in accordance with art-accepted methods, such as the peptides set forth in Tables VIII-XXI and XXII-XLIX. CTL epitopes can be determined using specific algorithms to identify peptides within a 282P1G3 protein that are capable of optionally binding to specified HLA alleles (e.g., Table IV; EpimatrixTM and EpimerTM, Brown University, and BIMAS). 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.
Also known in the art are principles for creating analogs of such epitopes in order to modulate immunogenicity. For example, one begins with an epitope that bears a CTL or HTL motif (see, e.g., the HLA Class I and HLA Class II motifs/supermotifs of Table IV): The epitope is analoged by substituting out an amino acid at one of the specified positions, and replacing it with another amino acid specified for that position. For example, on the basis of residues defined in Table IV, one can substitute out a deleterious residue in favor of any other residue, such as a preferred residue; substitute a less-preferred residue with a preferred residue; or substitute an originally-occurring preferred residue with another preferred residue. Substitutions can occur at primary anchor positions or at other positions in a peptide; see, e.g., Table IV.
A variety of references reflect the art regarding the identification and generation of epitopes in a protein of interest as well as analogs thereof. See, for example, WO 97/33602 to Chesnut et al.; Sette, Immunogenetics 1999 50(3-4): 201-212; Selle et al., J. Immunol. 2001 166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20; Kondo et al., Immunogenetics 1997 45(4): 249-258; Sidney et al., J. Immunol. 1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75 (1994)); Kast et al., 1994 152(8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3): 266-278; Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al., J. Immunol. 1991 147(8): 2663-2669; Alexander et al., Immunity 1994 1(9): 751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92.
Related embodiments of the invention include polypeptides comprising combinations of the different motifs set forth in Table VI, and/or, one or more of the predicted CTL epitopes of Tables VIII-XXI and XXII-XLIX, and/or, one or more of the predicted HTL epitopes of Tables XLVI-XLIX, and/or, one or more of the T cell binding motifs known in the art. Preferred embodiments contain no insertions, deletions or substitutions either within the motifs or within the intervening sequences of the polypeptides. In addition, embodiments which include a number of either N-terminal and/or C-terminal amino acid residues on either side of these motifs may be desirable (to, for example, include a greater portion of the polypeptide architecture in which the motif is located). Typically, the number of N-terminal and/or C-terminal amino acid residues on either side of a motif is between about 1 to about 100 amino acid residues, preferably 5 to about 50 amino acid residues.
282P1G3-related proteins are embodied in many forms, preferably in isolated form. A purified 282P1G3 protein molecule will be substantially free of other proteins or molecules that impair the binding of 282P1G3 to antibody, T cell or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a 282P1G3-related proteins include purified 282P1G3-related proteins and functional, soluble 282P1G3-related proteins. In one embodiment, a functional, soluble 282P1G3 protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.
The invention also provides 282P1G3 proteins comprising biologically active fragments of a 282P1G3 amino acid sequence shown in FIG. 2 or FIG. 3. Such proteins exhibit properties of the starting 282P1G3 protein, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the starting 282P1G3 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.
282P1G3-related polypeptides that contain particularly interesting structures can be predicted and/or identified using various analytical techniques well known in the art, including, for example, the methods of Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis, or based on immunogenicity. Fragments that contain such structures are particularly useful in generating subunit-specific anti-282P1G3 antibodies or T cells or in identifying cellular factors that bind to 282P1G3. 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.
CTL epitopes can be determined using specific algorithms to identify peptides within a 282P1G3 protein that are capable of optimally binding to specified HLA alleles (e.g., by using the SYFPEITHI site at the World Wide Web; the listings in Table IV(A)-(E); EpimatrixTM and EpimerTM, Brown University, and BIMAS. Illustrating this, peptide epitopes from 282P1G3 that are presented in the context of human MHC Class I molecules, e.g., HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (see, e.g., Tables VIII-XXI, XXII-XLIX). Specifically, the complete amino acid sequence of the 282P1G3 protein and relevant portions of other variants, i.e., for HLA Class I predictions 9 flanking residues on either side of a point mutation or exon junction, and for HLA Class II predictions 14 flanking residues on either side of a point mutation or exon junction corresponding to that variant, were entered into the HLA Peptide Motif Search algorithm found in the Bioinformatics and Molecular Analysis Section (BIMAS) web site on the World Wide Web; in addition to the site SYFPEITHI.
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 282P1G3 predicted binding peptides are shown in Tables VIII-XXI and XXII-XLIX herein. In Tables VIII-XXI and XXII-XLVII, selected candidates, 9-mers and 10-mers, for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score. In Tables XLVI-XLIX, selected candidates, 15-mers, for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score. The binding score corresponds to the estimated half time of dissociation of complexes containing the peptide at 37° 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.
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.
It is to be appreciated that every epitope predicted by the BIMAS site, EpimerTM and EpimatrixTM 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 for SYFPEITHI, or BIMAS) are to be “applied” to a 282P1G3 protein in accordance with the invention. As used in this context “applied” means that a 282P1G3 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 282P1G3 protein of 8, 9, 10, or 11 amino acid residues that bears an HLA Class I motif, or a subsequence of 9 or more amino acid residues that bear an HLA Class II motif are within the scope of the invention.
III.B.) Expression of 282P1G3-Related Proteins
In an embodiment described in the examples that follow, 282P1G3 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 282P1G3 with a C-terminal 6×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 282P1G3 protein in transfected cells. The secreted HIS-tagged 282P1G3 in the culture media can be purified, e.g., using a nickel column using standard techniques.
III.C.) Modifications of 282P1G3-Related Proteins
Modifications of 282P1G3-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 282P1G3 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of a 282P1G3 protein. Another type of covalent modification of a 282P1G3 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 282P1G3 comprises linking a 282P1G3 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.
The 282P1G3-related proteins of the present invention can also be modified to form a chimeric molecule comprising 282P1G3 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 282P1G3 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 282P1G3. A chimeric molecule can comprise a fusion of a 282P1G3-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 282P1G3 protein. In an alternative embodiment, the chimeric molecule can comprise a fusion of a 282P1G3-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 282P1G3 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.
II.D.) Uses of 282P1G3-Related Proteins
The proteins of the invention have a number of different specific uses. As 282P1G3 is highly expressed in prostate and other cancers, 282P1G3-related proteins are used in methods that assess the status of 282P1G3 gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of a 282P1G3 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 282P1G3-related proteins comprising the amino acid residues of one or more of the biological motifs contained within a 282P1G3 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, 282P1G3-related proteins that contain the amino acid residues of one or more of the biological motifs in a 282P1G3 protein are used to screen for factors that interact with that region of 282P1G3.
282P1G3 protein fragments/subsequences are particularly useful in generating and characterizing domain-specific antibodies (e.g., antibodies recognizing an extracellular or intracellular epitope of a 282P1G3 protein), for identifying agents or cellular factors that bind to 282P1G3 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.
Proteins encoded by the 282P1G3 genes, or by analogs, homologs or fragments thereof, have a variety of uses, including but not limited to generating antibodies and in methods for identifying ligands and other agents and cellular constituents that bind to a 282P1G3 gene product. Antibodies raised against a 282P1G3 protein or fragment thereof are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of 282P1G3 protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers. 282P1G3-related nucleic acids or proteins are also used in generating HTL or CTL responses.
Various immunological assays useful for the detection of 282P1G3 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 282P1G3-expressing cells (e.g., in radioscintigraphic imaging methods). 282P1G3 proteins are also particularly useful in generating cancer vaccines, as further described herein.
IV.) 282P1G3 Antibodies
Another aspect of the invention provides antibodies that bind to 282P1G3-related proteins. Preferred antibodies specifically bind to a 282P1G3-related protein and do not bind (or bind weakly) to peptides or proteins that are not 282P1G3-related proteins under physiological conditions. In this context, examples of physiological conditions include: 1) phosphate buffered saline; 2) Tris-buffered saline containing 25 mM Tris and 150 mM NaCl; or normal saline (0.9% NaCl); 4) animal serum such as human serum; or, 5) a combination of any of 1) through 4); these reactions preferably taking place at pH 7.5, alternatively in a range of pH 7.0 to 8.0, or alternatively in a range of pH 6.5 to 8.5; also, these reactions taking place at a temperature between 4° C. to 37° C. For example, antibodies that bind 282P1G3 can bind 282P1G3-related proteins such as the homologs or analogs thereof.
282P1G3 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 282P1G3 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 282P1G3 is involved, such as advanced or metastatic prostate cancers.
The invention also provides various immunological assays useful for the detection and quantification of 282P1G3 and mutant 282P1G3-related proteins. Such assays can comprise one or more 282P1G3 antibodies capable of recognizing and binding a 282P1G3-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.
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.
In addition, immunological imaging methods capable of detecting prostate cancer and other cancers expressing 282P1G3 are also provided by the invention, including but not limited to radioscintigraphic imaging methods using labeled 282P1G3 antibodies. Such assays are clinically useful in the detection, monitoring, and prognosis of 282P1G3 expressing cancers such as prostate cancer.
282P1G3 antibodies are also used in methods for purifying a 282P1G3-related protein and for isolating 282P1G3 homologues and related molecules. For example, a method of purifying a 282P1G3-related protein comprises incubating a 282P1G3 antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a 282P1G3-related protein under conditions that permit the 282P1G3 antibody to bind to the 282P1G3-related protein; washing the solid matrix to eliminate impurities; and eluting the 282P1G3-related protein from the coupled antibody. Other uses of 282P1G3 antibodies in accordance with the invention include generating anti-idiotypic antibodies that mimic a 282P1G3 protein.
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 282P1G3-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 282P1G3 can also be used, such as a 282P1G3 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 282P1G3-related protein is synthesized and used as an immunogen.
In addition, naked DNA immunization techniques known in the art are used (with or without purified 282P1G3-related protein or 282P1G3 expressing cells) to generate an immune response to the encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).
The amino acid sequence of a 282P1G3 protein as shown in FIG. 2 or FIG. 3 can be analyzed to select specific regions of the 282P1G3 protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of a 282P1G3 amino acid sequence are used to identify hydrophilic regions in the 282P1G3 structure. Regions of a 282P1G3 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-Doolitle, 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 282P1G3 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 282P1G3 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.
282P1G3 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 282P1G3-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.
The antibodies or fragments of the invention can also be produced, by recombinant means. Regions that bind specifically to the desired regions of a 282P1G3 protein can also be produced in the context of chimeric or complementarity-determining region (CDR) grafted antibodies of multiple species origin. Humanized or human 282P1G3 antibodies can also be produced, and are preferred for use in therapeutic contexts. Methods for humanizing murine and other non-human antibodies, by substituting one or more of the non-human antibody CDRs for corresponding human antibody sequences, are well known (see for example, Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen et al., 1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151: 2296.
Methods for producing fully human monoclonal antibodies include phage display and transgenic methods (for review, see Vaughan et al., 1998, Nature Biotechnology 16: 535-539). Fully human 282P1G3 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 282P1G3 monoclonal antibodies can also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application WO98/24893, Kucherlapati and Jakobovits et al., published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614; U.S. Pat. No. 6,162,963 issued 19 Dec. 2000; U.S. Pat. No. 6,150,584 issued 12 Nov. 2000; and, U.S. Pat. No. 6,114,598 issued 5 Sep. 2000). This method avoids the in vitro manipulation required with phage display technology and efficiently produces high affinity authentic human antibodies.
Reactivity of 282P1G3 antibodies with a 282P1G3-related protein can be established by a number of well known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses using, as appropriate, 282P1G3-related proteins, 282P1G3-expressing cells or extracts thereof. A 282P1G3 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 282P1G3 epitopes are generated using methods generally known in the art. Homodimeric antibodies can also be generated by cross-linking techniques known in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).
V.) 282P1G3 Cellular Immune Responses
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.
A complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A. and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev. Immunol. 11:403, 1993). Through the study of single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues that correspond to motifs required for specific binding to HLA antigen molecules have been identified and are set forth in Table IV (see also, e.g., Southwood, et al., J. Immunol. 160:3363, 1998; Rammensee, et al., Immunogenetics 41:178, 1995; Rammensee et al., SYFPEITHI, access via the World Wide Web; Sette, A. and Sidney, J. Cuff. 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; Selte, A. and Sidney, J. Immunogenetics 1999 November; 50(3-4):201-12, Review).
Furthermore, x-ray crystallographic analyses of HLA-peptide complexes have revealed pockets within the peptide binding cleft/groove of HLA molecules which accommodate, in an allele-specific mode, residues borne by peptide ligands; these residues in turn determine the HLA binding capacity of the peptides in which they are present. (See, e.g., Madden, D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203, 1996; Fremont et al., Immunity 8:305, 1998; Stern et al., Structure 2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H. et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci. USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M. L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927, 1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science 257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D. C., J. Mol. Biol. 219:277, 1991.)
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).
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.
Various strategies can be utilized to evaluate cellular immunogenicity, including:
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 51Cr-release assay involving peptide sensitized target cells.
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 51 Cr-release assay involving peptide sensitized target cells and target cells expressing endogenously generated antigen.
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 51Cr release involving peptide-sensitized targets, T cell proliferation, or lymphokine release.
VI.) 282P1G3 Transgenic Animals
Nucleic acids that encode a 282P1G3-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 282P1G3 can be used to clone genomic DNA that encodes 282P1G3. The cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode 282P1G3. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Pat. No. 4,736,866 issued 12 Apr. 1988, and U.S. Pat. No. 4,870,009 issued 26 Sep. 1989. Typically, particular cells would be targeted for 282P1G3 transgene incorporation with tissue-specific enhancers.
Transgenic animals that include a copy of a transgene encoding 282P1G3 can be used to examine the effect of increased expression of DNA that encodes 282P1G3. 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.
Alternatively, non-human homologues of 282P1G3 can be used to construct a 282P1G3 “knock out” animal that has a defective or altered gene encoding 282P1G3 as a result of homologous recombination between the endogenous gene encoding 282P1G3 and altered genomic DNA encoding 282P1G3 introduced into an embryonic cell of the animal. For example, cDNA that encodes 282P1G3 can be used to clone genomic DNA encoding 282P1G3 in accordance with established techniques. A portion of the genomic DNA encoding 282P1G3 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 282P1G3 polypeptide.
VII.) Methods for the Detection of 282P1G3
Another aspect of the present invention relates to methods for detecting 282P1G3 polynucleotides and 282P1G3-related proteins, as well as methods for identifying a cell that expresses 282P1G3. The expression profile of 282P1G3 makes it a diagnostic marker for metastasized disease. Accordingly, the status of 282P1G3 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 282P1G3 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.
More particularly, the invention provides assays for the detection of 282P1G3 polynucleotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, semen, cell preparations, and the like. Detectable 282P1G3 polynucleotides include, for example, a 282P1G3 gene or fragment thereof, 282P1G3 mRNA, alternative splice variant 282P1G3 mRNAs, and recombinant DNA or RNA molecules that contain a 282P1G3 polynucleotide. A number of methods for amplifying and/or detecting the presence of 282P1G3 polynucleotides are well known in the art and can be employed in the practice of this aspect of the invention.
In one embodiment, a method for detecting a 282P1G3 mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using a 282P1G3 polynucleotides as sense and antisense primers to amplify 282P1G3 cDNAs therein; and detecting the presence of the amplified 282P1G3 cDNA. Optionally, the sequence of the amplified 282P1G3 cDNA can be determined.
In another embodiment, a method of detecting a 282P1G3 gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using 282P1G3 polynucleotides as sense and antisense primers; and detecting the presence of the amplified 282P1G3 gene. Any number of appropriate sense and antisense probe combinations can be designed from a 282P1G3 nucleotide sequence (see, e.g., FIG. 2) and used for this purpose.
The invention also provides assays for detecting the presence of a 282P1G3 protein in a tissue or other biological sample such as serum, semen, bone, prostate, urine, cell preparations, and the like. Methods for detecting a 282P1G3-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 282P1G3-related protein in a biological sample comprises first contacting the sample with a 282P1G3 antibody, a 282P1G3-reactive fragment thereof, or a recombinant protein containing an antigen-binding region of a 282P1G3 antibody; and then detecting the binding of 282P1G3-related protein in the sample.
Methods for identifying a cell that expresses 282P1G3 are also within the scope of the invention. In one embodiment, an assay for identifying a cell that expresses a 282P1G3 gene comprises detecting the presence of 282P1G3 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 282P1G3 riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 282P1G3, 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 282P1G3 gene comprises detecting the presence of 282P1G3-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 282P1G3-related proteins and cells that express 282P1G3-related proteins.
282P1G3 expression analysis is also useful as a tool for identifying and evaluating agents that modulate 282P1G3 gene expression. For example, 282P1G3 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 282P1G3 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 282P1G3 expression by RT-PCR, nucleic acid hybridization or antibody binding.
VII.) Methods for Monitoring the Status of 282P1G3-Related Genes and Their Products
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 282P1G3 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 282P1G3 in a biological sample of interest can be compared, for example, to the status of 282P1G3 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 282P1G3 in the biological sample (as compared to the normal sample) provides evidence of dysregulated cellular growth. In addition to using a biological sample that is not affected by a pathology as a normal sample, one can also use a predetermined normative value such as a predetermined normal level of mRNA expression (see, e.g., Grever et al., J. Comp. Neurol. 1996 Dec. 9; 376(2): 306-14 and U.S. Pat. No. 5,837,501) to compare 282P1G3 status in a sample.
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 282P1G3 expressing cells) as well as the level, and biological activity of expressed gene products (such as 282P1G3 mRNA, polynucleotides and polypeptides). Typically, an alteration in the status of 282P1G3 comprises a change in the location of 282P1G3 and/or 282P1G3 expressing cells and/or an increase in 282P1G3 mRNA and/or protein expression.
282P1G3 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 282P1G3 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 282P1G3 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 282P1G3 gene), Northern analysis and/or PCR analysis of 282P1G3 mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of 282P1G3 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 282P1G3 proteins and/or associations of 282P1G3 proteins with polypeptide binding partners). Detectable 282P1G3 polynucleotides include, for example, a 282P1G3 gene or fragment thereof, 282P1G3 mRNA, alternative splice variants, 282P1G3 mRNAs, and recombinant DNA or RNA molecules containing a 282P1G3 polynucleotide.
The expression profile of 282P1G3 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 282P1G3 provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The invention provides methods and assays for determining 282P1G3 status and diagnosing cancers that express 282P1G3, such as cancers of the tissues listed in Table I. For example, because 282P1G3 mRNA is so highly expressed in prostate and other cancers relative to normal prostate tissue, assays that evaluate the levels of 282P1G3 mRNA transcripts or proteins in a biological sample can be used to diagnose a disease associated with 282P1G3 dysregulation, and can provide prognostic information useful in defining appropriate therapeutic options.
The expression status of 282P1G3 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 282P1G3 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.
As described above, the status of 282P1G3 in a biological sample can be examined by a number of well-known procedures in the art. For example, the status of 282P1G3 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 282P1G3 expressing cells (e.g. those that express 282P1G3 mRNAs or proteins). This examination can provide evidence of dysregulated cellular growth, for example, when 282P1G3-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 282P1G3 in a biological sample are often associated with dysregulated cellular growth. Specifically, one indicator of dysregulated cellular growth is the metastases of cancer cells from an organ of origin (such as the prostate) to a different area of the body (such as a lymph node). In this context, evidence of dysregulated cellular growth is important for example because occult lymph node metastases can be detected in a substantial proportion of patients with prostate cancer, and such metastases are associated with known predictors of disease progression (see, e.g., Murphy et al., Prostate 42(4): 315-317 (2000); Su et al., Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al., J Urol 1995 August 154(2 Pt 1):474-8).
In one aspect, the invention provides methods for monitoring 282P1G3 gene products by determining the status of 282P1G3 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 282P1G3 gene products in a corresponding normal sample. The presence of aberrant 282P1G3 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.
In another aspect, the invention provides assays useful in determining the presence of cancer in an individual, comprising detecting a significant increase in 282P1G3 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 282P1G3 mRNA can, for example, be evaluated in tissues including but not limited to those listed in Table I. The presence of significant 282P1G3 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 282P1G3 mRNA or express it at lower levels.
In a related embodiment, 282P1G3 status is determined at the protein level rather than at the nucleic acid level. For example, such a method comprises determining the level of 282P1G3 protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 282P1G3 expressed in a corresponding normal sample. In one embodiment, the presence of 282P1G3 protein is evaluated, for example, using immunohistochemical methods. 282P1G3 antibodies or binding partners capable of detecting 282P1G3 protein expression are used in a variety of assay formats well known in the art for this purpose.
In a further embodiment, one can evaluate the status of 282P1G3 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 282P1G3 may be indicative of the presence or promotion of a tumor. Such assays therefore have diagnostic and predictive value where a mutation in 282P1G3 indicates a potential loss of function or increase in tumor growth.
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 282P1G3 gene products are observed by the Northern, Southern, Western, PCR and DNA sequencing protocols discussed herein. In addition, other methods for observing perturbations in nucleotide and amino acid sequences such as single strand conformation polymorphism analysis are well known in the art (see, e.g., U.S. Pat. No. 5,382,510 issued 7 Sep. 1999, and U.S. Pat. No. 5,952,170 issued 17 Jan. 1995).
Additionally, one can examine the methylation status of a 282P1G3 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.
Gene amplification is an additional method for assessing the status of 282P1G3. 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.
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 282P1G3 expression. The presence of RT-PCR amplifiable 282P1G3 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).
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 282P1G3 mRNA or 282P1G3 protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of 282P1G3 mRNA expression correlates to the degree of susceptibility. In a specific embodiment, the presence of 282P1G3 in prostate or other tissue is examined, with the presence of 282P1G3 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 282P1G3 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 282P1G3 gene products in the sample is an indication of cancer susceptibility (or the emergence or existence of a tumor).
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 282P1G3 mRNA or 282P1G3 protein expressed by tumor cells, comparing the level so determined to the level of 282P1G3 mRNA or 282P1G3 protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of 282P1G3 mRNA or 282P1G3 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 282P1G3 is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors. Another embodiment is the evaluation of the integrity of 282P1G3 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.
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 282P1G3 mRNA or 282P1G3 protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of 282P1G3 mRNA or 282P1G3 protein expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of 282P1G3 mRNA or 282P1G3 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 282P1G3 expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer. Also, one can evaluate the integrity 282P1G3 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.
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 282P1G3 gene and 282P1G3 gene products (or perturbations in 282P1G3 gene and 282P1G3 gene products) and a factor that is associated with malignancy, as a means for diagnosing and prognosticating the status of a tissue sample. A wide variety of factors associated with malignancy can be utilized, such as the expression of genes associated with malignancy (e.g. PSA, PSCA and PSM expression for prostate cancer etc.) as well as gross cytological observations (see, e.g., Bocking et al., 1984, Anal. Quant. Cytol. 6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al., 1998, Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods for observing a coincidence between the expression of 282P1G3 gene and 282P1G3 gene products (or perturbations in 282P1G3 gene and 282P1G3 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.
In one embodiment, methods for observing a coincidence between the expression of 282P1G3 gene and 282P1G3 gene products (or perturbations in 282P1G3 gene and 282P1G3 gene products) and another factor associated with malignancy entails detecting the overexpression of 282P1G3 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 282P1G3 mRNA or protein and PSA mRNA or protein overexpression (or PSCA or PSM expression). In a specific embodiment, the expression of 282P1G3 and PSA mRNA in prostate tissue is examined, where the coincidence of 282P1G3 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.
Methods for detecting and quantifying the expression of 282P1G3 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 282P1G3 mRNA include in situ hybridization using labeled 282P1G3 riboprobes, Northern blot and related techniques using 282P1G3 polynucleotide probes, RT-PCR analysis using primers specific for 282P1G3, 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 282P1G3 mRNA expression. Any number of primers capable of amplifying 282P1G3 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 282P1G3 protein can be used in an immunohistochemical assay of biopsied tissue.
IX.) Identification of Molecules that Interact with 282P1G3
The 282P1G3 protein and nucleic acid sequences disclosed herein allow a skilled artisan to identify proteins, small molecules and other agents that interact with 282P1G3, as well as pathways activated by 282P1G3 via any one of a variety of art accepted protocols. For example, one can utilize one of the so-called interaction trap systems (also referred to as the “two-hybrid assay”). In such systems, molecules interact and reconstitute a transcription factor which directs expression of a reporter gene, whereupon the expression of the reporter gene is assayed. Other systems identify protein-protein interactions in vivo through reconstitution of a eukaryotic transcriptional activator, see, e.g., U.S. Pat. No. 5,955,280 issued 21 Sep. 1999, U.S. Pat. No. 5,925,523 issued 20 Jul. 1999, U.S. Pat. No. 5,846,722 issued 8 Dec. 1998 and U.S. Pat. No. 6,004,746 issued 21 Dec. 1999. Algorithms are also available in the art for genome-based predictions of protein function (see, e.g., Marcotte, et al., Nature 402: 4 Nov. 1999, 83-86).
Alternatively one can screen peptide libraries to identify molecules that interact with 282P1G3 protein sequences. In such methods, peptides that bind to 282P1G3 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 282P1G3 protein(s).
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 282P1G3 protein sequences are disclosed for example in U.S. Pat. No. 5,723,286 issued 3 Mar. 1998 and U.S. Pat. No. 5,733,731 issued 31 Mar. 1998.
Alternatively, cell lines that express 282P1G3 are used to identify protein-protein interactions mediated by 282P1G3. Such interactions can be examined using immunoprecipitation techniques (see, e.g., Hamilton B. J., et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51). 282P1G3 protein can be immunoprecipitated from 282P1G3-expressing cell lines using anti-282P1G3 antibodies. Alternatively, antibodies against His-tag can be used in a cell line engineered to express fusions of 282P1G3 and a His-tag (vectors mentioned above). The immunoprecipitated complex can be examined for protein association by procedures such as Western blotting, 35S-methionine labeling of proteins, protein microsequencing, silver staining and two-dimensional gel electrophoresis.
Small molecules and ligands that interact with 282P1G3 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 282P1G3'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 282P1G3-related ion channel, protein pump, or cell communication functions are identified and used to treat patients that have a cancer that expresses 282P1G3 (see, e.g., Hille, B., Ionic Channels of Excitable Membranes 2nd Ed., Sinauer Assoc., Sunderland, Mass., 1992). Moreover, ligands that regulate 282P1G3 function can be identified based on their ability to bind 282P1G3 and activate a reporter construct. Typical methods are discussed for example in U.S. Pat. No. 5,928,868 issued 27 Jul. 1999, and include methods for forming hybrid ligands in which at least one ligand is a small molecule. In an illustrative embodiment, cells engineered to express a fusion protein of 282P1G3 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 282P1G3.
An embodiment of this invention comprises a method of screening for a molecule that interacts with a 282P1G3 amino acid sequence shown in FIG. 2 or FIG. 3, comprising the steps of contacting a population of molecules with a 282P1G3 amino acid sequence, allowing the population of molecules and the 282P1G3 amino acid sequence to interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the 282P1G3 amino acid sequence, and then separating molecules that do not interact with the 282P1G3 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 282P1G3 amino acid sequence. The identified molecule can be used to modulate a function performed by 282P1G3. In a preferred embodiment, the 282P1G3 amino acid sequence is contacted with a library of peptides.
X)) Therapeutic Methods and Compositions
The identification of 282P1G3 as a protein that is normally expressed in a restricted set of tissues, but which is also expressed in cancers such as those listed in Table I, opens a number of therapeutic approaches to the treatment of such cancers.
Of note, targeted antitumor therapies have been useful even when the targeted protein is expressed on normal tissues, even vital normal organ tissues. A vital organ is one that is necessary to sustain life, such as the heart or colon. A non-vital organ is one that can be removed whereupon the individual is still able to survive. Examples of non-vital organs are ovary, breast, and prostate.
For example, Herceptin®) is an FDA approved pharmaceutical that has as its active ingredient an antibody which is immunoreactive with the protein variously known as HER2, HER2/neu, and erb-b-2. It is marketed by Genentech and has been a commercially successful antitumor agent. Herceptin sales reached almost $400 million in 2002. Herceptin is a treatment for HER2 positive metastatic breast cancer. However, the expression of HER2 is not limited to such tumors. The same protein is expressed in a number of normal tissues. In particular, it is known that HER2/neu is present in normal kidney and heart, thus these tissues are present in all human recipients of Herceptin. The presence of HER2/neu in normal kidney is also confirmed by Latif, Z., et al., B.J.U. International (2002) 89:5-9. As shown in this article (which evaluated whether renal cell carcinoma should be a preferred indication for anti-HER2 antibodies such as Herceptin) both protein and mRNA are produced in benign renal tissues. Notably, HER2/neu protein was strongly overexpressed in benign renal tissue.
Despite the fact that HER2/neu is expressed in such vital tissues as heart and kidney, Herceptin is a very useful, FDA approved, and commercially successful drug. The effect of Herceptin on cardiac tissue, i.e., “cardiotoxicity,” has merely been a side effect to treatment. When patients were treated with Herceptin alone, significant cardiotoxicity occurred in a very low percentage of patients.
Of particular note, although kidney tissue is indicated to exhibit normal expression, possibly even higher expression than cardiac tissue, kidney has no appreciable Herceptin side effect whatsoever. Moreover, of the diverse array of normal tissues in which HER2 is expressed, there is very little occurrence of any side effect. Only cardiac tissue has manifested any appreciable side effect at all. A tissue such as kidney, where HER2/neu expression is especially notable, has not been the basis for any side effect.
Furthermore, favorable therapeutic effects have been found for antitumor therapies that target epidermal growth factor receptor (EGFR). EGFR is also expressed in numerous normal tissues. There have been very limited side effects in normal tissues following use of anti-EGFR therapeutics.
Thus, expression of a target protein in normal tissue, even vital normal tissue, does not defeat the utility of a targeting agent for the protein as a therapeutic for certain tumors in which the protein is also overexpressed.
Accordingly, therapeutic approaches that inhibit the activity of a 282P1G3 protein are useful for patients suffering from a cancer that expresses 282P1G3. These therapeutic approaches generally fall into two classes. One class comprises various methods for inhibiting the binding or association of a 282P1G3 protein with its binding partner or with other proteins. Another class comprises a variety of methods for inhibiting the transcription of a 282P1G3 gene or translation of 282P1G3 mRNA.
X.A.) Anti-Cancer Vaccines
The invention provides cancer vaccines comprising a 282P1G3-related protein or 282P1G3-related nucleic acid. In view of the expression of 282P1G3, cancer vaccines prevent and/or treat 282P1G3-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).
Such methods can be readily practiced by employing a 282P1G3-related protein, or a 282P1G3-encoding nucleic acid molecule and recombinant vectors capable of expressing and presenting the 282P1G3 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., Heryin et al., Ann Med 1999 Feb. 31(1):66-78; Maruyama et al., Cancer Immunol Immunother 2000 June 49(3):123-32) Briefly, such methods of generating an immune response (e.g. humoral and/or cell-mediated) in a mammal, comprise the steps of: exposing the mammal's immune system to an immunoreactive epitope (e.g. an epitope present in a 282P1G3 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 282P1G3 immunogen contains a biological motif, see e.g., Tables VIII-XXI and XXII-XLIX, or a peptide of a size range from 282P1G3 indicated in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.
The entire 282P1G3 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 ofviral 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.
In patients with 282P1G3-associated cancer, the vaccine compositions of the inventon 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.
Cellular Vaccines:
CTL epitopes can be determined using specific algorithms to identify peptides within 282P1G3 protein that bind corresponding HLA alleles (see e.g., Table IV; EpimerTM and EpimatrixTM, Brown University, and BIMAS; SYFPEITHI on the World Wide Web. In a preferred embodiment, a 282P1G3 immunogen contains one or more amino acid sequences identified using techniques well known in the art, such as the sequences shown in Tables VIII-XXI and XXII-XLIX or a peptide of 8, 9, 10 or 11 amino acids specified by an HLA Class I motif/supermotif (e.g., Table IV (A), Table IV (D), or Table IV (E)) and/or a peptide of at least 9 amino acids that comprises an HLA Class II motif/supermotif (e.g., Table IV (B) or Table IV (C)). As is appreciated in the art, the HLA Class I binding groove is essentially closed ended so that peptides of only a particular size range can fit into the groove and be bound, generally HLA Class I epitopes are 8, 9, 10, or 11 amino acids long. In contrast, the HLA Class II binding groove is essentially open ended; therefore a peptide of about 9 or more amino acids can be bound by an HLA Class II molecule. Due to the binding groove differences between HLA Class I and 11, 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, Le., 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.
Antibody-Based Vaccines
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 282P1G3 protein) so that an immune response is generated. A typical embodiment consists of a method for generating an immune response to 282P1G3 in a host, by contacting the host with a sufficient amount of at least one 282P1G3 B cell or cytotoxic T-cell epitope or analog thereof; and at least one periodic interval thereafter re-contacting the host with the 282P1G3 B cell or cytotoxic T-cell epitope or analog thereof. A specific embodiment consists of a method of generating an immune response against a 282P1G3-related protein or a man-made multiepitopic peptide comprising: administering 282P1G3 immunogen (e.g. a 282P1G3 protein or a peptide fragment thereof, a 282P1G3 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 PADRETM 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 282P1G3 immunogen by: administering in vivo to muscle or skin of the individual's body a DNA molecule that comprises a DNA sequence that encodes a 282P1G3 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 282P1G3, in order to generate a response to the target antigen.
Nucleic Acid Vaccines:
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 282P1G3. Constructs comprising DNA encoding a 282P1G3-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 282P1G3 protein/immunogen. Alternatively, a vaccine comprises a 282P1G3-related protein. Expression of the 282P1G3-related protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells that bear a 282P1G3 protein. Various prophylactic and therapeutic genetic immunization techniques known in the art can be used (for review, see information and references published at Internet address genweb.com). Nucleic acid-based delivery is described, for instance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720. Examples of DNA-based delivery technologies include “naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687).
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, lentvirus, 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 282P1G3-related protein into the patient (e.g., intramuscularly or intradermally) to induce an anti-tumor response.
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.
Thus, gene delivery systems are used to deliver a 282P1G3-related nucleic acid molecule. In one embodiment, the full-length human 282P1G3 cDNA is employed. In another embodiment, 282P1G3 nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopes are employed.
Ex Vivo Vaccines
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 282P1G3 antigen to a patient's immune system. Dendritic cells express MHC class I and 11 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 282P1G3 peptides to T cells in the context of MHC class I or 11 molecules. In one embodiment, autologous dendritic cells are pulsed with 282P1G3 peptides capable of binding to MHC class I and/or class II molecules. In another embodiment, dendritic cells are pulsed with the complete 282P1G3 protein. Yet another embodiment involves engineering the overexpression of a 282P1G3 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 282P1G3 can also be engineered to express immune modulators, such as GM-CSF, and used as immunizing agents.
X.B.) 282P1G3 as a Target for Antibody-based Therapy
282P1G3 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 282P1G3 is expressed by cancer cells of various lineages relative to corresponding normal cells, systemic administration of 282P1G3-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 282P1G3 are useful to treat 282P1G3-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.
282P1G3 antibodies can be introduced into a patient such that the antibody binds to 282P1G3 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 282P1G3, inhibition of ligand binding or signal transduction pathways, modulation of tumor cell differentiation, alteration of tumor angiogenesis factor profiles, and/or apoptosis.
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 282P1G3 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. 282P1G3), the cytotoxic agent will exert its known biological effect (i.e. cytotoxicity) on those cells.
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-282P1G3 antibody) that binds to a marker (e.g. 282P1G3) 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 282P1G3, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 282P1G3 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.
Cancer immunotherapy using anti-282P1G3 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 Y91 or I131 to anti-CD20 antibodies (e.g., ZevalinTM, IDEC Pharmaceuticals Corp. or BexxarTM, Coulter Pharmaceuticals), while others involve co-administration of antibodies and other therapeutic agents, such as HerceptinTM (trastuzumab) with paclitaxel (Genentech, Inc.). The antibodies can be conjugated to a therapeutic agent. To treat prostate cancer, for example, 282P1G3 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., MylotargTM, Wyeth-Ayerst, Madison, N.J., a recombinant humanized IgG4 kappa antibody conjugated to antitumor antibiotic calicheamicin) or a maytansinoid (e.g., taxane-based Tumor-Activated Prodrug, TAP, platform, ImmunoGen, Cambridge, Mass., also see e.g., U.S. Pat. No. 5,416,064).
Although 282P1G3 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.
Although 282P1G3 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.
Cancer patients can be evaluated for the presence and level of 282P1G3 expression, preferably using immunohistochemical assessments of tumor tissue, quantitative 282P1G3 imaging, or other techniques that reliably indicate the presence and degree of 282P1G3 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.
Anti-282P1G3 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-282P1G3 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-282P1G3 mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express 282P1G3. 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-282P1G3 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.
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 282P1G3 antigen with high affinity but exhibit low or no antigenicity in the patient.
Therapeutic methods of the invention contemplate the administration of single anti-282P1G3 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-282P1G3 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-282P1G3 mAbs are administered in their “naked” or unconjugated form, or can have a therapeutic agent(s) conjugated to them.
Anti-282P1G3 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-282P1G3 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.
Based on clinical experience with the HerceptinTM 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-282P1G3 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 282P1G3 expression in the patient, the extent of circulating shed 282P1G3 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.
Optionally, patients should be evaluated for the levels of 282P1G3 in a given sample (e.g. the levels of circulating 282P1G3 antigen and/or 282P1G3 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).
Anti-idiotypic anti-282P1G3 antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a 282P1G3-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-282P1G3 antibodies that mimic an epitope on a 282P1G3-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.
X.C.) 282P1G3 as a Target for Cellular Immune Responses
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.
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-5-glycerylcysteinlyseryl-serine (P3CSS). Moreover, an adjuvant such as a synthetic cytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotides has been found to increase CTL responses 10- to 100-fold. (see, e.g. Davila and Celis, J. Immunol. 165:539-547 (2000))
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 282P1G3 antigen, or derives at least some therapeutic benefit when the antigen was tumor-associated.
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 PADRETM (Epimmune, San Diego, Calif.) molecule (described e.g., in U.S. Pat. No. 5,736,142).
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.
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.
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 TMs to produce a vaccine that targets tumors with varying expression patterns of frequently-expressed TMs.
2.) Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an IC50 of 500 nM or less, often 200 nM or less; and for Class II an IC50 of 1000 nM or less.
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.
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.
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.
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.
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.
X.C.1. Minigene Vaccines
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.
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 282P1G3, the PADRES universal helper T cell epitope or multiple HTL epitopes from 282P1G3 (see e.g., Tables VIII-XXI and XXII to XLIX), and an endoplasmic reticulum-translocating signal sequence can be engineered. A vaccine may also comprise epitopes that are derived from other TAAs.
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.
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.
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.
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.
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.
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.
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.
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., LelF), costimulatory molecules, or for HTL responses, pan-DR binding proteins (PADRETM, 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-β) may be beneficial in certain diseases.
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.
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.
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 (51Cr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by 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.
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, 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.
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.
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.
X.C.2. Combinations of CTL Peptides with Helper Peptides
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.
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.
In certain embodiments, the T helper peptide is one that is recognized by T helper cells present in a majority of a genetically diverse population. This can be accomplished by selecting peptides that bind to many, most, or all of the HLA class II molecules. Examples of such amino acid bind many HLA Class II molecules include sequences from antigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO: 37), Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398 (DIEKKIAKMEKASSVFNWNS; SEQ ID NO: 38), and Streptococcus 18 kD protein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO: 39). Other examples include peptides bearing a DR 1-4-7 supermotif, or either of the DR3 motifs.
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., PADRETM, Epimmune, Inc., San Diego, Calif.) are designed, most preferably, to bind most HLA-DR (human HLA class II) molecules. For instance, a pan-DR-binding epitope peptide having the formula: aKXVAAWTLKAa (SEQ ID NO: 40), where “X” is either cyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanine or L-alanine, has been found to bind to most HLA-DR alleles, and to stimulate the response of T helper lymphocytes from most individuals, regardless of their HLA type. An alternative of a pan-DR binding epitope comprises all “L” natural amino acids and can be provided in the form of nucleic acids that encode the epitope.
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.
X.C.3. Combinations of CTL Peptides with T Cell Priming Agents
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 ε- and α-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 ε- and α-amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.
As another example of lipid priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S-glycerylcysteinlyseryl-serine (P3CSS) can be used to prime virus specific CTL when covalently attached to an appropriate peptide (see, e.g., Deres, et al., Nature 342:561, 1989). Peptides of the invention can be coupled to P3CSS, for example, and the lipopeptide administered to an individual to prime specifically an immune response to the target antigen. Moreover, because the induction of neutralizing antibodies can also be primed with P3CSS-conjugated epitopes, two such compositions can be combined to more effectively elicit both humoral and cell-mediated responses.
X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and/or HTL Peptides
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 ProgenipoietinTM (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.
The DC can be pulsed ex vivo with a cocktail of peptides, some of which stimulate CTL responses to 282P1G3. 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 282P1G3.
X.D. Adoptive Immunotherapy
Antigenic 282P1G3-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.
X.E. Administration of Vaccines for Therapeutic or Prophylactic Purposes
Pharmaceutical and vaccine compositions of the invention are typically used to treat and/or prevent a cancer that expresses or overexpresses 282P1G3. 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.
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 282P1G3. 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.
For therapeutic use, administration should generally begin at the first diagnosis of 282P1G3-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 282P1G3, a vaccine comprising 282P1G3-specific CTL may be more efficacious in killing tumor cells in patient with advanced disease than alternative embodiments.
It is generally important to provide an amount of the peptide epitope delivered by a mode of administration sufficient to stimulate effectively a cytotoxic T cell response; compositions which stimulate helper T cell responses can also be given in accordance with this embodiment of the invention.
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 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0 μg to about 50,000 μg 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.
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.
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 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. This is followed by boosting dosages of between about 1.0 μg to about 50,000 μg 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.
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.
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.
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.
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.
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, 17th 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 μg, generally 100-5,000 μg, 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 μg) 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 5-107 to 5×109 pfu.
For antibodies, a treatment generally involves repeated administration of the anti-282P1G3 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-282P1G3 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 282P1G3 expression in the patient, the extent of circulating shed 282P1G3 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 μg-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 m2 of body area weekly; 1-600 mg m2 of body area weekly; 225-400 mg m2 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.
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.
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 104 cells to about 106 cells, about 106 cells to about 108 cells, about 108 to about 1011 cells, or about 108 to about 5×1010 cells. A dose may also about 106 cells/m2 to about 1010 cells/m2, or about 106 cells/m2 to about 108 cells/m2.
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.
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.
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%.
For aerosol administration, immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are about 0.01%-20% by weight, preferably about 1%-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from about 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute about 0.1%-20% by weight of the composition, preferably about 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.
XI.) Diagnostic and Prognostic Embodiments of 282P1G3.
As disclosed herein, 282P1G3 polynucleotides, polypeptides, reactive cytotoxic T cells (CTL), reactive helper T cells (HTL) and anti-polypeptide antibodies are used in well known diagnostic, prognostic and therapeutic assays that examine conditions associated with dysregulated cell growth such as cancer, in particular the cancers listed in Table I (see, e.g., both its specific pattern of tissue expression as well as its overexpression in certain cancers as described for example in the Example entitled “Expression analysis of 282P1G3 in normal tissues, and patient specimens”).
282P1G3 can be analogized to a prostate associated antigen PSA, the archetypal marker that has been used by medical practitioners for years to identify and monitor the presence of prostate cancer (see, e.g., Merrill et al., J. Urol. 163(2): 503-5120 (2000); Polascik et al., J. Urol. August; 162(2):293-306 (1999) and Fortier et al., J. Nat. Cancer Inst. 91(19): 1635-1640(1999)). A variety of other diagnostic markers are also used in similar contexts including p53 and K-ras (see, e.g., Tulchinsky et al., Int J Mol Med 1999 Jul. 4(1):99-102 and Minimoto et al., Cancer Detect Prev 2000; 24(1):1-12). Therefore, this disclosure of 282P1G3 polynucleotides and polypeptides (as well as 282P1G3 polynucleotide probes and anti-282P1G3 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.
Typical embodiments of diagnostic methods which utilize the 282P1G3 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 282P1G3 polynucleotides described herein can be utilized in the same way to detect 282P1G3 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 282P1G3 polypeptides described herein can be utilized to generate antibodies for use in detecting 282P1G3 overexpression or the metastasis of prostate cells and cells of other cancers expressing this gene.
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 282P1G3 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 282P1G3-expressing cells (lymph node) is found to contain 282P1G3-expressing cells such as the 282P1G3 expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis.
Alternatively 282P1G3 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 282P1G3 or express 282P1G3 at a different level are found to express 282P1G3 or have an increased expression of 282P1G3 (see, e.g., the 282P1G3 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 282P1G3) such as PSA, PSCA etc. (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)).
The use of immunohistochemistry to identify the presence of a 282P1G3 polypeptide within a tissue section can indicate an altered state of certain cells within that tissue. It is well understood in the art that the ability of an antibody to localize to a polypeptide that is expressed in cancer cells is a way of diagnosing presence of disease, disease stage, progression and/or tumor aggressiveness. Such an antibody can also detect an altered distribution of the polypeptide within the cancer cells, as compared to corresponding non-malignant tissue.
The 282P1G3 polypeptide and immunogenic compositions are also useful in view of the phenomena of altered subcellular protein localization in disease states. Alteration of cells from normal to diseased state causes changes in cellular morphology and is often associated with changes in subcellular protein localization/distribution. For example, cell membrane proteins that are expressed in a polarized manner in normal cells can be altered in disease, resulting in distribution of the protein in a non-polar manner over the whole cell surface.
The phenomenon of altered subcellular protein localization in a disease state has been demonstrated with MUC1 and Her2 protein expression by use of immunohistochemical means. Normal epithelial cells have a typical apical distribution of MUC1, in addition to some supranuclear localization of the glycoprotein, whereas malignant lesions often demonstrate an apolar staining pattern (Diaz et al, The Breast Journal, 7; 40-45 (2001); Zhang et al, Clinical Cancer Research, 4; 2669-2676 (1998): Cao, et al, The Journal of Histochemistry and Cytochemistry, 45: 1547-1557 (1997)). In addition, normal breast epithelium is either negative for Her2 protein or exhibits only a basolateral distribution whereas malignant cells can express the protein over the whole cell surface (De Potter, et al, International Journal of Cancer, 44; 969-974 (1989): McCormick, et al, 117; 935-943 (2002)). Alternatively, distribution of the protein may be altered from a surface only localization to include diffuse cytoplasmic expression in the diseased state. Such an example can be seen with MUC1 (Diaz, et al, The Breast Journal, 7: 40-45 (2001)).
Alteration in the localization/distribution of a protein in the cell, as detected by immunohistochemical methods, can also provide valuable information concerning the favorability of certain treatment modalities. This last point is illustrated by a situation where a protein may be intracellular in normal tissue, but cell surface in malignant cells; the cell surface location makes the cells favorably amenable to antibody-based diagnostic and treatment regimens. When such an alteration of protein localization occurs for 282P1G3, the 282P1G3 protein and immune responses related thereto are very useful. Accordingly, the ability to determine whether alteration of subcellular protein localization occurred for 24P4C12 make the 282P1G3 protein and immune responses related thereto very useful. Use of the 282P1G3 compositions allows those skilled in the art to make important diagnostic and therapeutic decisions.
Immunohistochemical reagents specific to 282P1G3 are also useful to detect metastases of tumors expressing 282P1G3 when the polypeptide appears in tissues where 282P1G3 is not normally produced.
Thus, 282P1G3 polypeptides and antibodies resulting from immune responses thereto are useful in a variety of important contexts such as diagnostic, prognostic, preventative and/or therapeutic purposes known to those skilled in the art.
Just as PSA polynucleotide fragments and polynucleotide variants are employed by skilled artisans for use in methods of monitoring PSA, 282P1G3 polynucleotide fragments and polynucleotide variants are used in an analogous manner. In particular, typical PSA polynucleotides used in methods of monitoring PSA are probes or primers which consist of fragments of the PSA cDNA sequence. Illustrating this, primers used to PCR amplify a PSA polynucleotide must include less than the whole PSA sequence to function in the polymerase chain reaction. In the context of such PCR reactions, skilled artisans generally create a variety of different polynucleotide fragments that can be used as primers in order to amplify different portions of a polynucleotide of interest or to optimize amplification reactions (see, e.g., Caetano-Anolles, G. Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., Methods Mol. Biol. 98:121-154 (1998)). An additional illustration of the use of such fragments is provided in the Example entitled “Expression analysis of 282P1G3 in normal tissues, and patient specimens,” where a 282P1G3 polynucleotide fragment is used as a probe to show the expression of 282P1G3 RNAs in cancer cells. In addition, variant polynucleotide sequences are typically used as primers and probes for the corresponding mRNAs in PCR and Northern analyses (see, e.g., Sawai et al., Fetal Diagn. Ther. 1996 Nov.-Dec. 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 282P1G3 polynucleotide shown in FIG. 2 or variant thereof) under conditions of high stringency.
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. 282P1G3 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 282P1G3 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 282P1G3 polypeptide shown in FIG. 3).
As shown herein, the 282P1G3 polynucleotides and polypeptides (as well as the 282P1G3 polynucleotide probes and anti-282P1G3 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 282P1G3 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 282P1G3 polynucleotides and polypeptides (as well as the 282P1G3 polynucleotide probes and anti-282P1G3 antibodies used to identify the presence of these molecules) need to be employed to confirm a metastases of prostatic origin.
Finally, in addition to their use in diagnostic assays, the 282P1G3 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 282P1G3 gene maps (see the Example entitled “Chromosomal Mapping of 282P1G3” below). Moreover, in addition to their use in diagnostic assays, the 282P1G3-related proteins and polynucleotides disclosed herein have other utilities such as their use in the forensic analysis of tissues of unknown origin (see, e.g., Takahama K Forensic Sci Int 1996 Jun. 28; 80(1-2): 63-9).
Additionally, 282P1G3-related proteins or polynucleotides of the invention can be used to treat a pathologic condition characterized by the over-expression of 282P1G3. 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 282P1G3 antigen. Antibodies or other molecules that react with 282P1G3 can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.
XII.) Inhibition of 282P1G3 Protein Function
The invention includes various methods and compositions for inhibiting the binding of 282P1G3 to its binding partner or its association with other protein(s) as well as methods for inhibiting 282P1G3 function.
XII.A.) Inhibition of 282P1G3 with Intracellular Antibodies
In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to 282P1G3 are introduced into 282P1G3 expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti-282P1G3 antibody is expressed intracellularly, binds to 282P1G3 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).
Single chain antibodies comprise the variable domains of the heavy and light chain joined by a flexible linker polypeptide, and are expressed as a single polypeptide. Optionally, single chain antibodies are expressed as a single chain variable region fragment joined to the light chain constant region. Well-known intracellular trafficking signals are engineered into recombinant polynucleotide vectors encoding such single chain antibodies in order to target precisely the intrabody to the desired intracellular compartment. For example, intrabodies targeted to the endoplasmic reticulum (ER) are engineered to incorporate a leader peptide and, optionally, a C-terminal ER retention signal, such as the KDEL amino acid motif. Intrabodies intended to exert activity in the nucleus are engineered to include a nuclear localization signal. Lipid moieties are joined to intrabodies in order to tether the intrabody to the cytosolic side of the plasma membrane. Intrabodies can also be targeted to exert function in the cytosol. For example, cytosolic intrabodies are used to sequester factors within the cytosol, thereby preventing them from being transported to their natural cellular destination.
In one embodiment, intrabodies are used to capture 282P1G3 in the nucleus, thereby preventing its activity within the nucleus. Nuclear targeting signals are engineered into such 282P1G3 intrabodies in order to achieve the desired targeting. Such 282P1G3 intrabodies are designed to bind specifically to a particular 282P1G3 domain. In another embodiment, cytosolic intrabodies that specifically bind to a 282P1G3 protein are used to prevent 282P1G3 from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus (e.g., preventing 282P1G3 from forming transcription complexes with other factors).
In order to specifically direct the expression of such intrabodies to particular cells, the transcription of the intrabody is placed under the regulatory control of an appropriate tumor-specific promoter and/or enhancer. In order to target intrabody expression specifically to prostate, for example, the PSA promoter and/or promoter/enhancer can be utilized (See, for example, U.S. Pat. No. 5,919,652 issued 6 Jul. 1999).
XII.B.) Inhibition of 282P1G3 with Recombinant Proteins
In another approach, recombinant molecules bind to 282P1G3 and thereby inhibit 282P1G3 function. For example, these recombinant molecules prevent or inhibit 282P1G3 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 282P1G3 specific antibody molecule. In a particular embodiment, the 282P1G3 binding domain of a 282P1G3 binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two 282P1G3 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 282P1G3, whereby the dimeric fusion protein specifically binds to 282P1G3 and blocks 282P1G3 interaction with a binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking technologies.
XII.C.) Inhibition of 282P1G3 Transcription or Translation
The present invention also comprises various methods and compositions for inhibiting the transcription of the 282P1G3 gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of 282P1G3 mRNA into protein.
In one approach, a method of inhibiting the transcription of the 282P1G3 gene comprises contacting the 282P1G3 gene with a 282P1G3 antisense polynucleotide. In another approach, a method of inhibiting 282P1G3 mRNA translation comprises contacting a 282P1G3 mRNA with an antisense polynucleotide. In another approach, a 282P1G3 specific ribozyme is used to cleave a 282P1G3 message, thereby inhibiting translation. Such antisense and ribozyme based methods can also be directed to the regulatory regions of the 282P1G3 gene, such as 282P1G3 promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a 282P1G3 gene transcription factor are used to inhibit 282P1G3 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.
Other factors that inhibit the transcription of 282P1G3 by interfering with 282P1G3 transcriptional activation are also useful to treat cancers expressing 282P1G3. Similarly, factors that interfere with 282P1G3 processing are useful to treat cancers that express 282P1G3. Cancer treatment methods utilizing such factors are also within the scope of the invention.
XII.D.) General Considerations for Therapeutic Strategies
Gene transfer and gene therapy technologies can be used to deliver therapeutic polynucleotide molecules to tumor cells synthesizing 282P1G3 (i.e., antisense, ribozyme, polynucleotides encoding intrabodies and other 282P1G3 inhibitory molecules). A number of gene therapy approaches are known in the art. Recombinant vectors encoding 282P1G3 antisense polynucleotides, ribozymes, factors capable of interfering with 282P1G3 transcription, and so forth, can be delivered to target tumor cells using such gene therapy approaches.
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.
The anti-tumor activity of a particular composition (e.g., antisense, ribozyme, intrabody), or a combination of such compositions, can be evaluated using various in vitro and in vivo assay systems. In vitro assays that evaluate therapeutic activity include cell growth assays, soft agar assays and other assays indicative of tumor promoting activity, binding assays capable of determining the extent to which a therapeutic composition will inhibit the binding of 282P1G3 to a binding partner, etc.
In vivo, the effect of a 282P1G3 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.
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.
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 16th Edition, A. Osal., Ed., 1980).
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.
Dosages and administration protocols for the treatment of cancers using the foregoing methods will vary with the method and the target cancer, and will generally depend on a number of other factors appreciated in the art.
XIII.) Identification, Characterization and Use of Modulators of 282P1G3
Methods to Identify and Use Modulators
In one embodiment, screening is performed to identify modulators that induce or suppress a particular expression profile, suppress or induce specific pathways, preferably generating the associated phenotype thereby. In another embodiment, having identified differentially expressed genes important in a particular state; screens are performed to identify modulators that alter expression of individual genes, either increase or decrease. In another embodiment, screening is performed to identify modulators that alter a biological function of the expression product of a differentially expressed gene. Again, having identified the importance of a gene in a particular state, screens are performed to identify agents that bind and/or modulate the biological activity of the gene product.
In addition, screens are done for genes that are induced in response to a candidate agent. After identifying a modulator (one that suppresses a cancer expression pattern leading to a normal expression pattern, or a modulator of a cancer gene that leads to expression of the gene as in normal tissue) a screen is performed to identify genes that are specifically modulated in response to the agent. Comparing expression profiles between normal tissue and agent-treated cancer tissue reveals genes that are not expressed in normal tissue or cancer tissue, but are expressed in agent treated tissue, and vice versa. These agent-specific sequences are identified and used by methods described herein for cancer genes or proteins. In particular these sequences and the proteins they encode are used in marking or identifying agent-treated cells. In addition, antibodies are raised against the agent-induced proteins and used to target novel therapeutics to the treated cancer tissue sample.
Modulator-Related Identification and Screening Assays:
Gene Expression-Related Assays
Proteins, nucleic acids, and antibodies of the invention are used in screening assays. The cancer-associated proteins, antibodies, nucleic acids, modified proteins and cells containing these sequences are used in screening assays, such as evaluating the effect of drug candidates on a “gene expression profile,” expression profile of polypeptides or alteration of biological function. In one embodiment, the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent (e.g., Davis, G F, et al., J Biol Screen 7:69 (2002); Zlokarnik, et al., Science 279:84-8 (1998); Heid, Genome Res 6:986-94, 1996).
The cancer proteins, antibodies, nucleic acids, modified proteins and cells containing the native or modified cancer proteins or genes are used in screening assays. That is, the present invention comprises methods for screening for compositions which modulate the cancer phenotype or a physiological function of a cancer protein of the invention. This is done on a gene itself or by evaluating the effect of drug candidates on a “gene expression profile” or biological function. In one embodiment, expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring after treatment with a candidate agent, see Zlokamik, supra.
A variety of assays are executed directed to the genes and proteins of the invention. Assays are run on an individual nucleic acid or protein level. That is, having identified a particular gene as up regulated in cancer, test compounds are screened for the ability to modulate gene expression or for binding to the cancer protein of the invention. “Modulation” in this context includes an increase or a decrease in gene expression. The preferred amount of modulation will depend on the original change of the gene expression in normal versus tissue undergoing cancer, with changes of at least 10%, preferably 50%, more preferably 100-300%, and in some embodiments 300-1000% or greater. Thus, if a gene exhibits a 4-fold increase in cancer tissue compared to normal tissue, a decrease of about four-fold is often desired; similarly, a 10-fold decrease in cancer tissue compared to normal tissue a target value of a 10-fold increase in expression by the test compound is often desired. Modulators that exacerbate the type of gene expression seen in cancer are also useful, e.g., as an upregulated target in further analyses.
The amount of gene expression is monitored using nucleic acid probes and the quantification of gene expression levels, or, alternatively, a gene product itself is monitored, e.g., through the use of antibodies to the cancer protein and standard immunoassays. Proteomics and separation techniques also allow for quantification of expression.
Expression Monitoring to Identify Compounds that Modify Gene Expression
In one embodiment, gene expression monitoring, i.e., an expression profile, is monitored simultaneously for a number of entities. Such profiles will typically involve one or more of the genes of FIG. 2. In this embodiment, e.g., cancer nucleic acid probes are attached to biochips to detect and quantify cancer sequences in a particular cell. Alternatively, PCR can be used. Thus, a series, e.g., wells of a microtiter plate, can be used with dispensed primers in desired wells. A PCR reaction can then be performed and analyzed for each well.
Expression monitoring is performed to identify compounds that modify the expression of one or more cancer-associated sequences, e.g., a polynucleotide sequence set out in FIG. 2. Generally, a test modulator is added to the cells prior to analysis. Moreover, screens are also provided to identify agents that modulate cancer, modulate cancer proteins of the invention, bind to a cancer protein of the invention, or interfere with the binding of a cancer protein of the invention and an antibody or other binding partner.
In one embodiment, high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such “combinatorial chemical libraries” are then screened in one or more assays to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional “lead compounds,” as compounds for screening, or as therapeutics.
In certain embodiments, combinatorial libraries of potential modulators are screened for an ability to bind to a cancer polypeptide or to modulate activity. Conventionally, new chemical entities with useful properties are generated by identifying a chemical compound (called a “lead compound”) with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis.
As noted above, gene expression monitoring is conveniently used to test candidate modulators (e.g., protein, nucleic acid or small molecule). After the candidate agent has been added and the cells allowed to incubate for a period, the sample containing a target sequence to be analyzed is, e.g., added to a biochip.
If required, the target sequence is prepared using known techniques. For example, a sample is treated to lyse the cells, using known lysis buffers, electroporation, etc., with purification and/or amplification such as PCR performed as appropriate. For example, an in vitro transcription with labels covalently attached to the nucleotides is performed. Generally, the nucleic acids are labeled with biotin-FITC or PE, or with cy3 or cy5.
The target sequence can be labeled with, e.g., a fluorescent, a chemiluminescent, a chemical, or a radioactive signal, to provide a means of detecting the target sequence's specific binding to a probe. The label also can be an enzyme, such as alkaline phosphatase or horseradish peroxidase, which when provided with an appropriate substrate produces a product that is detected. Alternatively, the label is a labeled compound or small molecule, such as an enzyme inhibitor, that binds but is not catalyzed or altered by the enzyme. The label also can be a moiety or compound, such as, an epitope tag or biotin which specifically binds to streptavidin. For the example of biotin, the streptavidin is labeled as described above, thereby, providing a detectable signal for the bound target sequence. Unbound labeled streptavidin is typically removed prior to analysis.
As will be appreciated by those in the art, these assays can be direct hybridization assays or can comprise “sandwich assays”, which include the use of multiple probes, as is generally outlined in U.S. Pat. Nos. 5,681,702; 5,597,909; 5,545,730; 5,594,117; 5,591,584; 5,571,670; 5,580,731; 5,571,670; 5,591,584; 5,624,802; 5,635,352; 5,594,118; 5,359,100; 5,124, 246; and 5,681,697. In this embodiment, in general, the target nucleic acid is prepared as outlined above, and then added to the biochip comprising a plurality of nucleic acid probes, under conditions that allow the formation of a hybridization complex.
A variety of hybridization conditions are used in the present invention, including high, moderate and low stringency conditions as outlined above. The assays are generally run under stringency conditions which allow formation of the label probe hybridization complex only in the presence of target. Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration pH, organic solvent concentration, etc. These parameters may also be used to control non-specific binding, as is generally outlined in U.S. Pat. No. 5,681,697. Thus, it can be desirable to perform certain steps at higher stringency conditions to reduce non-specific binding.
The reactions outlined herein can be accomplished in a variety of ways. Components of the reaction can be added simultaneously, or sequentially, in different orders, with preferred embodiments outlined below. In addition, the reaction may include a variety of other reagents. These include salts, buffers, neutral proteins, e.g. albumin, detergents, etc. which can be used to facilitate optimal hybridization and detection, and/or reduce nonspecific or background interactions. Reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may also be used as appropriate, depending on the sample preparation methods and purity of the target. The assay data are analyzed to determine the expression levels of individual genes, and changes in expression levels as between states, forming a gene expression profile.
Biological Activity-Related Assays
The invention provides methods identify or screen for a compound that modulates the activity of a cancer-related gene or protein of the invention. The methods comprise adding a test compound, as defined above, to a cell comprising a cancer protein of the invention. The cells contain a recombinant nucleic acid that encodes a cancer protein of the invention. In another embodiment, a library of candidate agents is tested on a plurality of cells.
In one aspect, the assays are evaluated in the presence or absence or previous or subsequent exposure of physiological signals, e.g. hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (i.e., cell-cell contacts). In another example, the determinations are made at different stages of the cell cycle process. In this way, compounds that modulate genes or proteins of the invention are identified. Compounds with pharmacological activity are able to enhance or interfere with the activity of the cancer protein of the invention. Once identified, similar structures are evaluated to identify critical structural features of the compound.
In one embodiment, a method of modulating (e.g., inhibiting) cancer cell division is provided; the method comprises administration of a cancer modulator. In another embodiment, a method of modulating (e.g., inhibiting) cancer is provided; the method comprises administration of a cancer modulator. In a further embodiment, methods of treating cells or individuals with cancer are provided; the method comprises administration of a cancer modulator.
In one embodiment, a method for modulating the status of a cell that expresses a gene of the invention is provided. As used herein status comprises such art-accepted parameters such as growth, proliferation, survival, function, apoptosis, senescence, location, enzymatic activity, signal transduction, etc. of a cell. In one embodiment, a cancer inhibitor is an antibody as discussed above. In another embodiment, the cancer inhibitor is an antisense molecule. A variety of cell growth, proliferation, and metastasis assays are known to those of skill in the art, as described herein.
High Throughput Screening to Identify Modulators
The assays to identify suitable modulators are amenable to high throughput screening. Preferred assays thus detect enhancement or inhibition of cancer gene transcription, inhibition or enhancement of polypeptide expression, and inhibition or enhancement of polypeptide activity.
In one embodiment, modulators evaluated in high throughput screening methods are proteins, often naturally occurring proteins or fragments of naturally occurring proteins. Thus, e.g., cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, are used. In this way, libraries of proteins are made for screening in the methods of the invention. Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred. Particularly useful test compound will be directed to the class of proteins to which the target belongs, e.g., substrates for enzymes, or ligands and receptors.
Use of Soft Agar Growth and Colony Formation to Identify and Characterize Modulators
Normal cells require a solid substrate to attach and grow. When cells are transformed, they lose this phenotype and grow detached from the substrate. For example, transformed cells can grow in stirred suspension culture or suspended in semi-solid media, such as semi-solid or soft agar. The transformed cells, when transfected with tumor suppressor genes, can regenerate normal phenotype and once again require a solid substrate to attach to and grow. Soft agar growth or colony formation in assays are used to identify modulators of cancer sequences, which when expressed in host cells, inhibit abnormal cellular proliferation and transformation. A modulator reduces or eliminates the host cells' ability to grow suspended in solid or semisolid media, such as agar.
Techniques for soft agar growth or colony formation in suspension assays are described in Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd ed., 1994). See also, the methods section of Garkavtsev et al. (1996), supra.
Evaluation of Contact Inhibition and Growth Density Limitation to Identify and Characterize Modulators
Normal cells typically grow in a flat and organized pattern in cell culture until they touch other cells. When the cells touch one another, they are contact inhibited and stop growing. Transformed cells, however, are not contact inhibited and continue to grow to high densities in disorganized foci. Thus, transformed cells grow to a higher saturation density than corresponding normal cells. This is detected morphologically by the formation of a disoriented monolayer of cells or cells in foci. Alternatively, labeling index with (3H)-thymidine at saturation density is used to measure density limitation of growth, similarly an MTT or Alamar blue assay will reveal proliferation capacity of cells and the ability of modulators to affect same. See Freshney (1994), supra. Transformed cells, when transfected with tumor suppressor genes, can regenerate a normal phenotype and become contact inhibited and would grow to a lower density.
In this assay, labeling index with 3H)-thymidine at saturation density is a preferred method of measuring density limitation of growth. Transformed host cells are transfected with a cancer-associated sequence and are grown for 24 hours at saturation density in non-limiting medium conditions. The percentage of cells labeling with (3H)-thymidine is determined by incorporated cpm.
Contact independent growth is used to identify modulators of cancer sequences, which had led to abnormal cellular proliferation and transformation. A modulator reduces or eliminates contact independent growth, and returns the cells to a normal phenotype.
Evaluation of Growth Factor or Serum Dependence to Identify and Characterize Modulators
Transformed cells have lower serum dependence than their normal counterparts (see, e.g., Temin, J. Natl. Cancer Inst. 37:167-175 (1966); Eagle et al., J. Exp. Med 131:836-879 (1970)); Freshney, supra. This is in part due to release of various growth factors by the transformed cells. The degree of growth factor or serum dependence of transformed host cells can be compared with that of control. For example, growth factor or serum dependence of a cell is monitored in methods to identify and characterize compounds that modulate cancer-associated sequences of the invention.
Use of Tumor-Specific Marker Levels to Identify and Characterize Modulators
Tumor cells release an increased amount of certain factors (hereinafter “tumor specific markers”) than their normal counterparts. For example, plasminogen activator (PA) is released from human glioma at a higher level than from normal brain cells (see, e.g., Gullino, Angiogenesis, Tumor Vascularization, and Potential Interference with Tumor Growth, in Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985)). Similarly, Tumor Angiogenesis Factor (TAF) is released at a higher level in tumor cells than their normal counterparts. See, e.g., Folkman, Angiogenesis and Cancer, Sem Cancer Biol. (1992)), while bFGF is released from endothelial tumors (Ensoli, B et al.).
Various techniques which measure the release of these factors are described in Freshney (1994), supra. Also, see, Unkless et al., J. Biol. Chem. 249:4295-4305 (1974); Strickland & Beers, J. Biol. Chem. 251:5694-5702 (1976); Whur et al., Br. J. Cancer 42:305 312 (1980); Gullino, Angiogenesis, Tumor Vascularization, and Potential Interference with Tumor Growth, in Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985); Freshney, Anticancer Res. 5:111-130 (1985). For example, tumor specific marker levels are monitored in methods to identify and characterize compounds that modulate cancer-associated sequences of the invention.
Invasiveness into Matrigel to Identify and Characterize Modulators
The degree of invasiveness into Matrigel or an extracellular matrix constituent can be used as an assay to identify and characterize compounds that modulate cancer associated sequences. Tumor cells exhibit a positive correlation between malignancy and invasiveness of cells into Matrigel or some other extracellular matrix constituent. In this assay, tumorigenic cells are typically used as host cells. Expression of a tumor suppressor gene in these host cells would decrease invasiveness of the host cells. Techniques described in Cancer Res. 1999; 59:6010; Freshney (1994), supra, can be used. Briefly, the level of invasion of host cells is measured by using filters coated with Matrigel or some other extracellular matrix constituent. Penetration into the gel, or through to the distal side of the filter, is rated as invasiveness, and rated histologically by number of cells and distance moved, or by prelabeling the cells with 1251 and counting the radioactivity on the distal side of the filter or bottom of the dish. See, e.g., Freshney (1984), supra.
Evaluation of Tumor Growth In Vivo to Identify and Characterize Modulators
Effects of cancer-associated sequences on cell growth are tested in transgenic or immune-suppressed organisms. Transgenic organisms are prepared in a variety of art-accepted ways. For example, knock-out transgenic organisms, e.g., mammals such as mice, are made, in which a cancer gene is disrupted or in which a cancer gene is inserted. Knock-out transgenic mice are made by insertion of a marker gene or other heterologous gene into the endogenous cancer gene site in the mouse genome via homologous recombination. Such mice can also be made by substituting the endogenous cancer gene with a mutated version of the cancer gene, or by mutating the endogenous cancer gene, e.g., by exposure to carcinogens.
To prepare transgenic chimeric animals, e.g., mice, a DNA construct is introduced into the nuclei of embryonic stem cells. Cells containing the newly engineered genetic lesion are injected into a host mouse embryo, which is re-implanted into a recipient female. Some of these embryos develop into chimeric mice that possess germ cells some of which are derived from the mutant cell line. Therefore, by breeding the chimeric mice it is possible to obtain a new line of mice containing the introduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288 (1989)). Chimeric mice can be derived according to U.S. Pat. No. 6,365,797, issued 2 Apr. 2002; U.S. Pat. No. 6,107,540 issued 22 Aug. 2000; Hogan et al., Manipulating the Mouse Embryo: A laboratory Manual, Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington, D.C., (1987).
Alternatively, various immune-suppressed or immune-deficient host animals can be used. For example, a genetically athymic “nude” mouse (see, e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921 (1974)), a SCID mouse, a thymectornized mouse, or an irradiated mouse (see, e.g., Bradley et al., Br. J. Cancer 38:263 (1978); Selby et al., Br. J. Cancer 41:52 (1980)) can be used as a host. Transplantable tumor cells (typically about 106 cells) injected into isogenic hosts produce invasive tumors in a high proportion of cases, while normal cells of similar origin will not. In hosts which developed invasive tumors, cells expressing cancer-associated sequences are injected subcutaneously or orthotopically. Mice are then separated into groups, including control groups and treated experimental groups) e.g. treated with a modulator). After a suitable length of time, preferably 4-8 weeks, tumor growth is measured (e.g., by volume or by its two largest dimensions, or weight) and compared to the control. Tumors that have statistically significant reduction (using, e.g., Student's T test) are said to have inhibited growth.
In Vitro Assays to Identify and Characterize Modulators
Assays to identify compounds with modulating activity can be performed in vitro. For example, a cancer polypeptide is first contacted with a potential modulator and incubated for a suitable amount of time, e.g., from 0.5 to 48 hours. In one embodiment, the cancer polypeptide levels are determined in vitro by measuring the level of protein or mRNA. The level of protein is measured using immunoassays such as Western blotting, ELISA and the like with an antibody that selectively binds to the cancer polypeptide or a fragment thereof. For measurement of mRNA, amplification, e.g., using PCR, LCR, or hybridization assays, e.g., Northern hybridization, RNAse protection, dot blotting, are preferred. The level of protein or mRNA is detected using directly or indirectly labeled detection agents, e.g., fluorescently or radioactively labeled nucleic acids, radioactively or enzymatically labeled antibodies, and the like, as described herein.
Alternatively, a reporter gene system can be devised using a cancer protein promoter operably linked to a reporter gene such as luciferase, green fluorescent protein, CAT, or P-gal. The reporter construct is typically transfected into a cell. After treatment with a potential modulator, the amount of reporter gene transcription, translation, or activity is measured according to standard techniques known to those of skill in the art (Davis G F, supra; Gonzalez, J. & Negulescu, P. Curr. Opin. Biotechnol. 1998: 9:624).
As outlined above, in vitro screens are done on individual genes and gene products. That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of the expression of the gene or the gene product itself is performed.
In one embodiment, screening for modulators of expression of specific gene(s) is performed. Typically, the expression of only one or a few genes is evaluated. In another embodiment, screens are designed to first find compounds that bind to differentially expressed proteins. These compounds are then evaluated for the ability to modulate differentially expressed activity. Moreover, once initial candidate compounds are identified, variants can be further screened to better evaluate structure activity relationships.
Binding Assays to Identify and Characterize Modulators
In binding assays in accordance with the invention, a purified or isolated gene product of the invention is generally used. For example, antibodies are generated to a protein of the invention, and immunoassays are run to determine the amount and/or location of protein. Alternatively, cells comprising the cancer proteins are used in the assays.
Thus, the methods comprise combining a cancer protein of the invention and a candidate compound such as a ligand, and determining the binding of the compound to the cancer protein of the invention. Preferred embodiments utilize the human cancer protein; animal models of human disease of can also be developed and used. Also, other analogous mammalian proteins also can be used as appreciated by those of skill in the art. Moreover, in some embodiments variant or derivative cancer proteins are used.
Generally, the cancer protein of the invention, or the ligand, is non-diffusibly bound to an insoluble support. The support can, e.g., be one having isolated sample receiving areas (a microtiter plate, an array, etc.). The insoluble supports can be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports can be solid or porous and of any convenient shape.
Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharide, nylon, nitrocellulose, or TeflonTM, etc. Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. The particular manner of binding of the composition to the support is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is nondiffusable. Preferred methods of binding include the use of antibodies which do not sterically block either the ligand binding site or activation sequence when attaching the protein to the support, direct binding to “sticky” or ionic supports, chemical crosslinking, the synthesis of the protein or agent on the surface, etc. Following binding of the protein or ligand/binding agent to the support, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.
Once a cancer protein of the invention is bound to the support, and a test compound is added to the assay. Alternatively, the candidate binding agent is bound to the support and the cancer protein of the invention is then added. Binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc.
Of particular interest are assays to identify agents that have a low toxicity for human cells. A wide variety of assays can be used for this purpose, including proliferation assays, cAMP assays, labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like.
A determination of binding of the test compound (ligand, binding agent, modulator, etc.) to a cancer protein of the invention can be done in a number of ways. The test compound can be labeled, and binding determined directly, e.g., by attaching all or a portion of the cancer protein of the invention to a solid support, adding a labeled candidate compound (e.g., a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support. Various blocking and washing steps can be utilized as appropriate.
In certain embodiments, only one of the components is labeled, e.g., a protein of the invention or ligands labeled. Alternatively, more than one component is labeled with different labels, e.g., I125, for the proteins and a fluorophor for the compound. Proximity reagents, e.g., quenching or energy transfer reagents are also useful.
Competitive Binding to Identify and Characterize Modulators
In one embodiment, the binding of the “test compound” is determined by competitive binding assay with a “competitor.” The competitor is a binding moiety that binds to the target molecule (e.g., a cancer protein of the invention). Competitors include compounds such as antibodies, peptides, binding partners, ligands, etc. Under certain circumstances, the competitive binding between the test compound and the competitor displaces the test compound. In one embodiment, the test compound is labeled. Either the test compound, the competitor, or both, is added to the protein for a time sufficient to allow binding. Incubations are performed at a temperature that facilitates optimal activity, typically between four and 40° C. Incubation periods are typically optimized, e.g., to facilitate rapid high throughput screening; typically between zero and one hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.
In one embodiment, the competitor is added first, followed by the test compound. Displacement of the competitor is an indication that the test compound is binding to the cancer protein and thus is capable of binding to, and potentially modulating, the activity of the cancer protein. In this embodiment, either component can be labeled. Thus, e.g., if the competitor is labeled, the presence of label in the post-test compound wash solution indicates displacement by the test compound. Alternatively, if the test compound is labeled, the presence of the label on the support indicates displacement.
In an alternative embodiment, the test compound is added first, with incubation and washing, followed by the competitor. The absence of binding by the competitor indicates that the test compound binds to the cancer protein with higher affinity than the competitor. Thus, if the test compound is labeled, the presence of the label on the support, coupled with a lack of competitor binding, indicates that the test compound binds to and thus potentially modulates the cancer protein of the invention.
Accordingly, the competitive binding methods comprise differential screening to identity agents that are capable of modulating the activity of the cancer proteins of the invention. In this embodiment, the methods comprise combining a cancer protein and a competitor in a first sample. A second sample comprises a test compound, the cancer protein, and a competitor. The binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the cancer protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the cancer protein.
Alternatively, differential screening is used to identify drug candidates that bind to the native cancer protein, but cannot bind to modified cancer proteins. For example the structure of the cancer protein is modeled and used in rational drug design to synthesize agents that interact with that site, agents which generally do not bind to site-modified proteins. Moreover, such drug candidates that affect the activity of a native cancer protein are also identified by screening drugs for the ability to either enhance or reduce the activity of such proteins.
Positive controls and negative controls can be used in the assays. Preferably control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples occurs for a time sufficient to allow for the binding of the agent to the protein. Following incubation, samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples can be counted in a scintillation counter to determine the amount of bound compound.
A variety of other reagents can be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc. which are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., can be used. The mixture of components is added in an order that provides for the requisite binding.
Use of Polynucleotides to Down-Regulate or Inhibit a Protein of the Invention.
Polynucleotide modulators of cancer can be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand-binding molecule, as described in WO 91/04753. Suitable ligand-binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell. Alternatively, a polynucleotide modulator of cancer can be introduced into a cell containing the target nucleic acid sequence, e.g., by formation of a polynucleotide-lipid complex, as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models may also be used in screening assays as discussed above, in addition to methods of treatment.
Inhibitory and Antisense Nucleotides
In certain embodiments, the activity of a cancer-associated protein is down-regulated, or entirely inhibited, by the use of antisense polynucleotide or inhibitory small nuclear RNA (snRNA), i.e., a nucleic acid complementary to, and which can preferably hybridize specifically to, a coding mRNA nucleic acid sequence, e.g., a cancer protein of the invention, mRNA, or a subsequence thereof. Binding of the antisense polynucleotide to the mRNA reduces the translation and/or stability of the mRNA.
In the context of this invention, antisense polynucleotides can comprise naturally occurring nucleotides, or synthetic species formed from naturally occurring subunits or their close homologs. Antisense polynucleotides may also have altered sugar moieties or inter-sugar linkages. Exemplary among these are the phosphorothioate and other sulfur containing species which are known for use in the art. Analogs are comprised by this invention so long as they function effectively to hybridize with nucleotides of the invention. See, e.g., Isis Pharmaceuticals, Carlsbad, Calif.; Sequitor, Inc., Natick, Mass.
Such antisense polynucleotides can readily be synthesized using recombinant means, or can be synthesized in vitro. Equipment for such synthesis is sold by several vendors, including Applied Biosystems. The preparation of other oligonucleotides such as phosphorothioates and alkylated derivatives is also well known to those of skill in the art.
Antisense molecules as used herein include antisense or sense oligonucleotides. Sense oligonucleotides can, e.g., be employed to block transcription by binding to the anti-sense strand. The antisense and sense oligonucleotide comprise a single stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences for cancer molecules. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment generally at least about 12 nucleotides, preferably from about 12 to 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, e.g., Stein &Cohen (Cancer Res. 48:2659 (1988 and van der Krol et al. (BioTechniques 6:958 (1988)).
Ribozymes
In addition to antisense polynucleotides, ribozymes can be used to target and inhibit transcription of cancer-associated nucleotide sequences. A ribozyme is an RNA molecule that catalytically cleaves other RNA molecules. Different kinds of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, RNase P, and axhead ribozymes (see, e.g., Castanotto et al., Adv. in Pharmacology 25: 289-317 (1994) for a general review of the properties of different ribozymes).
The general features of hairpin ribozymes are described, e.g., in Hampel et al., Nucl. Acids Res. 18:299-304 (1990); European Patent Publication No. 0360257; U.S. Pat. No. 5,254,678. Methods of preparing are well known to those of skill in the art (see, e.g., WO 94/26877; Ojwang et al, Proc. Nat. Acad. Sci. USA 90:6340-6344 (1993); Yamada et al., Human Gene Therapy 1:39-45 (1994); Leavitt et al., Proc. Natl. Acad. Sci. USA 92:699-703 (1995); Leavitt et al., Human Gene Therapy 5: 1151-120 (1994); and Yamada et al., Virology 205: 121-126 (1994)).
Use of Modulators in Phenotypic Screening
In one embodiment, a test compound is administered to a population of cancer cells, which have an associated cancer expression profile. By “administration” or “contacting” herein is meant that the modulator is added to the cells in such a manner as to allow the modulator to act upon the cell, whether by uptake and intracellular action, or by action at the cell surface. In some embodiments, a nucleic acid encoding a proteinaceous agent (i.e., a peptide) is put into a viral construct such as an adenoviral or retroviral construct, and added to the cell, such that expression of the peptide agent is accomplished, e.g., PCT US97/01019. Regulatable gene therapy systems can also be used. Once the modulator has been administered to the cells, the cells are washed if desired and are allowed to incubate under preferably physiological conditions for some period. The cells are then harvested and a new gene expression profile is generated. Thus, e.g., cancer tissue is screened for agents that modulate, e.g., induce or suppress, the cancer phenotype. A change in at least one gene, preferably many, of the expression profile indicates that the agent has an effect on cancer activity. Similarly, altering a biological function or a signaling pathway is indicative of modulator activity. By defining such a signature for the cancer phenotype, screens for new drugs that alter the phenotype are devised. With this approach, the drug target need not be known and need not be represented in the original gene/protein expression screening platform, nor does the level of transcript for the target protein need to change. The modulator inhibiting function will serve as a surrogate marker
As outlined above, screens are done to assess genes or gene products. That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of either the expression of the gene or the gene product itself is performed.
Use of Modulators to Affect Peptides of the Invention
Measurements of cancer polypeptide activity, or of the cancer phenotype are performed using a variety of assays. For example, the effects of modulators upon the function of a cancer polypeptide(s) are measured by examining parameters described above. A physiological change that affects activity is used to assess the influence of a test compound on the polypeptides of this invention. When the functional outcomes are determined using intact cells or animals, a variety of effects can be assesses such as, in the case of a cancer associated with solid tumors, tumor growth, tumor metastasis, neovascularization, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., by Northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as cGNIP.
Methods of Identifying Characterizing Cancer-Associated Sequences
Expression of various gene sequences is correlated with cancer. Accordingly, disorders based on mutant or variant cancer genes are determined. In one embodiment, the invention provides methods for identifying cells containing variant cancer genes, e.g., determining the presence of, all or part, the sequence of at least one endogenous cancer gene in a cell. This is accomplished using any number of sequencing techniques. The invention comprises methods of identifying the cancer genotype of an individual, e.g., determining all or part of the sequence of at least one gene of the invention in the individual. This is generally done in at least one tissue of the individual, e.g., a tissue set forth in Table I, and may include the evaluation of a number of tissues or different samples of the same tissue. The method may include comparing the sequence of the sequenced gene to a known cancer gene, i.e., a wild-type gene to determine the presence of family members, homologies, mutations or variants. The sequence of all or part of the gene can then be compared to the sequence of a known cancer gene to determine if any differences exist. This is done using any number of known homology programs, such as BLAST, Bestfit, etc. The presence of a difference in the sequence between the cancer gene of the patient and the known cancer gene correlates with a disease state or a propensity for a disease state, as outlined herein.
In a preferred embodiment, the cancer genes are used as probes to determine the number of copies of the cancer gene in the genome. The cancer genes are used as probes to determine the chromosomal localization of the cancer genes. Information such as chromosomal localization finds use in providing a diagnosis or prognosis in particular when chromosomal abnormalities such as translocations, and the like are identified in the cancer gene locus.
XIV.) Kits/Articles of Manufacture
For use in the laboratory, prognostic, prophylactic, diagnostic and therapeutic applications described herein, kits are within the scope of the invention. Such kits can comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method, along with a label or insert comprising instructions for use, such as a use described herein. For example, the container(s) can comprise a probe that is or can be detectably labeled. Such probe can be an antibody or polynucleotide specific for a protein or a gene or message of the invention, respectively. Where the method utilizes nucleic acid hybridization to detect the target nucleic acid, the kit can also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence. Kits can comprise a container comprising a reporter, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, fluorescent, or radioisotope label; such a reporter can be used with, e.g., a nucleic acid or antibody. The kit can include all or part of the amino acid sequences in FIG. 2 or FIG. 3 or analogs thereof, or a nucleic acid molecule that encodes such amino acid sequences.
The kit of the invention will typically comprise the container described above and one or more other containers associated therewith that comprise materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes; carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package inserts with instructions for use.
A label can be present on or with the container to indicate that the composition is used for a specific therapy or non-therapeutic application, such as a prognostic, prophylactic, diagnostic or laboratory application, and can also indicate directions for either in vivo or in vitro use, such as those described herein. Directions and or other information can also be included on an insert(s) or label(s) which is included with or on the kit. The label can be on or associated with the container. A label a can be on a container when letters, numbers or other characters forming the label are molded or etched into the container itself; a label can be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. The label can indicate that the composition is used for diagnosing, treating, prophylaxing or prognosing a condition, such as a neoplasia of a tissue set forth in Table I.
The terms “kit” and “article of manufacture” can be used as synonyms.
In another embodiment of the invention, an article(s) of manufacture containing compositions, such as amino acid sequence(s), small molecule(s), nucleic acid sequence(s), and/or antibody(s), e.g., materials useful for the diagnosis, prognosis, prophylaxis and/or treatment of neoplasias of tissues such as those set forth in Table I is provided. The article of manufacture typically comprises at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass, metal or plastic. The container can hold amino acid sequence(s), small molecule(s), nucleic acid sequence(s), cell population(s) and/or antibody(s). In one embodiment, the container holds a polynucleotide for use in examining the mRNA expression profile of a cell, together with reagents used for this purpose. In another embodiment a container comprises an antibody, binding fragment thereof or specific binding protein for use in evaluating protein expression of 282P1G3 in cells and tissues, or for relevant laboratory, prognostic, diagnostic, prophylactic and therapeutic purposes; indications and/or directions for such uses can be included on or with such container, as can reagents and other compositions or tools used for these purposes. In another embodiment, a container comprises materials for eliciting a cellular or humoral immune response, together with associated indications and/or directions. In another embodiment, a container comprises materials for adoptive immunotherapy, such as cytotoxic T cells (CTL) or helper T cells (HTL), together with associated indications and/or directions; reagents and other compositions or tools used for such purpose can also be included.
The container can alternatively hold a composition that is effective for treating, diagnosis, prognosing or prophylaxing a condition and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agents in the composition can be an antibody capable of specifically binding 282P1G3 and modulating the function of 282P1G3.
The article of manufacture can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and/or dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, stirrers, needles, syringes, and/or package inserts with indications and/or instructions for use.
EXAMPLES
Various aspects of the invention are further described and illustrated by way of the several examples that follow, none of which are intended to limit the scope of the invention.
Example 1
SSH-Generated Isolation of cDNA Fragment of the 282P1G3 Gene
To isolate genes that are over-expressed in pancreatic cancer we used the Suppression Subtractive Hybridization (SSH) procedure using cDNA derived from pancreatic cancer tissues. The 282P1G3 SSH cDNA sequence was derived from pancreatic tumor minus cDNAs derived from normal pancreas. The 282P1G3 cDNA was identified as highly expressed in the pancreas cancer.
Materials and Methods
Human Tissues:
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.
RNA Isolation:
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.
Oligonucleotides:
The following HPLC purified oligonucleotides were used:








DPNCDN (cDNA synthesis primer):









(SEQ ID NO: 41)









5′TTTTGATCAAGCTT303′



 


Adaptor 1:








(SEQ ID NO: 42)









5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′



 








(SEQ ID NO: 43)









3′GGCCCGTCCTAG5′



 


Adaptor 2:








(SEQ ID NO: 44)









5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′



 








(SEQ ID NO: 45)









3′CGGCTCCTAG5′



 


PCR primer 1:








(SEQ ID NO: 46)









5′CTAATACGACTCACTATAGGGC3′



 


Nested primer (NP)1:








(SEQ ID NO: 47)









5′TCGAGCGGCCGCCCGGGCAGGA3′



 


Nested primer (NP)2:








(SEQ ID NO: 48)










5′AGCGTGGTCGCGGCCGAGGA3′






Suppression Subtractive Hybridization:
Suppression Subtractive Hybridization (SSH) was used to identify cDNAs corresponding to genes that may be differentially expressed in pancreas cancer. The SSH reaction utilized cDNA from pancreas cancer and normal tissues.
The gene 282P1G3 sequence was derived from pancreas cancer minus normal pancreas cDNA subtraction. The SSH DNA sequence (FIG. 1) was identified.
The cDNA derived from normal pancreas mixed with a pool of 9 normal tissues was used as the source of the “driver” cDNA, while the cDNA from pancreas cancer was used as the source of the “tester” cDNA. Double stranded cDNAs corresponding to tester and driver cDNAs were synthesized from 2 μg 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° C. Digested cDNA was extracted with phenol/chloroform (1:1) and ethanol precipitated.
Driver cDNA was generated by combining in a 1:1 ratio Dpn II digested cDNA from normal pancreas with a mix of digested cDNAs derived from the nine normal tissues: stomach, skeletal muscle, lung, brain, liver, kidney, pancreas, small intestine, and heart.
Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA from the relevant tissue source (see above) (400 ng) in 5 μl of water. The diluted cDNA (2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 and Adaptor 2 (10 μM), in separate ligation reactions, in a total volume of 10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH). Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C. for 5 min.
The first hybridization was performed by adding 1.5 μl (600 ng) of driver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1- and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, the samples were overlaid with mineral oil, denatured in an MJ Research thermal cycler at 98° C. for 1.5 minutes, and then were allowed to hybridize for 8 hrs at 68° C. The two hybridizations were then mixed together with an additional 1 μl of fresh denatured driver cDNA and were allowed to hybridize overnight at 68° C. The second hybridization was then diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA, heated at 70° C. for 7 min. and stored at −20° C.
PCR Amplification, Cloning and Sequencing of Gene Fragments Generated from SSH:
To amplify gene fragments resulting from SSH reactions, two PCR amplifications were performed. In the primary PCR reaction 1 μl of the diluted final hybridization mix was added to 1 μl of PCR primer 1 (10 μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and 0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5 min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C. for 30 sec, 72° 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 μl 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 μM) were used instead of PCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10 sec, 68° C. for 30 sec, and 72° C. for 1.5 minutes. The PCR products were analyzed using 2% agarose gel electrophoresis.
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 μl of bacterial culture using the conditions of PCR1 and NP1 and NP2 as primers. PCR products were analyzed using 2% agarose gel electrophoresis.
Bacterial clones were stored in 20% glycerol in a 96 well format. Plasmid DNA was prepared, sequenced, and subjected to nucleic acid homology searches of the GenBank, dBest, and NCI-CGAP databases.
RT-PCR Expression Analysis:
First strand cDNAs can be generated from 1 μg 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° C. with reverse transcriptase followed by RNAse H treatment at 37° C. for 20 min. After completing the reaction, the volume can be increased to 200 μl with water prior to normalization. First strand cDNAs from 16 different normal human tissues can be obtained from Clontech.
Normalization of the first strand cDNAs from multiple tissues was performed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID NO: 49) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 50) to amplify-actin. First strand cDNA (5 μl) were amplified in a total volume of 50 μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgCl2, 50 mM KCl, pH8.3) and 1× Klentaq DNA polymerase (Clontech). Five μl 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° C. for 15 sec, followed by a 18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C. for 5 sec. A final extension at 72° C. was carried out for 2 min. After agarose gel electrophoresis, the band intensities of the 283 bp β-actin bands from multiple tissues were compared by visual inspection. Dilution factors for the first strand cDNAs were calculated to result in equal β-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.
To determine expression levels of the 282P1G3 gene, 5 μl 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 282P1G3 SSH sequence and are listed below:









282P1G3.1




5′-TAAGGTCTCAGCTGTAAACCAAAAG-3′
(SEQ ID NO: 51)


 


282P1G3.2


5′-CTGTTTTAAGATTGTTGGAACCTGT-3′
(SEQ ID NO: 52)






A typical RT-PCR expression analysis is shown in FIG. 14. First strand cDNA was prepared from vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), normal pancreas, ovary cancer pool, and pancreas cancer pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 282P1G3, was performed at 26 and 30 cycles of amplification. Expression of 282P1G3 was detected in ovary cancer pool, pancreas cancer pool vital pool 1, but not in vital pool 2 nor in normal pancreas.
Example 2
Isolation of Full Length 282P1G3 Encoding cDNA
The 282P1G3 SSH cDNA sequence was derived from a subtraction consisting of pancreas cancer minus a normal pancreas. The SSH cDNA sequence of 321 bp (FIG. 1) was designated 282P1G3.
282P1G3 v.2 of 3464 bp was cloned from a pool of normal tissue cDNA library, revealing an ORF of 1171 amino acids (FIG. 2 and FIG. 3). Other variants of 282P1G3 were also identified and these are listed in FIG. 2 and FIG. 3.
282P1G3 v.1, v.9, v.10, v.11, v.24 and v.25 proteins are 1224 amino acids in length and differ from each other by one amino acid as shown in FIG. 11. 282P1G3 v.12 through v.23, v.26 and v.27 are SNP variants and code for the same protein as 282P1G3 v.1. 282P1G3 v.2, v.3, v.4, v.5, v.6, v.7, and v.8 are splice variants of 282P1G3 v.1 and code for proteins of 1171, 893, 1117, 1208, 1183, 1236, and 1195 amino acids, respectively. 282P1G3 v.28 is a splice variant identified by the 282P1G3 SSH, and deletes the second exon of v.1.
282P1G3 v.1 shows 99% identity over 7650 nucleotides to cell adhesion molecule with homology to L1CAM (close homolog of L1) (CHL1), accession number NM—006614. It is a neural recognition molecule that may be involved in signal transduction pathways. 282P1G3 v.2 is a novel splice variant of 282P1G3 and has not been previously described.
Example 3
Chromosomal Mapping of 282P1G3
Chromosomal localization can implicate genes in disease pathogenesis. Several chromosome mapping approaches are available including fluorescent in situ hybridization (FISH), human/hamster radiation hybrid (RH) panels (Walter et al., 1994; Nature Genetics 7:22; Research Genetics, Huntsville Ala.), human-rodent somatic cell hybrid panels such as is available from the Cornell Institute (Camden, N.J.), and genomic viewers utilizing BLAST homologies to sequenced and mapped genomic clones (NCBI, Bethesda, Md.).
282P1G3 maps to chromosome 3p26.1 using 282P1G3 sequence and the NCBI BLAST tool located on the World Wide Web.
Example 4
Expression Analysis of 282P1G3 in Normal Tissues and Patient Specimens
Expression analysis by RT-PCR demonstrated that 282P1G3 is strongly expressed in pancreas cancer and ovary cancer patient specimens (FIG. 14). First strand cDNA was prepared from (A) vital pool 1 (liver, lung and kidney), vital pool 2 (pancreas, colon and stomach), normal pancreas, ovary cancer pool, and pancreas cancer pool; (B) normal stomach, normal brain, normal heart, normal liver, normal skeletal muscle, normal testis, normal prostate, normal bladder, normal kidney, normal colon, normal lung, normal pancreas, and a pool of cancer specimens from pancreas cancer patients, ovary cancer patients, and cancer metastasis specimens. Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primers to 282P1G3, was performed at 26 and 30 cycles of amplification. (A) Expression of 282P1G3 was detected in ovary cancer pool, pancreas cancer pool vital pool 1, but not in vital pool 2 nor in normal pancreas. (B) Samples were run on an agarose gel, and PCR products were quantitated using the Alphalmager software. Results show strong expression in pancreas cancer, ovary cancer, cancer metastasis, and normal brain compared to all other normal tissues tested.
Extensive expression of 282P1G3 in normal tissues is shown in FIG. 15. Two multiple tissue northern blots (Clontech) both with 2 μg of mRNA/lane were probed with the 282P1G3 sequence. Size standards in kilobases (kb) are indicated on the side. Results show expression of an approximately 9-10 kb transcript in normal but not in any other normal tissue tested.
Expression of 282P1G3 in pancreas cancer patient specimens is shown in FIG. 16. RNA was extracted from pancreas cancer cell lines (CL), normal pancreas (N), and pancreas cancer patient tumor (T). Northern blots with 10 μg of total RNA were probed with the 282P1G3 SSH fragment. Size standards in kilobases are on the side. Results show expression of 282P1G3 in pancreas cancer patient tumor specimen but not in the cell lines nor in the normal pancreas.
Expression of 282P1G3 was also detected in ovary cancer patient specimens (FIG. 17). RNA was extracted from ovary cancer cell lines (CL), normal ovary (N), and ovary cancer patient tumor (T). Northern blots with 10 μg of total RNA were probed with the 282P1G3 DNA probe. Size standards in kilobases are on the side. Results show expression of 282P1G3 in ovary cancer patient tumor specimen but not in the cell lines nor in the normal ovary.
FIG. 18 shows expression of 282P1G3 in lymphoma cancer patient specimens. RNA was extracted from peripheral blood lymphocytes, cord blood isolated from normal individuals, and from lymphoma patient cancer specimens. Northern blots with 10 μg of total RNA were probed with the 282P1G3 sequence. Size standards in kilobases are on the side. Results show expression of 282P1G3 in lymphoma patient specimens but not in the normal blood cells tested.
The restricted expression of 282P1G3 in normal tissues and the expression detected in cancer patient specimens suggest that 282P1G3 is a potential therapeutic target and a diagnostic marker for human cancers.
Example 5
Transcript Variants of 282P1G3
Transcript variants are variants of mature mRNA from the same gene which arise by alternative transcription or alternative splicing. Alternative transcripts are transcripts from the same gene but start transcription at different points. Splice variants are mRNA variants spliced differently from the same transcript. In eukaryotes, when a multi-exon gene is transcribed from genomic DNA, the initial RNA is spliced to produce functional mRNA, which has only exons and is used for translation into an amino acid sequence. Accordingly, a given gene can have zero to many alternative transcripts and each transcript can have zero to many splice variants. Each transcript variant has a unique exon makeup, and can have different coding and/or non-coding (5′ or 3′ end) portions, from the original transcript. Transcript variants can code for similar or different proteins with the same or a similar function or can encode proteins with different functions, and can be expressed in the same tissue at the same time, or in different tissues at the same time, or in the same tissue at different times, or in different tissues at different times. Proteins encoded by transcript variants can have similar or different cellular or extracellular localizations, e.g., secreted versus intracellular.
Transcript variants are identified by a variety of art-accepted methods. For example, alternative transcripts and splice variants are identified by full-length cloning experiment, or by use of full-length transcript and EST sequences. First, all human ESTs were grouped into clusters which show direct or indirect identity with each other. Second, ESTs in the same cluster were further grouped into sub-clusters and assembled into a consensus sequence. The original gene sequence is compared to the consensus sequence(s) or other full-length sequences. Each consensus sequence is a potential splice variant for that gene. Even when a variant is identified that is not a full-length clone, that portion of the variant is very useful for antigen generation and for further cloning of the full-length splice variant, using techniques known in the art.
Moreover, computer programs are available in the art that identify transcript variants based on genomic sequences. Genomic-based transcript variant identification programs include FgenesH (A. Salamov and V. Solovyev, “Ab initio gene finding in Drosophila genomic DNA,” Genome Research. 2000 April; 10(4):516-22); Grail and GenScan on the World Wide Web. For a general discussion of splice variant identification protocols see., e.g., Southan, C., A genomic perspective on human proteases, FEBS Lett. 2001 Jun. 8; 498(2-3):214-8; de Souza, S. J., et al., Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags, Proc. Natl Acad Sci USA. 2000 Nov. 7; 97(23):12690-3.
To further confirm the parameters of a transcript variant, a variety of techniques are available in the art, such as full-length cloning, proteomic validation, PCR-based validation, and 5′ RACE validation, etc. (see e.g., Proteomic Validation: Brennan, S. O., et al., Albumin banks peninsula: a new termination variant characterized by electrospray mass spectrometry, Biochem Biophys Acta. 1999 Aug. 17; 1433(1-2):321-6; Ferranti P, et al., Differential splicing of pre-messenger RNA produces multiple forms of mature caprine alpha(s1)-casein, Eur J. Biochem. 1997 Oct. 1; 249(1):1-7. For PCR-based Validation: Wellmann S, et al., Specific reverse transcription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCycler technology, Clin Chem. 2001 April; 47(4):654-60; Jia, H. P., et al., Discovery of new human beta-defensins using a genomics-based approach, Gene. 2001 Jan. 24; 263(1-2):211-8. For PCR-based and 5′ RACE Validation: Brigle, K. E., et al., Organization of the murine reduced folate carrier gene and identification of variant splice forms, Biochem Biophys Acta. 1997 Aug. 7; 1353(2): 191-8).
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 282P1G03 has a particular expression profile related to cancer. Alternative transcripts and splice variants of 282P1G03 may also be involved in cancers in the same or different tissues, thus serving as tumor-associated markers/antigens.
Using the full-length gene and EST sequences, eight additional transcript variants were identified, designated as 282P1G03 v.2, v.3, v.4, v.5, v.6, v.7, v.8 and v.28. The boundaries of exons in the original transcript, 282P1G03 v.1 were shown in Table LI. FIG. 12 shows the structures of the transcript variants. Theoretically, each different combination of exons in spatial order (aligned on the genomic sequence), e.g. exons 2, 3, 5, 7, and 9-28 of v. 1, is a potential splice variant.
Tables LII(a)-(h) through LV(a)-(h) are set forth on a variant-by-variant bases. Tables LII(a)-(h) show the nucleotide sequence of the transcript variant. Tables LIII(a)-(h) show the alignment of the transcript variant with nucleic acid sequence of 282P1G03 v.1. Tables LIV(a)-(h) show the amino acid translation of the transcript variant for the identified reading frame orientation. Tables LV(a)-(h) display alignments of the amino acid sequence encoded by the splice variant with that of 282P1G03 v. 1.
Example 6
Single Nucleotide Polymorphisms of 282P1G3
A Single Nucleotide Polymorphism (SNP) is a single base pair variation in a nucleotide sequence at a specific location. At any given point of the genome, there are four possible nucleotide base pairs: A/T, C/G, G/C and T/A. Genotype refers to the specific base pair sequence of one or more locations in the genome of an individual. Haplotype refers to the base pair sequence of more than one location on the same DNA molecule (or the same chromosome in higher organisms), often in the context of one gene or in the context of several tightly linked genes. SNP that occurs on a cDNA is called cSNP. This cSNP may change amino acids of the protein encoded by the gene and thus change the functions of the protein. Some SNP cause inherited diseases; others contribute to quantitative variations in phenotype and reactions to environmental factors including diet and drugs among individuals. Therefore, SNP and/or combinations of alleles (called haplotypes) have many applications, including diagnosis of inherited diseases, determination of drug reactions and dosage, identification of genes responsible for diseases, and analysis of the genetic relationship between individuals (P. Nowotny, J. M. Kwon and A. M. Goate, “SNP analysis to dissect human traits,” Curr. Opin. Neurobiol. 2001 October; 11(5):637-641; M. Pirmohamed and B. K. Park, “Genetic susceptibility to adverse drug reactions,” Trends Pharmacol. Sci. 2001 June; 22(6):298-305; J. H. Riley, C. J. Allan, E. Lai and A. Roses, “The use of single nucleotide polymorphisms in the isolation of common disease genes,” Pharmacogenomics. 2000 February; 1(1):39-47; R. Judson, J. C. Stephens and A. Windemuth, “The predictive power of haplotypes in clinical response,” Pharmacogenomics. 2000 February; 1(1):15-26).
SNP are identified by a variety of art-accepted methods (P. Bean, “The promising voyage of SNP target discovery,” Am. Clin. Lab. 2001 October-November; 20(9):18-20; K. M. Weiss, “In search of human variation,” Genome Res. 1998 July; 8(7):691-697; M. M. She, “Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies,” Clin. Chem. 2001 February; 47(2):164-172). For example, SNP can be identified by sequencing DNA fragments that show polymorphism by gel-based methods such as restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). They can also be discovered by direct sequencing of DNA samples pooled from different individuals or by comparing sequences from different DNA samples. With the rapid accumulation of sequence data in public and private databases, one can discover SNP 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). SNP can be verified and genotype or haplotype of an individual can be determined by a variety of methods including direct sequencing and high throughput microarrays (P. Y. Kwok, “Methods for genotyping single nucleotide polymorphisms,” Annu. Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K. Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft, “High-throughput SNP genotyping with the Masscode system,” Mol. Diagn. 2000 December; 5(4):329-340).
Using the methods described above, 19 SNP were identified in the original transcript, 282P1G03 v.1, at positions 320 (c/t), 668 (c/t), 1178 (a/g), 3484 (c/t), 4615 (g/a), 4636 (−/t), 5078 (c/t), 5530 (t/a), 5812 (c/t), 6114 (a/g), 6229 (c/t), 6383 (g/a), 6626 (c/t), 6942 (c/t), 7085 (c/t), 2684 (a/g), 3864 (t/c), 5768 (t/c) and 6125 (c/t). The transcripts or proteins with alternative allele were designated as variant 282P1G03 v.9 through v.25, as shown in FIG. 10. FIG. 11 shows the schematic alignment of protein variants, corresponding to nucleotide variants. Nucleotide variants that code for the same amino acid sequence as v.1 are not shown in FIG. 11. These alleles of the SNP, though shown separately here, can occur in different combinations (haplotypes) and in any one of the transcript variants (such as 282P1G03 v.2) that contains the site of the SNP.
Example 7
Production of Recombinant 282P1G3 in Prokaryotic Systems
To express recombinant 282P1G3 and 282P1G3 variants in prokaryotic cells, the full or partial length 282P1G3 and 282P1G3 variant cDNA sequences are cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 282P1G3 variants are expressed: the full length sequence presented in FIGS. 2 and 3, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 282P1G3, variants, or analogs thereof.
A. In vitro Transcription and Translation Constructs:
pCRII: To generate 282P1G3 sense and ant-sense RNA probes for RNA in situ investigations, pCRII constructs (Invitrogen, Carlsbad Calif.) are generated encoding either all or fragments of the 282P1G3 cDNA. The pCR11 vector has Sp6 and T7 promoters flanking the insert to drive the transcription of 282P1G3 RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze the cell and tissue expression of 282P1G3 at the RNA level. Transcribed 282P1G3 RNA representing the cDNA amino acid coding region of the 282P1G3 gene is used in in vitro translation systems such as the TnTTM Coupled Reticulolysate System (Promega, Corp., Madison, Wis.) to synthesize 282P1G3 protein.
B. Bacterial Constructs:
pGEX Constructs: To generate recombinant 282P1G3 proteins in bacteria that are fused to the Glutathione S-transferase (GST) protein, all or parts of the 282P1G3 cDNA protein coding sequence are cloned into the pGEX family of GST-fusion vectors (Amersham Pharmacia Biotech, Piscataway, N.J.). These constructs allow controlled expression of recombinant 282P1G3 protein sequences with GST fused at the amino-terminus and a six histidine epitope (6×His) at the carboxyl-terminus. The GST and 6×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×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 PreScissionTM recognition site in pGEX-6P-1, may be employed such that it permits cleavage of the GST tag from 282P1G3-related protein. The ampicillin resistance gene and pBR322 origin permits selection and maintenance of the pGEX plasmids in E. coli. 
pMAL Constructs: To generate, in bacteria, recombinant 282P1G3 proteins that are fused to maltose-binding protein (MBP), all or parts of the 282P1G3 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 282P1G3 protein sequences with MBP fused at the amino-terminus and a 6×His epitope tag at the carboxyl-terminus. The MBP and 6×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×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 282P1G3. 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.
pET Constructs: To express 282P1G3 in bacterial cells, all or parts of the 282P1G3 cDNA protein coding sequence are cloned into the pET family of vectors (Novagen, Madison, Wis.). These vectors allow tightly controlled expression of recombinant 282P1G3 protein in bacteria with and without fusion to proteins that enhance solubility, such as NusA and thioredoxin (Trx), and epitope tags, such as 6×His and S-TagTM 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 282P1G3 protein are expressed as amino-terminal fusions to NusA.
C. Yeast Constructs:
pESC Constructs: To express 282P1G3 in the yeast species Saccharomyces cerevisiae for generation of recombinant protein and functional studies, all or parts of the 282P1G3 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 FlagTM or Myc epitope tags in the same yeast cell. This system is useful to confirm protein-protein interactions of 282P1G3. In addition, expression in yeast yields similar post-translational modifications, such as glycosylations and phosphorylations that are found when expressed in eukaryotic cells.
pESP Constructs: To express 282P1G3 in the yeast species Saccharomyces pombe, all or parts of the 282P1G3 cDNA protein coding sequence are cloned into the pESP family of vectors. These vectors allow controlled high level of expression of a 282P1G3 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 epitope tag allows detection of the recombinant protein with anti-FlagTM antibody.
Example 8
Production of Recombinant 282P1G3 in Higher Eukaryotic Systems
A. Mammalian Constructs:
To express recombinant 282P1G3 in eukaryotic cells, the full or partial length 282P1G3 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 282P1G3 were expressed in these constructs, amino acids 1 to 1224, 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 282P1G3 v.1, and v.9 through v.25; amino acids 1 to 1171, 1 to 893, 1 to 1117, 1 to 1208, 1 to 1183, 1 to 1236, 1 to 1195 of v.2, v.3, v.4, v.5, v.6, v.7, and v.8 respectively; 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 282P1G3 variants, or analogs thereof.
The constructs can be transfected into any one of a wide variety of mammalian cells such as 293T cells. Transfected 293T cell lysates can be probed with the anti-282P1G3 polyclonal serum, described herein.
pcDNA4/HisMax Constructs: To express 282P1G3 in mammalian cells, a 282P1G3 ORF, or portions thereof, of 282P1G3 are cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter and the SP16 translational enhancer. The recombinant protein has XpressTM and six histidine (6×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. 
pcDNA3.1/MycHis Constructs: To express 282P1G3 in mammalian cells, a 282P1G3 ORF, or portions thereof, of 282P1G3 with a consensus Kozak translation initiation site is cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the myc epitope and 6×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. 
The complete ORF of 282P1G3 v.2 was cloned into the pcDNA3.1/MycHis construct to generate 282P1G3.pcDNA3.1/MycHis. FIG. 19 shows expression of 282P1G3.pcDNA3.1/MycHis following transfection into 293T cells. 293T cells were transfected with either 282P1G3.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 282P1G3 from the 282P1G3.pcDNA3.1/MycHis construct in the lysates of transfected cells.
pcDNA3.1/CT-GFP-TOPO Construct: To express 282P1G3 in mammalian cells and to allow detection of the recombinant proteins using fluorescence, a 282P1G3 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 282P1G3 protein.
PAPtag: A 282P1G3 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 282P1G3 protein while fusing the IgGκ signal sequence to the amino-terminus. Constructs are also generated in which alkaline phosphatase with an amino-terminal IgGκ signal sequence is fused to the amino-terminus of a 282P1G3 protein. The resulting recombinant 282P1G3 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 282P1G3 proteins. Protein expression is driven from the CMV promoter and the recombinant proteins also contain myc and 6×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 282P1G3 ORF, or portions thereof, were cloned into pTag-5. This vector is similar to pAPtag but without the alkaline phosphatase fusion. This construct generates 282P1G3 protein with an amino-terminal IgGκ signal sequence and myc and 6×His epitope tags at the carboxyl-terminus that facilitate detection and affinity purification. The resulting recombinant 282P1G3 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 282P1G3 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. 
The extracellular domain, amino acids 26-1043, of 282P1G3 v.2 was cloned into the pTag5 construct to generate 282P1G3.pTag5. FIG. 20 shows expression and secretion of the extracellular domain of 282P1G3 following 282P1G3.pTag5 vector transfection into 293T cells. 293T cells were transfected with 282P1G3.pTag5 construct. Forty hours later, supernatant as well as 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 and secretion of 282P1G3 from the 282P1G3.pTag5 transfected cells.
PsecFc: A 282P1G3 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 282P1G3 proteins, while fusing the IgGK signal sequence to N-terminus. 282P1G3 fusions utilizing the murine IgG1 Fc region are also used. The resulting recombinant 282P1G3 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 282P1G3 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. 
pSRα Constructs: To generate mammalian cell lines that express 282P1G3 constitutively, 282P1G3 ORF, or portions thereof, of 282P1G3 were cloned into pSRα constructs. Amphotropic and ecotropic retroviruses were generated by transfection of pSRα constructs into the 293T-10A1 packaging line or co-transfection of pSRα 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, 282P1G3, 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.
Additional pSRα constructs are made that fuse an epitope tag such as the FLAGTM tag to the carboxyl-terminus of 282P1G3 sequences to allow detection using anti-Flag antibodies. For example, the FLAGTM sequence 5′ gat tac aag gat gac gac gat aag 3′ (SEQ ID NO: 53) is added to cloning primer at the 3′ end of the ORF. Additional pSRα constructs are made to produce both amino-terminal and carboxyl-terminal GFP and myc/6×His fusion proteins of the full-length 282P1G3 proteins.
Additional Viral Vectors: Additional constructs are made for viral-mediated delivery and expression of 282P1G3. High virus titer leading to high level expression of 282P1G3 is achieved in viral delivery systems such as adenoviral vectors and herpes amplicon vectors. A 282P1G3 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, 282P1G3 coding sequences or fragments thereof are cloned into the HSV-1 vector (Imgenex) to generate herpes viral vectors. The viral vectors are thereafter used for infection of various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
Regulated Expression Systems: To control expression of 282P1G3 in mammalian cells, coding sequences of 282P1G3, 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 282P1G3. These vectors are thereafter used to control expression of 282P1G3 in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
B. Baculovirus Expression Systems
To generate recombinant 282P1G3 proteins in a baculovirus expression system, 282P1G3 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-282P1G3 is co-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda) insect cells to generate recombinant baculovirus (see Invitrogen instruction manual for details). Baculovirus is then collected from cell supernatant and purified by plaque assay.
Recombinant 282P1G3 protein is then generated by infection of HighFive insect cells (Invitrogen) with purified baculovirus. Recombinant 282P1G3 protein can be detected using anti-282P1G3 or anti-His-tag antibody. 282P1G3 protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 282P1G3.
Example 9
Antigenicity Profiles and Secondary Structure
FIG. 5(A-C), FIG. 6(A-C), FIG. 7(A-C), FIG. 8(A-C), and FIG. 9(A-C) depict graphically five amino acid profiles of 282P1G3 variants 1, 3, and 7, each assessment available by accessing the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) on the ExPasy molecular biology server.
These profiles: FIG. 5(A-C), Hydrophilicity, (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 6(A-C), Hydropathicity, (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132); FIG. 7(A-C), Percentage Accessible Residues (Janin J., 1979 Nature 277:491-492); FIG. 8(A-C), Average Flexibility, (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255); FIG. 9(A-C), Beta-turn (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294); and optionally others available in the art, such as on the ProtScale website, were used to identify antigenic regions of each of the 282P1G3 variant proteins. Each of the above amino acid profiles of 282P1G3 variants were generated using the following ProtScale parameters for analysis: 1) A window size of 9; 2) 100% weight of the window edges compared to the window center; and, 3) amino acid profile values normalized to lie between 0 and 1.
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.
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.
Antigenic sequences of the 282P1G3 variant proteins indicated, e.g., by the profiles set forth in FIG. 5(A-C), FIG. 6(A-C), FIG. 7(A-C), FIG. 8(A-C), and/or FIG. 9(A-C) are used to prepare immunogens, either peptides or nucleic acids that encode them, to generate therapeutic and diagnostic anti-282P1G3 antibodies. The immunogen can be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50 contiguous amino acids, or the corresponding nucleic acids that encode them, from the 282P1G3 protein variants listed in FIGS. 2 and 3, In particular, peptide immunogens of the invention can comprise, a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profiles of FIG. 5; a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of FIG. 6; a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profiles of FIG. 7; a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profiles on FIG. 8; and, a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of FIG. 9. Peptide immunogens of the invention can also comprise nucleic acids that encode any of the forgoing.
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.
The secondary structure of 282P1G3 protein variants 1 through 8, 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, accessed from the ExPasy molecular biology server located on the World Wide Web at (.expasy.ch/tools/). The analysis indicates that 282P1G3 variant 1 is composed of 15.77% alpha helix, 26.14% extended strand, and 58.09% random coil (FIG. 13A). Variant 2 is composed of 14.86% alpha helix, 26.39% extended strand, and 58.75% random coil (FIG. 13B). Variant 3 is composed of 14.00% alpha helix, 29.34% extended strand, and 56.66% random coil (FIG. 13C). Variant 4 is composed of 15.94% alpha helix, 26.14% extended strand, and 57.92% random coil (FIG. 13D). Variant 5 is composed of 15.73% alpha helix, 26.32% extended strand, and 57.95% random coil (FIG. 13E). Variant 6 is composed of 16.99% alpha helix, 25.36% extended strand, and 57.65% random coil (FIG. 13F). Variant 7 is composed of 15.78% alpha helix, 26.13% extended strand, and 58.09% random coil (FIG. 13G). Variant 8 is composed of 16.99% alpha helix, 25.36% extended strand, and 57.66% random coil (FIG. 13H).
Analysis for the potential presence of transmembrane domains in the 282P1G3 variant proteins was carried out using a variety of transmembrane prediction algorithms accessed from the ExPasy molecular biology server located on the World Wide Web at (.expasy.ch/tools/). Shown graphically in FIG. 13I and 13J are the results of analysis of variant 1 depicting the presence and location of 1 transmembrane domain using the TMpred program (FIG. 13I) and 1 transmembrane domain using the TMHMM program (FIG. 13J). Shown graphically in FIGS. 13K and 13L are the results of analysis of variant 2 depicting the presence and location of 1 transmembrane domains using the TMpred program (FIG. 13K) and 1 transmembrane domain using the TMHMM program (FIG. 13L). Shown graphically in FIGS. 13M and 13N are the results of analysis of variant 3 depicting no transmembrane domain using both the TMpred program (FIG. 13M) and TMHMM program (FIG. 13N). Shown graphically in FIG. 13O and 13P are the results of analysis of variant 4 depicting the presence and location of 1 transmembrane domain using the TMpred program (FIG. 13O) and 1 transmembrane domain using the TMHMM program (FIG. 13P). Shown graphically in FIGS. 13Q and 13R are the results of analysis of variant 5 depicting the presence and location of 1 transmembrane domain using the TMpred program (FIG. 13Q) and 1 transmembrane domain using the TMHMM program (FIG. 13R). Shown graphically in FIGS. 13S and 13T are the results of analysis of variant 6 depicting the presence and location of 1 transmembrane domain using the TMpred program (FIG. 13S) and 1 transmembrane domain using the TMHMM program (FIG. 13T). Shown graphically in FIGS. 13U and 13V are the results of analysis of variant 7 depicting the presence and location of 1 transmembrane domain using the TMpred program (FIG. 13U) and 1 transmembrane domain using the TMHMM program (FIG. 13V). Shown graphically in FIGS. 13W and 13X are the results of analysis of variant 8 depicting the presence and location of 1 transmembrane domain using the TMpred program (FIG. 13W) and 1 transmembrane domain using the TMHMM program (FIG. 13X). The results of each program, namely the amino acids encoding the transmembrane domains are summarized in Table VI.
Example 10
Generation of 282P1G3 Polyclonal Antibodies
Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. In addition to immunizing with a full length 282P1G3 protein variant, computer algorithms are employed in design of immunogens that, based on amino acid sequence analysis contain characteristics of being antigenic and available for recognition by the immune system of the immunized host (see the Example entitled “Antigenicity Profiles and Secondary Structure”). Such regions would be predicted to be hydrophilic, flexible, in beta-turn conformations, and be exposed on the surface of the protein (see, e.g., FIG. 5(A-C), FIG. 6(A & C), FIG. 7(A-C), FIG. 8(A-C), or FIG. 9(A-C) for amino acid profiles that indicate such regions of 282P1G3 protein variants).
For example, recombinant bacterial fusion proteins or peptides containing hydrophilic, flexible, beta-turn regions of 282P1G3 protein variants are used as antigens to generate polyclonal antibodies in New Zealand White rabbits or monoclonal antibodies as described in Example 11. For example, in 282P1G3 variant 1, such regions include, but are not limited to, amino acids 57-75, amino acids 131-135, amino acids 210-265, amino acids 550-588, and amino acids 662-688. In sequence unique to variant 3, such regions include, but are not limited to, amino acids 855-872 and amino acids 856-886. In sequence specific for variant 7, such regions include, but are not limited to, amino acids 345-356. 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 57-75 of 282P1G3 variant 1 was conjugated to KLH and used to immunize a rabbit. Alternatively the immunizing agent may include all or portions of the 282P1G3 variant proteins, analogs or fusion proteins thereof. For example, the 282P1G3 variant 1 amino acid sequence can be fused using recombinant DNA techniques to any one of a variety of fusion protein partners that are well known in the art, such as glutathione-5-transferase (GST) and HIS tagged fusion proteins. In another embodiment, amino acids 26-265 of 282P1G3 variant 1 was fused to GST using recombinant techniques and the pGEX expression vector, expressed, purified and used to immunize a rabbit. Such fusion proteins are purified from induced bacteria using the appropriate affinity matrix.
Other recombinant bacterial fusion proteins that may be employed include maltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see the section entitled “Production of 282P1G3 in Prokaryotic Systems” and Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, P. S., Brady, W., Urnes, M., Grosmaire, L., Damle, N., and Ledbetter, L. (1991) J.Exp. Med. 174, 561-566).
In addition to bacterial derived fusion proteins, mammalian expressed protein antigens are also used. These antigens are expressed from mammalian expression vectors such as the Tag5 and Fc-fusion vectors (see the section entitled “Production of Recombinant 282P1G3 in Eukaryotic Systems”), and retains post-translational modifications such as glycosylations found in native protein. In one embodiment, amino acids 26-1,043 of variant 2, encoding the extracellular domain, was cloned into the Tag5 mammalian secretion vector, and expressed in 293T cells. The recombinant protein is purified by metal chelate chromatography from tissue culture supernatants of 293T cells stably expressing the recombinant vector. The purified Tag5 282P1G3 protein is then used as immunogen.
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).
In a typical protocol, rabbits are initially immunized subcutaneously with up to 200 μg, typically 100-200 μg, 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 μg, typically 100-200 μg, 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.
To test reactivity and specificity of immune serum, such as the rabbit serum derived from immunization with the Tag5-282P1G3 variant 2 protein, the full-length 282P1G3 variant 1 cDNA is cloned into pcDNA 3.1 myc-his expression vector (Invitrogen, see the Example entitled “Production of Recombinant 282P1G3 in Eukaryotic Systems”). After transfection of the constructs into 293T cells, cell lysates are probed with the anti-282P1G3 serum and with anti-His antibody (See FIG. 19; Santa Cruz Biotechnologies, Santa Cruz, Calif.) to determine specific reactivity to denatured 282P1G3 protein using the Western blot technique. In addition, the immune serum is tested by fluorescence microscopy, flow cytometry and immunoprecipitation against 293T and other recombinant 282P1G3-expressing cells to determine specific recognition of native protein. Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometric techniques using cells that endogenously express 282P1G3 are also carried out to test reactivity and specificity.
Anti-serum from rabbits immunized with 282P1G3 variant fusion proteins, such as GST and MBP fusion proteins, are purified by depletion of antibodies reactive to the fusion partner sequence by passage over an affinity column containing the fusion partner either alone or in the context of an irrelevant fusion protein. For example, antiserum derived from a GST-282P1G3 variant 1 fusion protein is first purified by passage over a column of GST protein covalently coupled to AffiGel matrix (BioRad, Hercules, Calif.). The antiserum is then affinity purified by passage over a column composed of a MBP-282P1G3 fusion protein covalently coupled to Affigel matrix. The serum is then further purified by protein G affinity chromatography to isolate the IgG fraction. Sera from other His-tagged antigens and peptide immunized rabbits as well as fusion partner depleted sera are affinity purified by passage over a column matrix composed of the original protein immunogen or free peptide.
Example 11
Generation of 282P1G3 Monoclonal Antibodies (mAbs)
In one embodiment, therapeutic mAbs to 282P1G3 variants comprise those that react with epitopes specific for each variant protein or specific to sequences in common between the variants that would disrupt or modulate the biological function of the 282P1G3 variants, for example those that would disrupt the interaction with ligands and binding partners. Immunogens for generation of such mAbs include those designed to encode or contain the entire 282P1G3 protein variant sequence, regions of the 282P1G3 protein variants predicted to be antigenic from computer analysis of the amino acid sequence (see, e.g., FIG. 5(A-C), FIG. 6(A-C), FIG. 7(A-C), FIG. 8(A-C), or FIG. 9(A-C), and the Example entitled “Antigenicity Profiles and Secondary Structure”). Immunogens include peptides, recombinant bacterial proteins, and mammalian expressed Tag 5 proteins and human and murine IgG FC fusion proteins. In addition, cells engineered to express high levels of a respective 282P1G3 variant, such as 293T-282P1G3 variant 1 or 300.19-282P1G3 variant 1 murine Pre-B cells, are used to immunize mice.
To generate mAbs to a 282P1G3 variant, mice are first immunized intraperitoneally (IP) with, typically, 10-50 μg of protein immunogen or 107 282P1G3-expressing cells mixed in complete Freund's adjuvant. Mice are then subsequently immunized IP every 2-4 weeks with, typically, 10-50 μg of protein immunogen or 107 cells mixed in incomplete Freund's adjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. In addition to the above protein and cell-based immunization strategies, a DNA-based immunization protocol is employed in which a mammalian expression vector encoding a 282P1G3 variant sequence is used to immunize mice by direct injection of the plasmid DNA. For example, amino acids 26-1,043 of variant 2 was cloned into the Tag5 mammalian secretion vector and the recombinant vector will then be used as immunogen. In another example the same amino acids are cloned into an Fc-fusion secretion vector in which the 282P1G3 variant 2 sequence is fused at the amino-terminus to an IgK leader sequence and at the carboxyl-terminus to the coding sequence of the human or murine IgG Fc region. This recombinant vector is then used as immunogen. The plasmid immunization protocols are used in combination with purified proteins expressed from the same vector and with cells expressing the respective 282P1G3 variant.
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).
In one embodiment for generating 282P1G3 monoclonal antibodies, a Tag5-282P1G3 variant 2 antigen encoding amino acids 26-1,043, was expressed (FIG. 20) and then purified from stably transfected 293T cells. Balb C mice are initially immunized intraperitoneally with 25 μg of the Tag5-282P1G3 variant 2 protein mixed in complete Freund's adjuvant. Mice are subsequently immunized every two weeks with 25 μg 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 282P1G3 variant 2 protein is monitored by Western blotting, immunoprecipitation and flow cytometry using 293T cells transfected with an expression vector encoding the 282P1G3 variant 2 cDNA (see e.g., the Example entitled “Production of Recombinant 282P1G3 in Eukaryotic Systems” and FIG. 19). Other recombinant 282P1G3 variant 2-expressing cells or cells endogenously expressing 282P1G3 variant 2 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 282P1G3 specific antibody-producing clones.
To generate monoclonal antibodies that are specific for each 282P1G3 variant protein, immunogens are designed to encode sequences unique for each variant. For example, peptides or recombinant protein antigens (i.e. Tag5 fusion proteins) encompassing the unique sequence derived from alternate exon usage in splice variants 2, 3, 4, 5, 6, and 7 are used as immunogens. In one embodiment, a Tag5 protein encoding amino acids 838-893 unique to 282P1G3 variant 3 is produced, purified, and used as immunogen to derive monoclonal antibodies specific to 282P1G3 variant 3. In another embodiment, an antigenic peptide composed of amino acids 1025-1037 of 282P1G3 variant 2 is coupled to KLH and used as immunogen. In another embodiment, an antigenic peptide composed of amino acids 817-829 of 282P1G3 variant 4 is coupled to KLH and used as immunogen. In another embodiment, an antigenic peptide composed of amino acids 220-232 of 282P1G3 variant 5 is coupled to KLH and used as immunogen. In another embodiment, an antigenic peptide composed of amino acids 122-134 of 282P1G3 variant 6 is coupled to KLH and used as immunogen. In another embodiment, an antigenic peptide composed of amino acids 339-362 of 282P1G3 variant 7 is coupled to KLH and used as immunogen. Hybridoma supernatants are then screened on the respective antigen and then further screened on cells expressing the specific variant and cross-screened on cells expressing the other variants to derive variant-specific monoclonal antibodies.
The binding affinity of a 282P1G3 variant monoclonal antibody is determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which 282P1G3 variant monoclonal antibodies preferred for diagnostic or therapeutic use, as appreciated by one of skill in the art. The BIAcore system (Uppsala, Sweden) is a preferred method for determining binding affinity. The BIAcore system uses surface plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Morton and Myszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecular interactions in real time. BIAcore analysis conveniently generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants.
Example 12
HLA Class I and Class II Binding Assays
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 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.
Since under these conditions [label]<[HLA] and IC50≧[HLA], the measured IC50 values are reasonable approximations of the true KD values. Peptide inhibitors are typically tested at concentrations ranging from 120 μg/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 IC50 of a positive control for inhibition by the IC50 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 IC50 nM values by dividing the IC50 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.
Binding assays as outlined above may be used to analyze HLA supermotif and/or HLA motif-bearing peptides (see Table IV).
Example 13
Identification of HLA Supermotif- and Motif-Bearing CTL Candidate Epitopes
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.
Computer Searches and Algorithms for Identification of Supermotif and/or Motif-bearing Epitopes
The searches performed to identify the motif-bearing peptide sequences in the Example entitled “Antigenicity Profiles” and Tables VIII-XXI and XXII-XLIX employ the protein sequence data from the gene product of 282P1G3 set forth in FIGS. 2 and 3, the specific search peptides used to generate the tables are listed in Table VII.
Computer searches for epitopes bearing HLA Class I or Class II supermotifs or motifs are performed as follows. All translated 282P1G3 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.
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 ΔG) of peptide-HLA molecule interactions can be approximated as a linear polynomial function of the type:

“ΔG”=a1i×a2i×a3i . . . ×ani 

where aji 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 ji to the free energy of binding of the peptide irrespective of the sequence of the rest of the peptide.
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 ji. 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.
Selection of HLA-A2 Supertype Cross-reactive Peptides
Protein sequences from 282P1G3 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).
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.
Selection of HLA-A3 Supermotif-bearing Epitopes
The 282P1G3 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.
Selection of HLA-B7 Supermotif Bearing Epitopes
The 282P1G3 protein(s) scanned above is also analyzed for the presence of 8-, 9-10-, or 11-mer peptides with the HLA-B7-supermotif. Corresponding peptides are synthesized and tested for binding to HLA-B*0702, the molecule encoded by the most common B7-supertype allele (i.e., the prototype B7 supertype allele). Peptides binding B*0702 with IC50 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.
Selection of A1 and A24 Motif-bearing Epitopes
To further increase population coverage, HLA-A1 and -A24 epitopes can also be incorporated into vaccine compositions. An analysis of the 282P1G3 protein can also be performed to identify HLA-A1- and A24-motif-containing sequences.
High affinity and/or cross-reactive binding epitopes that bear other motif and/or supermotifs are identified using analogous methodology.
Example 14
Confirmation of Immunogenicity
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:
Target Cell Lines for Cellular Screening:
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.
Primary CTL Induction Cultures:
Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI with 30 μg/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 10×106 PBMC/well in a 6-well plate. After 2 hours at 37° 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. TNFα is added to the DCs on day 6 at 75 ng/ml and the cells are used for CTL induction cultures on day 7.
Induction of CTL with DC and Peptide: CD8+ T-cells are isolated by positive selection with Dynal immunomagnetic beads (Dynabeads® M-450) and the detacha-bead® reagent. Typically about 200-250×106 PBMC are processed to obtain 24×106 CD8+ T-cells (enough for a 48-well plate culture). Briefly, the PBMCs are thawed in RPMI with 30 μg/ml DNAse, washed once with PBS containing 1% human AB serum and resuspended in PBS/1% AB serum at a concentration of 20×106 cells/ml. The magnetic beads are washed 3 times with PBS/AB serum, added to the cells (140 μl beads/20×106 cells) and incubated for 1 hour at 4° C. with continuous mixing. The beads and cells are washed 4× with PBS/AB serum to remove the nonadherent cells and resuspended at 100×106 cells/ml (based on the original cell number) in PBS/AB serum containing 100 μl/ml detacha-bead® reagent and 30 μg/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 μg/ml of peptide at a cell concentration of 1-2×106/ml in the presence of 3 μg/ml β2-microglobulin for 4 hours at 20° C. The DC are then irradiated (4,200 rads), washed 1 time with medium and counted again.
Setting up induction cultures: 0.25 ml cytokine-generated DC (at 1×105 cells/ml) are co-cultured with 0.25 ml of CD8+ T-cells (at 2×106 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.
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 5×106 cells/ml and irradiated at −4200 rads. The PBMCs are plated at 2×106 in 0.5 ml complete medium per well and incubated for 2 hours at 37° 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 μg/ml of peptide in the presence of 3 μg/ml β2 microglobulin in 0.25 ml RPMI/5% AB per well for 2 hours at 37° 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 51Cr release assay. In some experiments the cultures are assayed for peptide-specific recognition in the in situ IFNγ 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.
Measurement of CTL Lytic Activity by 51Cr Release.
Seven days after the second restimulation, cytotoxicity is determined in a standard (5 hr) 51Cr release assay by assaying individual wells at a single E:T. Peptide-pulsed targets are prepared by incubating the cells with 10 μg/ml peptide overnight at 37° C.
Adherent target cells are removed from culture flasks with trypsin-EDTA. Target cells are labeled with 200 μCi of 51Cr sodium chromate (Dupont, Wilmington, Del.) for 1 hour at 37° C. Labeled target cells are resuspended at 106 per ml and diluted 1:10 with K562 cells at a concentration of 3.3×106/ml (an NK-sensitive erythroblastoma cell line used to reduce non-specific lysis). Target cells (100 μl) and effectors (100 μl) are plated in 96 well round-bottom plates and incubated for 5 hours at 37° C. At that time, 100 μl 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 51Cr release sample)/(cpm of the maximal 51Cr release sample−cpm of the spontaneous 51Cr release sample)]×100.

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.
In situ Measurement of Human IFNγ Production as an Indicator of Peptide-Specific and Endogenous Recognition
Immulon 2 plates are coated with mouse anti-human IFNγ monoclonal antibody (4 μg/ml 0.1M NaHCO3, pH8.2) overnight at 4° C. The plates are washed with Ca2+, Mg2+-free PBS/0.05% Tween 20 and blocked with PBS/10% FCS for two hours, after which the CTLs (100 μl/well) and targets (100 μl/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 1×106 cells/ml. The plates are incubated for 48 hours at 37° C. with 5% CO2.
Recombinant human IFN-gamma is added to the standard wells starting at 400 pg or 1200 pg/100 microliter/well and the plate incubated for two hours at 37° C. The plates are washed and 100 μl 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× 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 H3PO4 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.
CTL Expansion.
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, 5×104 CD8+ cells are added to a T25 flask containing the following: 1×106 irradiated (4,200 rad) PBMC (autologous or allogeneic) per ml, 2×105 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 μM 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 1×106/ml and the cultures are assayed between days 13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the 51Cr release assay or at 1×106/ml in the in situ IFNγ assay using the same targets as before the expansion.
Cultures are expanded in the absence of anti-CD3+ as follows. Those cultures that demonstrate specific lytic activity against peptide and endogenous targets are selected and 5×104 CD8+ cells are added to a T25 flask containing the following: 1×106 autologous PBMC per ml which have been peptide-pulsed with 10 μg/ml peptide for two hours at 37° C. and irradiated (4,200 rad); 2×105 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.
Immunogenicity of A2 Supermotif-bearing Peptides
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.
Immunogenicity can also be confirmed using PBMCs isolated from patients bearing a tumor that expresses 282P1G3. 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.
Evaluation of A*03/A11 Immunogenicity
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.
Evaluation of B7 Immunogenicity
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.
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
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.
Analoging at Primary Anchor Residues
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.
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.
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.
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 IC50 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).
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.
Analoging of HLA-A3 and B7-Supermotif-bearing Peptides
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.
The analog peptides are then tested for the ability to bind A*03 and A*11 (prototype A3 supertype alleles). Those peptides that demonstrate ≦500 nM binding capacity are then confirmed as having A3-supertype cross-reactivity.
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).
Analoging at primary anchor residues of other motif and/or supermotif-bearing epitopes is performed in a like manner.
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.
Analoging at Secondary Anchor Residues
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.
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 282P1G3-expressing tumors.
Other Analoging Strategies
Another form of peptide analoging, unrelated to anchor positions, involves the substitution of a cysteine with α-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 α-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 1. Chen, John Wiley & Sons, England, 1999).
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 282P1G3-Derived Sequences with HLA-DR Binding Motifs
Peptide epitopes bearing an HLA class II supermotifor motif are identified and confirmed as outlined below using methodology similar to that described for HLA Class I peptides.
Selection of HLA-DR-Supermotif-bearing Epitopes.
To identify 282P1G3-derived, HLA class II HTL epitopes, a 282P1G3 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).
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.
The 282P1G3-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 β1, DR2w2 β2, 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. 282P1G3-derived peptides found to bind common HLA-DR alleles are of particular interest.
Selection of DR3 Motif Peptides
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.
To efficiently identify peptides that bind DR3, target 282P1G3 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 μM or better, i.e., less than 1 μM. Peptides are found that meet this binding criterion and qualify as HLA class II high affinity binders.
DR3 binding epitopes identified in this manner are included in vaccine compositions with DR supermotif-bearing peptide epitopes.
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 282P1G3-derived HTL Epitopes
This example determines immunogenic DR supermotif- and DR3 motif-bearing epitopes among those identified using the methodology set forth herein.
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 282P1G3-expressing tumors.
Example 18
Calculation of Phenotypic Frequencies of HLA-supertypes in Various Ethnic Backgrounds to Determine Breadth of Population Coverage
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.
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)2].
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).
Population coverage achieved by combining the A2-, A3- and B7-supertypes is approximately 86% in five major ethnic groups. Coverage may be extended by including peptides bearing the A1 and A24 motifs. On average, A1 is present in 12% and A24 in 29% of the population across five different major ethnic groups (Caucasian, North American Black, Chinese, Japanese, and Hispanic). Together, these alleles are represented with an average frequency of 39% in these same ethnic populations. The total coverage across the major ethnicities when A1 and A24 are combined with the coverage of the A2-, A3- and B7-supertype alleles is >95%, see, e.g., Table IV (G). An analogous approach can be used to estimate population coverage achieved with combinations of class II motif-bearing epitopes.
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.
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
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.
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 51Cr labeled Jurkat-A2.1/Kb target cells in the absence or presence of peptide, and also tested on 51Cr labeled target cells bearing the endogenously synthesized antigen, i.e. cells that are stably transfected with 282P1G3 expression vectors.
The results demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized 282P1G3 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/Kb 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
This example illustrates the induction of CTLs and HTLs in transgenic mice, by use of a 282P1G3-derived CTL and HTL peptide vaccine compositions. The vaccine composition used herein comprise peptides to be administered to a patient with a 282P1G3-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.
Immunization procedures: Immunization of transgenic mice is performed as described (Alexander et al., J. Immunol. 159:4753-4761, 1997). For example, A2/Kb 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.
Cell lines: Target cells for peptide-specific cytotoxicity assays are Jurkat cells transfected with the HLA-A2.1/Kb chimeric gene (e.g., Vitiello et al., J. Exp. Med. 173:1007, 1991)
In vitro CTL activation: One week after priming, spleen cells (30×106 cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (10×106 cells/flask) in 10 ml of culture medium/T25 flask. After six days, effector cells are harvested and assayed for cytotoxic activity.
Assay for cytotoxic activity: Target cells (1.0 to 1.5×106) are incubated at 37° C. in the presence of 200 μl of 51Cr. After 60 minutes, cells are washed three times and resuspended in R10 medium. Peptide is added where required at a concentration of 1 μg/ml. For the assay, 104 51Cr-labeled target cells are added to different concentrations of effector cells (final volume of 200 μl) in U-bottom 96-well plates. After a six hour incubation period at 37° 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×(experimental release−spontaneous release)/(maximum release−spontaneous release). To facilitate comparison between separate CTL assays run under the same conditions, % 51Cr release data is expressed as lytic units/106 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 51Cr release assay. To obtain specific lytic units/106, the lytic units/106 obtained in the absence of peptide is subtracted from the lytic units/106 obtained in the presence of peptide. For example, if 30% 51Cr release is obtained at the effector (E): target (T) ratio of 50:1 (i.e., 5×105 effector cells for 10,000 targets) in the absence of peptide and 5:1 (i.e., 5×104 effector cells for 10,000 targets) in the presence of peptide, the specific lytic units would be: [(1/50,000)−(1/500,000)]×106=18 LU.
The results are analyzed to assess the magnitude of the CTL responses of animals injected with the immunogenic CTL/HTL conjugate vaccine preparation and are compared to the magnitude of the CTL response achieved using, for example, CTL epitopes as outlined above in the Example entitled “Confirmation of Immunogenicity.” Analyses similar to this may be performed to confirm the immunogenicity of peptide conjugates containing multiple CTL epitopes and/or multiple HTL epitopes. In accordance with these procedures, it is found that a CTL response is induced, and concomitantly that an HTL response is induced upon administration of such compositions.
Example 21
Selection of CTL and HTL Epitopes for Inclusion in a 282P1G3-specific Vaccine
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.
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.
Epitopes are selected which, upon administration, mimic immune responses that are correlated with 282P1G3 clearance. The number of epitopes used depends on observations of patients who spontaneously clear 282P1G3. For example, if it has been observed that patients who spontaneously clear 282P1G3-expressing cells generate an immune response to at least three (3) epitopes from 282P1G3 antigen, then at least three epitopes should be included for HLA class I. A similar rationale is used to determine HLA class II epitopes.
Epitopes are often selected that have a binding affinity of an IC50 of 500 nM or less for an HLA class I molecule, or for class II, an IC50 of 1000 nM or less; or HLA Class I peptides with high binding scores from the BIMAS web site on the World Wide Web.
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.
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 282P1G3, 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.
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 282P1G3.
Example 22
Construction of “Minigene” Multi-Epitope DNA Plasmids
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.
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 282P1G3, are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from 282P1G3 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.
Such a construct may additionally include sequences that direct the HTL epitopes to the endoplasmic reticulum. For example, the li protein may be fused to one or more HTL epitopes as described in the art, wherein the CLIP sequence of the li protein is removed and replaced with an HLA class II epitope sequence so that HLA class II epitope is directed to the endoplasmic reticulum, where the epitope binds to an HLA class II molecules.
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.
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.
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° C. for 15 sec, annealing temperature (5° below the lowest calculated Tm of each primer pair) for 30 sec, and 72° C. for 1 min.
For example, a minigene is prepared as follows. For a first PCR reaction, 5 μg 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 μl reactions containing Pfu polymerase buffer (1x=10 mM KCL, 10 mM (NH4)2SO4, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO4, 0.1% Triton X-100, 100 μg/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
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).
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.
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/Kb transgenic mice, for example, are immunized intramuscularly with 100 μg 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.
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 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.
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.
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-restricted mice, for example, are immunized intramuscularly with 100 μg of plasmid DNA. As a means of comparing the level of HTLs induced by DNA immunization, a group of control animals is also immunized with an actual peptide composition emulsified in complete Freund's adjuvant. CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunized animals and stimulated with each of the respective compositions (peptides encoded in the minigene). The HTL response is measured using a 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.
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).
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/Kb transgenic mice are immunized IM with 100 μg 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 107 pfu/mouse of a recombinant vaccinia virus expressing the same sequence encoded by the DNA minigene. Control mice are immunized with 100 μg 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.
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-AL 1 or HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 or HLA-B7 motif or supermotif epitopes. The use of prime boost protocols in humans is described below in the Example entitled “Induction of CTL Responses Using a Prime Boost Protocol.”
Example 24
Peptide Compositions for Prophylactic Uses
Vaccine compositions of the present invention can be used to prevent 282P1G3 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 282P1G3-associated tumor.
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 μg, generally 100-5,000 μg, 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 282P1G3-associated disease.
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 282P1G3 Sequences
A native 282P1G3 polyprotein sequence is analyzed, preferably using computer algorithms defined for each class I and/or class II supermotif or motif, to identify “relatively short” regions of the polyprotein that comprise multiple epitopes. The “relatively short” regions are preferably less in length than an entire native antigen. This relatively short sequence that contains multiple distinct or overlapping, “nested” epitopes can be used to generate a minigene construct. The construct is engineered to express the peptide, which corresponds to the native protein sequence. The “relatively short” peptide is generally less than 250 amino acids in length, often less than 100 amino acids in length, preferably less than 75 amino acids in length, and more preferably less than 50 amino acids in length. The protein sequence of the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, i.e., it has a high concentration of epitopes. As noted herein, epitope motifs may be nested or overlapping (i.e., frame shifted relative to one another). For example, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes.
The vaccine composition will include, for example, multiple CTL epitopes from 282P1G3 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.
The embodiment of this example provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally, such an embodiment provides for the possibility of motif-bearing epitopes for an HLA makeup(s) that is presently unknown. Furthermore, this embodiment (excluding an analoged embodiment) directs the immune response to multiple peptide sequences that are actually present in native 282P1G3, 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.
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
The 282P1G3 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 282P1G3 and such other antigens. For example, a vaccine composition can be provided as a single polypeptide that incorporates multiple epitopes from 282P1G3 as well as tumor-associated antigens that are often expressed with a target cancer associated with 282P1G3 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
Peptides of the invention may be used to analyze an immune response for the presence of specific antibodies, CTL or HTL directed to 282P1G3. 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.
In this example highly sensitive human leukocyte antigen tetrameric complexes (“tetramers”) are used for a cross-sectional analysis of, for example, 282P1G3 HLA-A*0201-specific CTL frequencies from HLA A*0201-positive individuals at different stages of disease or following immunization comprising a 282P1G3 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 β2-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, β2-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.
For the analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 μl 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 282P1G3 epitope, and thus the status of exposure to 282P1G3, or exposure to a vaccine that elicits a protective or therapeutic response.
Example 28
Use of Peptide Epitopes to Evaluate Recall Responses
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 282P1G3-associated disease or who have been vaccinated with a 282P1G3 vaccine.
For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any 282P1G3 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.
PBMC from vaccinated individuals are separated on Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis, Mo.), washed three times in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 μg/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 μg/ml to each well and HBV core 128-140 epitope is added at 1 μg/ml to each well as a source of T cell help during the first week of stimulation.
In the microculture format, 4×105 PBMC are stimulated with peptide in 8 replicate cultures in 96-well round bottom plate in 100 μl/well of complete RPMI. On days 3 and 10, 100 μl of complete RPMI and 20 U/ml final concentration of rlL-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 105 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 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).
Target cell lines are autologous and allogeneic EBV-transformed B-LCL that are either purchased from the American Society for Histocompatibility and Immunogenetics (ASH I, Boston, Mass.) or established from the pool of patients as described (Guilhot, et al. J. Virol. 66:2670-2678, 1992).
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 μM, and labeled with 100 μCi of 51Cr (Amersham Corp., Arlington Heights, Ill.) for 1 hour after which they are washed four times with HBSS.
Cytolytic activity is determined in a standard 4-h, split well 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×[(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.
The results of such an analysis indicate the extent to which HLA-restricted CTL populations have been stimulated by previous exposure to 282P1G3 or a 282P1G3 vaccine.
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 1.5×105 cells/well and are stimulated with 10 μg/ml synthetic peptide of the invention, whole 282P1G3 antigen, or PHA. Cells are routinely plated in replicates of 4-6 wells for each condition. After seven days of culture, the medium is removed and replaced with fresh medium containing 10 U/ml IL-2. Two days later, 1 μCi 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 3H-thymidine incorporation. Antigen-specific T cell proliferation is calculated as the ratio of 3H-thymidine incorporation in the presence of antigen divided by the 3H-thymidine incorporation in the absence of antigen.
Example 29
Induction Of Specific CTL Response in Humans
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:
A total of about 27 individuals are enrolled and divided into 3 groups:
Group I: 3 subjects are injected with placebo and 6 subjects are injected with 5 μg of peptide composition;
Group II: 3 subjects are injected with placebo and 6 subjects are injected with 50 μg peptide composition;
Group III: 3 subjects are injected with placebo and 6 subjects are injected with 500 μg of peptide composition.
After 4 weeks following the first injection, all subjects receive a booster inoculation at the same dosage.
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.
Safety: The incidence of adverse events is monitored in the placebo and drug treatment group and assessed in terms of degree and reversibility.
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.
The vaccine is found to be both safe and efficacious.
Example 30
Phase II Trials in Patients Expressing 282P1G3
Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients having cancer that expresses 282P1G3. The main objectives of the trial are to determine an effective dose and regimen for inducing CTLs in cancer patients that express 282P1G3, 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:
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.
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 282P1G3.
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 282P1G3-associated disease.
Example 31
Induction of CTL Responses Using a Prime Boost Protocol
A prime boost protocol similar in its underlying principle to that used to confirm the efficacy of a DNA vaccine in transgenic mice, such as described above in the Example entitled “The Plasmid Construct and the Degree to Which It Induces Immunogenicity,” can also be used for the administration of the vaccine to humans. Such a vaccine regimen can include an initial administration of, for example, naked DNA followed by a boost using recombinant virus encoding the vaccine, or recombinant protein/polypeptide or a peptide mixture administered in an adjuvant.
For example, the initial immunization may be performed using an expression vector, such as that constructed in the Example entitled “Construction of “Minigene” Multi-Epitope DNA Plasmids” in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 μg) 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 5-107 to 5×109 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.
Analysis of the results indicates that a magnitude of response sufficient to achieve a therapeutic or protective immunity against 282P1G3 is generated.
Example 32
Administration of Vaccine Compositions Using Dendritic Cells (DC)
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 282P1G3 protein from which the epitopes in the vaccine are derived.
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 ProgenipoietinTM (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.
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 2-50×106 DC per patient are typically administered, larger number of DC, such as 107 or 108 can also be provided. Such cell populations typically contain between 50-90% DC.
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 ProgenipoietinTM are injected into patients without purification of the DC. The total number of PBMC that are administered often ranges from 108 to 1010. 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 ProgenipoietinTM mobilizes 2% DC in the peripheral blood of a given patient, and that patient is to receive 5×106 DC, then the patient will be injected with a total of 2.5×108 peptide-loaded PBMC. The percent DC mobilized by an agent such as ProgenipoietinTM is typically estimated to be between 2-10%, but can vary as appreciated by one of skill in the art.
Ex Vivo Activation of CTL/HTL Responses
Alternatively, ex vivo CTL or HTL responses to 282P1G3 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
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. 282P1G3. 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.
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 282P1G3 to isolate peptides corresponding to 282P1G3 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.
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
Sequences complementary to the 282P1G3-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring 282P1G3. 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 282P1G3. 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 282P1G3-encoding transcript.
Example 35
Purification of Naturally-occurring or Recombinant 282P1G3 Using 282P1G3-Specific Antibodies
Naturally occurring or recombinant 282P1G3 is substantially purified by immunoaffinity chromatography using antibodies specific for 282P1G3. An immunoaffinity column is constructed by covalently coupling anti-282P1G3 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.
Media containing 282P1G3 are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of 282P1G3 (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/282P1G3 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 282P1G3
282P1G3, 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 282P1G3, washed, and any wells with labeled 282P1G3 complex are assayed. Data obtained using different concentrations of 282P1G3 are used to calculate values for the number, affinity, and association of 282P1G3 with the candidate molecules.
Example 37
In Vivo Assay for 282P1G3 Tumor Growth Promotion
The effect of the 282P1G3 protein on tumor cell growth is evaluated in vivo by evaluating tumor development and growth of cells expressing or lacking 282P1G3. For example, SCID mice are injected subcutaneously on each flank with 1×106 of either 3T3, ovarian (e.g. PA-1 cells), pancreatic (e.g. Panc-1 cells) or lymphoma (e.g. Daudi cells) cancer cell lines containing tkNeo empty vector or 282P1G3. At least two strategies may be used: (1) Constitutive 282P1G3 expression under regulation of a promoter such as a constitutive promoter obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, provided such promoters are compatible with the host cell systems, and (2) Regulated expression under control of an inducible vector system, such as ecdysone, tetracycline, etc., provided such promoters are compatible with the host cell systems. Tumor volume is then monitored by caliper measurement at the appearance of palpable tumors and followed over time to determine if 282P1G3-expressing cells grow at a faster rate and whether tumors produced by 282P1G3-expressing cells demonstrate characteristics of altered aggressiveness (e.g. enhanced metastasis, vascularization, reduced responsiveness to chemotherapeutic drugs).
Additionally, mice can be implanted with 1×105 of the same cells orthotopically to determine if 282P1G3 has an effect on local growth in the pancreas, and whether 282P1G3 affects the ability of the cells to metastasize, specifically to lymph nodes, and bone (Miki, T et al., Oncol Res. 2001; 12:209; Fu X et al., Int J Cancer. 1991, 49:938). The effect of 282P1G3 on bone tumor formation and growth may be assessed by injecting tumor cells intratibially.
The assay is also useful to determine the 282P1G3 inhibitory effect of candidate therapeutic compositions, such as, 282P1G3 intrabodies, 282P1G3 antisense molecules and ribozymes.
Example 38
282P1G3 Monoclonal Antibody-mediated Inhibition of Tumors in Vivo
The significant expression of 282P1G3 in cancer tissues and surface localization, together with its restrictive expression in normal tissues makes 282P1G3 a good target for antibody therapy. Similarly, 282P1G3 is a target for T cell-based immunotherapy. Thus, the therapeutic efficacy of anti-282P1G3 mAbs in human cancer xenograft mouse models, including ovarian, pancreatic or lymphoma and other -282P1G3 cancers listed in Table I, is evaluated by using recombinant cell lines such as Pa-1-282P1G3, Panc1-282P1G3, Daudi-282P1G3, and 3T3-282P1G3 (see, e.g., Kaighn, M. E., et al., Invest Urol, 1979. 17(1): 16-23), as well as human xenograft models (Saffran et al. PNAS 1999, 10:1073-1078).
Antibody efficacy on tumor growth and metastasis formation is studied, e.g., in a mouse orthotopic ovary, pancreas, or blood cancer xenograft models. The antibodies can be unconjugated, as discussed in this Example, or can be conjugated to a therapeutic modality, as appreciated in the art. Anti-282P1G3 mAbs inhibit formation of tumors in mouse xenografts. Anti-282P1G3 mAbs also retard the growth of established orthotopic tumors and prolonged survival of tumor-bearing mice. These results indicate the utility of anti-282P1G3 mAbs in the treatment of local and advanced stages several solid tumors. (See, e.g., Saffran, D., et al., PNAS 10:1073-1078 or on the World Wide Web.
Administration of the anti-282P1G3 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 282P1G3 as an attractive target for immunotherapy and demonstrate the therapeutic potential of anti-282P1G3 mAbs for the treatment of local and metastatic cancer. This example indicates that unconjugated 282P1G3 monoclonal antibodies are effective to inhibit the growth of human pancreatic, ovarian and lymphomas tumor xenografts grown in SCID mice; accordingly a combination of such efficacious monoclonal antibodies is also effective.
Tumor Inhibition Using Multiple Unconjugated 282P1G3 mAbs
Materials and Methods:
282P1G3 Monoclonal Antibodies:
Monoclonal antibodies are raised against 282P1G3 as described in the Example entitled “Generation of 282P1G3 Monoclonal Antibodies (mAbs).” The antibodies are characterized by ELISA, Western blot, FACS, and immunoprecipitation for their capacity to bind 282P1G3. Epitope mapping data for the anti-282P1G3 mAbs, as determined by ELISA and Western analysis, recognize epitopes on the 282P1G3 protein. Immunohistochemical analysis of cancer tissues and cells with these antibodies is performed.
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° C. Protein determinations are performed by a Bradford assay (Bio-Rad, Hercules, Calif.). A therapeutic monoclonal antibody or a cocktail comprising a mixture of individual monoclonal antibodies is prepared and used for the treatment of mice receiving subcutaneous or orthotopic injections of PC3, UM-UC3, CaKi and A427 tumor xenografts.
Cell Lines and Xenografts
The cancer cell lines PA-1, Panc1, Daudi cell lines, as well as the fibroblast line NIH 3T3 (American Type Culture Collection) are maintained in DMEM supplemented with L-glutamine and 10% FBS.
PA1-282P1G3, Panc1-282P1G3, Daudi-282P1G3 and 3T3-282P1G3 cell populations are generated by retroviral gene transfer as described in Hubert, R. S., et al., Proc Natl Acad Sci USA, 1999. 96(25): 14523.
Human patient-derived xenografts are passaged in 6- to 8-week-old male ICR-severe combined immunodeficient (SCID) mice (Taconic Farms) by s.c. trocar implant (Craft, N., et al., Nat Med. 1999, 5:280). Single-cell suspensions of tumor cells are prepared as described in Craft, et al.
Xenograft Mouse Models.
Subcutaneous (s.c.) tumors are generated by injection of 2×10 6 cancer cells mixed at a 1:1 dilution with Matrigel (Collaborative Research) in the right flank of male SCID mice. To test antibody efficacy on tumor formation, i.e. antibody injections are started on the same day as tumor-cell injections. As a control, mice are injected with either purified mouse IgG (ICN) or PBS; or a purified monoclonal antibody that recognizes an irrelevant antigen not expressed in human cells. In preliminary studies, no difference is found between mouse IgG or PBS on tumor growth. Tumor sizes are determined by caliper measurements, and the tumor volume is calculated as length×width×height. Mice with Subcutaneous tumors greater than 1.5 cm in diameter are sacrificed.
Orthotopic injections are performed under anesthesia by using ketamine/xylazine. Following tumor implantation, the mice are segregated into groups for the appropriate treatments, with anti-282P1G3 or control mAbs being injected i.p. To monitor tumor growth, mice are palpated and blood is collected on a weekly basis to measure hCG levels.
Anti-282P1G3 mAbs Inhibit Growth of 282P1G3-Expressing Xenograft-Cancer Tumors
The effect of anti-282P1G3 mAbs on tumor formation is tested by using cell line (e.g. PA-1, Panc1, Daudi and 3T3) and patient-derived tumor orthotopic models. As compared with the s.c. tumor model, the orthotopic model, which requires injection of tumor cells directly in the mouse organ results in a local tumor growth, development of metastasis in distal sites, deterioration of mouse health, and subsequent death (Saffran, D., et al., PNAS supra). The features make the orthotopic model more representative of human disease progression and allowed us to follow the therapeutic effect of mAbs on clinically relevant end points.
A major advantage of the orthotopic cancer models is the ability to study the development of metastases. Formation of metastasis in mice bearing established orthotopic tumors is studies by IHC analysis on lung sections using an antibody against a tumor-specific cell-surface protein such as anti-CK20 for prostate cancer (Lin S et al., Cancer Detect Prev. 2001; 25:202).
Another advantage of xenograft cancer models is the ability to study neovascularization and angiogenesis. Tumor growth is partly dependent on new blood vessel development. Although the capillary system and developing blood network is of host origin, the initiation and architecture of the neovasculature is regulated by the xenograft tumor (Davidoff A M et al., Clin Cancer Res. 2001; 7:2870; Solesvik O et al., Eur J Cancer Clin Oncol. 1984, 20:1295). The effect of antibody and small molecule on neovascularization is studied in accordance with procedures known in the art, such as by IHC analysis of tumor tissues and their surrounding microenvironment.
Mice bearing established orthotopic tumors are administered 1000 μp injections of either anti-282P1G3 mAb or PBS over a 4-week period. Mice in both groups are allowed to establish a high tumor burden, to ensure a high frequency of metastasis formation in mouse lungs. Mice then are killed and their bladders, livers, bone and lungs are analyzed for the presence of tumor cells by IHC analysis. These studies demonstrate a broad anti-tumor efficacy of anti-282P1G3 antibodies on initiation and progression of prostate cancer in xenograft mouse models. Anti-282P1G3 antibodies inhibit tumor formation of tumors as well as retarding the growth of already established tumors and prolong the survival of treated mice. Moreover, anti-282P1G3 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-282P1G3 mAbs are efficacious on major clinically relevant end points (tumor growth), prolongation of survival, and health.
Example 39
Therapeutic and Diagnostic Use of Anti-282P1G3 Antibodies in Humans
Anti-282P1G3 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-282P1G3 mAb show strong extensive staining in carcinoma but significantly lower or undetectable levels in normal tissues. Detection of 282P1G3 in carcinoma and in metastatic disease demonstrates the usefulness of the mAb as a diagnostic and/or prognostic indicator. Anti-282P1G3 antibodies are therefore used in diagnostic applications such as immunohistochemistry of kidney biopsy specimens to detect cancer from suspect patients.
As determined by flow cytometry, anti-282P1G3 mAb specifically binds to carcinoma cells. Thus, anti-282P1G3 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 282P1G3. Shedding or release of an extracellular domain of 282P1G3 into the extracellular milieu, such as that seen for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology 27:563-568 (1998)), allows diagnostic detection of 282P1G3 by anti-282P1G3 antibodies in serum and/or urine samples from suspect patients.
Anti-282P1G3 antibodies that specifically bind 282P1G3 are used in therapeutic applications for the treatment of cancers that express 282P1G3. Anti-282P1G3 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-282P1G3 antibodies are tested for efficacy of tumor prevention and growth inhibition in the SCID mouse cancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6, (see, e.g., the Example entitled “282P1G3 Monoclonal Antibody-mediated Inhibition of Bladder and Lung Tumors In Vivo”). Either conjugated and unconjugated anti-282P1G3 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-282P1G3 Antibodies In vivo
Antibodies are used in accordance with the present invention which recognize an epitope on 282P1G3, and are used in the treatment of certain tumors such as those listed in Table I. Based upon a number of factors, including 282P1G3 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.
I.) Adjunctive therapy: In adjunctive therapy, patients are treated with anti-282P1G3 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-282P1G3 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-282P1G3 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).
II.) Monotherapy: In connection with the use of the anti-282P1G3 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.
III.) Imaging Agent: Through binding a radionuclide (e.g., iodine or yttrium (I131, Y90) to anti-282P1G3 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 282P1G3. In connection with the use of the anti-282P1G3 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 (111In)-282P1G3 antibody is used as an imaging agent in a Phase I human clinical trial in patients having a carcinoma that expresses 282P1G3 (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.
Dose and Route of Administration
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-282P1G3 antibodies can be administered with doses in the range of 5 to 400 mg/m2, with the lower doses used, e.g., in connection with safety studies. The affinity of anti-282P1G3 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-282P1G3 antibodies that are fully human antibodies, as compared to the chimeric antibody, have slower clearance; accordingly, dosing in patients with such fully human anti-282P1G3 antibodies can be lower, perhaps in the range of 50 to 300 mg/m2, and still remain efficacious. Dosing in mg/m2, 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.
Three distinct delivery approaches are useful for delivery of anti-282P1G3 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.
Clinical Development Plan (CDP)
Overview: The CDP follows and develops treatments of anti-282P1G3 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-282P1G3 antibodies. As will be appreciated, one criteria that can be utilized in connection with enrollment of patients is 282P1G3 expression levels in their tumors as determined by biopsy.
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 282P1G3. Standard tests and follow-up are utilized to monitor each of these safety concerns. Anti-282P1G3 antibodies are found to be safe upon human administration.
Example 41
Human Clinical Trial Adjunctive Therapy with Human Anti-282P1G3 Antibody and Chemotherapeutic Agent
A phase I human clinical trial is initiated to assess the safety of six intravenous doses of a human anti-282P1G3 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-282P1G3 antibodies when utilized as an adjunctive therapy to an antineoplastic or chemotherapeutic agent as defined herein, such as, without limitation: cisplatin, topotecan, doxorubicin, adriamycin, taxol, or the like, is assessed. The trial design includes delivery of six single doses of an anti-282P1G3 antibody with dosage of antibody escalating from approximately about 25 mg/m 2 to about 275 mg/m2 over the course of the treatment in accordance with the following schedule:


















Day 0
Day 7
Day 14
Day 21
Day 28
Day 35






















mAb Dose
25 mg/m2
75 mg/m2
125 mg/m2
175 mg/m2
225 mg/m2
275 mg/m2


Chemotherapy
+
+
+
+
+
+


(standard dose)









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 282P1G3. 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.
The anti-282P1G3 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-282P1G3 Antibody
Anti-282P1G3 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-282P1G3 antibodies.
Example 43
Human Clinical Trial: Diagnostic Imaging with Anti-282P1G3 Antibody
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-282P1G3 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
Comparison of 282P1G3 to Known Sequences
The human 282P1G3 protein exhibit a high degree of homology to a known human protein, cell adhesion molecule with homology to Li CAM precursor (gi 27894376), also known Close Homolog of L1 (CHL1) or CALL. Human CHL1 shows 99% identity to 282P1G3 at the protein level (FIG. 4A). The mouse homolog of 282P1G3 has been identified as murine CHL1 (gi 6680936), and shows 82% identity and 89% homology to 282P1G3 (FIG. 4B). CHL1 has been reported to regulate neuronal development by altering cell adhesion and axonal projections (Montag-Sallaz M et al., Mol. Cell. Biol. 2002, 22:7967). In addition, CHL1 was found to play a role in neurite growth and survival (Dong L et al., J. Neurosci. Res, 2002; Chaisuksunt V et al., J. Comp. Neurol 2000, 425:382). Mutations in CHL1 have been associated with schizophrenia and metal disorders (Sakurai et al., Mol Psychiatry 2002, 7:412; We H et al., Hum Genet 1998, 103:355).
The prototype member of the 282P1G3 family, 282P1G3v.1, is a 1224 amino acids protein. Initial bioinformatics analysis using topology prediction programs suggested that 191P2D14 may contain 2 transmembranes based on hydrophobicity profile. However, the first hydrophobic domain was identified as a signal sequence, rendering 191P2D12 a single transmembrane protein.
The 282P1G3 gene has several variants, including 5 SNP represented by 282P1G3 v.9, v.10, v.11, v.24 and v.25. In addition, several splice variants have been identified, including deletion variants such as 282P1G3 v.2, v.4, v.5 and v.6, as well as insertion mutants such as 282P1G3 v.7 and v.8, and a splice variant at aa 838 of 282P1G3 v.1, namely 282P1G3 v.3.
Motif analysis revealed the presence of several protein functional motifs in the 282P1G3 protein (Table L). Six immunoglobulin domains have been identified in addition to four fibronectin type III repeats. Immunoglobulin domains are found in numerous proteins and participate in protein-protein such including protein-ligand interactions (Weismann et al., J Mol Med 2000, 78:247). In addition, Ig-domains function in cell adhesion, allowing the interaction of leukocytes and blood-born cells with the endothelium (Wang and Springer, Immunol Rev 1998, 163:197). Fibronectin type III repeats are 100 amino acid domains with binding sites for various molecules, including DNA, heparin, basement membrane and cell surface proteins (Kimizuka et al., J Biol. Chem. 1991, 266:3045; Yokosaki et al., J Biol. Chem. 1998, 273:11423). The majority for proteins containing fibronectin III motifs participate in cell surface binding, binding to specific substrates including heparin, collagen, DNA, actin and fibrin, or are involved in binding to fibronectin receptors. Fibronectins have been reported to function in wound healing; cell adhesion, cell differentiation, cell migration and tumour metastasis (Bloom et al., Mol Biol Cell. 1999, 10:1521; Brodt P. Cancer Met Rev 1991, 10:23). The motifs found in 282P1G3 as well as its similarity to CHL1 indicate that 282P1G3 can participate in tumor growth and progression by enhancing the initial stages of tumorigenesis, including tumor establishment and tumor growth, by allowing adhesion to basement membranes and surrounding cells, by mediating cell migration and metastasis.
Accordingly, when 282P1G3 functions as a regulator of tumor establishment, tumor formation, tumor growth, survival or cell signaling, 282P1G3 is used for therapeutic, diagnostic, prognostic and/or preventative purposes. In addition, when a molecule, such as a splice variant or SNP of 282P1G3 is expressed in cancerous tissues, such as those listed in Table I, they are used for therapeutic, diagnostic, prognostic and/or preventative purposes.
Example 45
Regulation of Transcription
The cell surface localization of 282P1G3 coupled to the presence of Ig-domains within its sequence indicate that 282P1G3 modulates signal transduction and the transcriptional regulation of eukaryotic genes. Regulation of gene expression is confirmed, e.g., by studying gene expression in cells expressing or lacking 282P1G3. For this purpose, two types of experiments are performed.
In the first set of experiments, RNA from parental and 282P1G3-expressing cells are extracted and hybridized to commercially available gene arrays (Clontech) (Smid-Koopman E et al. Br J Cancer. 2000. 83:246). Resting cells as well as cells treated with FBS, androgen or growth factors are compared. Differentially expressed genes are identified in accordance with procedures known in the art. The differentially expressed genes are then mapped to biological pathways (Chen K et al. Thyroid. 2001. 11:41.).
In the second set of experiments, specific transcriptional pathway activation is evaluated using commercially available (Stratagene) luciferase reporter constructs including: NFkB-luc, SRE-luc, ELK1-luc, ARE-luc, p53-luc, and CRE-luc. These transcriptional reporters contain consensus binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways, and represent a good tool to ascertain pathway activation and screen for positive and negative modulators of pathway activation.
Thus, 282P1G3 plays a role in gene regulation, and it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 46
Identification and Confirmation of Potential Signal Transduction Pathways
Many mammalian proteins have been reported to interact with signaling molecules and to participate in regulating signaling pathways. (J. Neurochem. 2001; 76:217-223). Immunoglobulin-like molecules in particular has been associated with several tyrpsine kinases including Lyc, Blk, syk (Tamir and Cambier, Oncogene. 1998, 17:1353), the MAPK signaling cascade that control cell mitogenesis and calcium flux (Vilen J et al., J Immunol 1997, 159:231; Jiang F, Jia Y, Cohen I. Blood. 2002, 99:3579). In addition, the 282P1G3 protein contains several phosphorylation sites (see Table VI) indicating an association with specific signaling cascades. Using immunoprecipitation and Western blotting techniques, proteins are identified that associate with 282P1G3 and mediate signaling events. Several pathways known to play a role in cancer biology can be regulated by 282P1G3, including phospholipid pathways such as P13K, AKT, etc, adhesion and migration pathways, including FAK, Rho, Rac-1, catenin, etc, as well as mitogenic/survival cascades such as ERK, p38, etc (Cell Growth Differ. 2000, 11:279; J Biol. Chem. 1999, 274:801; Oncogene. 2000, 19:3003, J. Cell Biol. 1997, 138:913.).). In order to determine whether expression of 282P1G3 is sufficient to regulate specific signaling pathways not otherwise active in resting cancer cells, the effect of 282P1G3 on the activation of the signaling cascade is investigated in the cancer cell lines PA-1, Panc1 and Daudi. Cancer cells stably expressing 282P1G3 or neo are stimulated with growth factor, FBS or other activating molecules. Whole cell lysates are analyzed by western blotting.
To confirm that 282P1G3 directly or indirectly activates known signal transduction pathways in cells, luciferase (luc) based transcriptional reporter assays are carried out in cells expressing individual genes. These transcriptional reporters contain consensus-binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways. The reporters and examples of these associated transcription factors, signal transduction pathways, and activation stimuli are listed below.
1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress
2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation
3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress
4. ARE-luc, androgen receptor; steroids/MAPK; growth/differentiation/apoptosis
5. p53-luc, p53; SAPK; growth/differentiation/apoptosis
6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress
7. TCF-luc, TCF/Lef; -catenin, Adhesion/invasion
Gene-mediated effects can be assayed in cells showing mRNA expression. Luciferase reporter plasmids can be introduced by lipid-mediated transfection (TFX-50, Promega). Luciferase activity, an indicator of relative transcriptional activity, is measured by incubation of cell extracts with luciferin substrate and luminescence of the reaction is monitored in a luminometer.
Signaling pathways activated by 282P1G3 are mapped and used for the identification and validation of therapeutic targets. When 282P1G3 is involved in cell signaling, it is used as target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 47
Involvement in Tumor Progression
Based on the role of Ig-domains and fibronectin motifs in cell growth and signal transduction, the 282P1G3 gene can contribute to the growth, invasion and transformation of cancer cells. The role of 282P1G3 in tumor growth is confirmed in a variety of primary and transfected cell lines including prostate cell lines, as well as NIH 3T3 cells engineered to stably express 282P1G3. Parental cells lacking 282P1G3 and cells expressing 282P1G3 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).
To confirm the role of 282P1G3 in the transformation process, its effect in colony forming assays is investigated. Parental NIH-3T3 cells lacking 282P1G3 are compared to NIH-3T3 cells expressing 282P1G3, using a soft agar assay under stringent and more permissive conditions (Song Z. et al. Cancer Res. 2000; 60:6730).
To confirm the role of 282P1G3 in invasion and metastasis of cancer cells, a well-established assay is used, e.g., a Transwell Insert System assay (Becton Dickinson) (Cancer Res. 1999; 59:6010). Control cells, including prostate, breast and kidney cell lines lacking 282P1G3 are compared to cells expressing 282P1G3. 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.
282P1G3 can also play a role in cell cycle and apoptosis. Parental cells and cells expressing 282P1G3 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 282P1G3, including normal and tumor prostate cells. Engineered and parental cells are treated with various chemotherapeutic agents, such as etoposide, taxol, etc, and protein synthesis inhibitors, such as cycloheximide. Cells are stained with annexin V-FITC and cell death is measured by FACS analysis. The modulation of cell death by 282P1G3 can play a critical role in regulating tumor progression and tumor load.
When 282P1G3 plays a role in cell growth, transformation, invasion or apoptosis, it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 48
Involvement in Angiogenesis
Angiogenesis or new capillary blood vessel formation is necessary for tumor growth (Hanahan D, Folkman J. Cell. 1996, 86:353; Folkman J. Endocrinology. 1998 139:441). Based on the effect of fibronectins on tumor cell adhesion and their interaction with endothelial cells, 282P1G3 plays a role in angiogenesis (Mareel and Leroy: Physiol Rev, 83:337; DeFouw L et al., Microvasc Res 2001, 62:263). Several assays have been developed to measure angiogenesis in vitro and in vivo, such as the tissue culture assays endothelial cell tube formation and endothelial cell proliferation. Using these assays as well as in vitro neo-vascularization, the role of 282P1G3 in angiogenesis, enhancement or inhibition, is confirmed.
For example, endothelial cells engineered to express 282P1G3 are evaluated using tube formation and proliferation assays. The effect of 282P1G3 is also confirmed in animal models in vivo. For example, cells either expressing or lacking 282P1G3 are implanted subcutaneously in immunocompromised mice. Endothelial cell migration and angiogenesis are evaluated 5-15 days later using immunohistochemistry techniques. 282P1G3 affects angiogenesis, and it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 49
Involvement in Protein-Protein Interactions
Ig-domains and fibronectin motifs have been shown to mediate interaction with other proteins, including cell surface protein. Using immunoprecipitation techniques as well as two yeast hybrid systems, proteins are identified that associate with 282P1G3. Immunoprecipitates from cells expressing 282P1G3 and cells lacking 282P1G3 are compared for specific protein-protein associations.
Studies are performed to confirm the extent of association of 282P1G3 with effector molecules, such as nuclear proteins, transcription factors, kinases, phosphates etc. Studies comparing 282P1G3 positive and 282P1G3 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.
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 282P1G3-DNA-binding domain fusion protein and a reporter construct. Protein-protein interaction is detected by colorimetric reporter activity. Specific association with effector molecules and transcription factors directs one of skill to the mode of action of 282P1G3, 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 282P1G3.
Thus, it is found that 282P1G3 associates with proteins and small molecules. Accordingly, 282P1G3 and these proteins and small molecules are used for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 50
Involvement of 282P1G3 in Cell-cell Communication
Cell-cell communication is essential in maintaining organ integrity and homeostasis, both of which become deregulated during tumor formation and progression. Based on the presence of a fibronectin motif in 282P1G3, a motif known to be involved in cell interaction and cell-cell adhesion, 282P1G3 can regulate cell communication. Intercellular communications can be measured using two types of assays (J. Biol. Chem. 2000, 275:25207). In the first assay, cells loaded with a fluorescent dye are incubated in the presence of unlabeled recipient cells and the cell populations are examined under fluorescent microscopy. This qualitative assay measures the exchange of dye between adjacent cells. In the second assay system, donor and recipient cell populations are treated as above and quantitative measurements of the recipient cell population are performed by FACS analysis. Using these two assay systems, cells expressing 282P1G3 are compared to controls that do not express 282P1G3, and it is found that 282P1G3 enhances cell communications. Small molecules and/or antibodies that modulate cell-cell communication mediated by 282P1G3 are used as therapeutics for cancers that express 282P1G3. When 282P1G3 functions in cell-cell communication and small molecule transport, it is used as a target or marker for diagnostic, prognostic, preventative and/or therapeutic purposes.
Throughout this application, various website data content, publications, patent applications and patents are referenced. (Websites are referenced by their Uniform Resource Locator, or on the World Wide Web.) The disclosures of each of these references are hereby incorporated by reference herein in their entireties.
The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any that are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention.
TABLES:







TABLE I





Tissues that Express 282P1G3:


















a.
Malignant Tissues




Pancreas




Ovary




Lymph node

















TABLE II







Amino Acid Abbreviations











SINGLE LETTER
THREE LETTER
FULL NAME







F
Phe
phenylalanine



L
Leu
leucine



S
Ser
serine



Y
Tyr
tyrosine



C
Cys
cysteine



W
Trp
tryptophan



P
Pro
proline



H
His
histidine



Q
Gln
glutamine



R
Arg
arginine



I
Ile
isoleucine



M
Met
methionine



T
Thr
threonine



N
Asn
asparagine



K
Lys
lysine



V
Val
valine



A
Ala
alanine



D
Asp
aspartic acid



E
Glu
glutamic acid



G
Gly
glycine

















TABLE III







Amino Acid Substitution Matrix


Adapted from the GCG Software 9.0 BLOSUM62 amino acid substitution matrix (block


substitution matrix). The higher the value, the more likely a substitution is found in related,


natural proteins. (See world wide web URL ikp.unibe.ch/manual/blosum62.html)



























A
C
D
E
F
G
H
I
K
L
M
N
P
Q
R
S
T
V
W
Y
.






























4
0
−2
−1
−2
0
−2
−1
−1
−1
−1
−2
−1
−1
−1
1
0
0
−3
−2
A



9
−3
−4
−2
−3
−3
−1
−3
−1
−1
−3
−3
−3
−3
−1
−1
−1
−2
−2
C




6
2
−3
−1
−1
−3
−1
−4
−3
1
−1
0
−2
0
−1
−3
−4
−3
D





5
−3
−2
0
−3
1
−3
−2
0
−1
2
0
0
−1
−2
−3
−2
E






6
−3
−1
0
−3
0
0
−3
−4
−3
−3
−2
−2
−1
1
3
F







6
−2
−4
−2
−4
−3
0
−2
−2
−2
0
−2
−3
−2
−3
G








8
−3
−1
−3
−2
1
−2
0
0
−1
−2
−3
−2
2
H









4
−3
2
1
−3
−3
−3
−3
−2
−1
3
−3
−1
I










5
−2
−1
0
−1
1
2
0
−1
−2
−3
−2
K











4
2
−3
−3
−2
−2
−2
−1
1
−2
−1
L












5
−2
−2
0
−1
−1
−1
1
−1
−1
M













6
−2
0
0
1
0
−3
−4
−2
N














7
−1
−2
−1
−1
−2
−4
−3
P















5
1
0
−1
−2
−2
−1
Q
















5
−1
−1
−3
−3
−2
R

















4
1
−2
−3
−2
S


















5
0
−2
−2
T



















4
−3
−1
V




















11
2
W





















7
Y
















TABLE IV





HLA Class I/II Motifs/Supermotifs







TABLE IV (A): HLA Class I Supermotifs/Motifs











POSITION 2 (Primary Anchor)
POSITION 3 (Primary Anchor)
POSITION C Terminus (Primary Anchor)





SUPERMOTIF


A1
TILVMS

FWY


A2
LIVMATQ

IVMATL


A3
VSMATLI

RK


A24
YFWIVLMT

FIYWLM


B7
P

VILFMWYA


B27
RHK

FYLWMIVA


B44
ED

FWYLIMVA


B58
ATS

FWYLIVMA


B62
QLIVMP

FWYMIVLA


MOTIFS


A1
TSM

Y


A1

DEAS
Y


A2.1
LMVQIAT

VLIMAT


A3
LMVISATFCGD

KYRHFA


A11
VTMLISAGNCDF

KRYH


A24
YFWM

FLIW


A*3101
MVTALIS

RK


A*3301
MVALFIST

RK


A*6801
AVTMSLI

RK


SUPERMOTIF


B*0702
P

LMFWYAIV


B*3501
P

LMFWYIVA


B51
P

LIVFWYAM


B*5301
P

IMFWYALV


B*5401
P

ATIVLMFWY





Bolded residues are preferred, italicized residues are less preferred: A peptide is considered motif-bearing if it has primary anchors at each primary anchor position for a motif or supermotif as specified in the above table.







TABLE IV (B): HLA Class II Supermotif









1
6
9





W, F, Y, V, I, L
A, V, I, L, P, C, S, T
A, V, I, L, C, S, T, M, Y










TABLE IV (C): HLA Class II Motifs

















MOTIFS

1° anchor 1
2
3
4
5
1° anchor 6
7
8
9





DR4
preferred
FMYLIVW
M
T

I
VSTCPALIM
MH

MH



deleterious



W


R

WDE


DR1
preferred
MFLIVWY


PAMQ

VMATSPLIC
M

AVM



deleterious

C
CH
FD
CWD

GDE
D


DR7
preferred
MFLIVWY
M
W
A

IVMSACTPL
M

IV



deleterious

C

G


GRD
N
G


DR3
MOTIFS
1° anchor 1
2
3
1° anchor 4
5
1° anchor 6


Motif a preferred

LIVMFY


D


Motif b preferred

LIVMFAY


DNQEST

KRH


DR Supermotif

MFLIVWY




VMSTACPLI





Italicized residues indicate less preferred or “tolerated” residues







TABLE IV (D): HLA Class I Supermotifs









SUPER-
POSITION:


















MOTIFS

1
2
3
4
5
6
7
8
C-terminus





A1


1° Anchor






1° Anchor





TILVMS






FWY


A2


1° Anchor






1° Anchor





LIVMATQ






LIVMAT


A3
Preferred

1° Anchor
YFW


YFW
YFW
P
1° Anchor





VSMATLI
(4/5)


(3/5)
(4/5)
(4/5)
RK



deleterious
DE (3/5);

DE




P (5/5)

(4/5)


A24


1° Anchor






1° Anchor





YFWIVLMT






FIYWLM


B7
Preferred
FWY (5/5)
1° Anchor
FWY




FWY
1° Anchor




LIVM (3/5)
P
(4/5)




(3/5)
VILFMWYA



deleterious
DE (3/5);



DE
G
QN
DE




P(5/5);



(3/5)
(4/5)
(4/5)
(4/5)




G(4/5);




A(3/5);




QN(3/5)


B27


1° Anchor






1° Anchor





RHK






FYLWMIVA


B44


1° Anchor






1° Anchor





ED






FWYLIMVA


B58


1° Anchor






1° Anchor





ATS






FWYLIVMA


B62


1° Anchor






1° Anchor





QLIVMP






FWYMIVLA





Italicized residues indicate less preferred or “tolerated” residues







TABLE IV (E): HLA Class I Motifs









POSITION




























9













or
C-




1
2
3
4
5
6
7
8
C-terminus
terminus





A1
preferred
GFYW
1° Anchor
DEA
YFW

P
DEQN
YFW
1° Anchor


9-mer


STM






Y



deleterious
DE

RHKLIVMP
A
G
A


A1
preferred
GRHK
ASTCLIVM
1° Anchor
GSTC

ASTC
LIVM
DE
1° Anchor


9-mer



DEAS





Y



deleterious
A
RHKDEPYFW

DE
PQN
RHK
PG
GP


A1
preferred
YFW
1° Anchor
DEAQN
A
YFWQN

PASTC
GDE
P
1° Anchor


10-


STM







Y


mer



deleterious
GP

RHKGLIVM
DE
RHK
QNA
RHKYFW
RHK
A


A1
preferred
YFW
STCLIVM
1° Anchor
A
YFW

PG
G
YFW
1° Anchor


10-



DEAS






Y


mer



deleterious
RHK
RHKDEPYFW


P
G

PRHK
QN


A2.1
preferred
YFW
1° Anchor
YFW
STC
YFW

A
P
1° Anchor


9-mer


LMIVQAT






VLIMAT



deleterious
DEP

DERKH


RKH
DERKH












POSITION:





























C-




1
2
3
4
5
6
7
8
9
Terminus





A2.1
preferred
AYFW
1° Anchor
LVIM
G

G

FYWL

1° Anchor


10-


LMIVQAT





VIM

VLIMAT


mer

















deleterious
DEP

DE
RKHA
P

RKH
DERKHRKH


















A3
preferred
RHK
1° Anchor
YFW
PRHKYF
A
YFW

P
1° Anchor












LMVISATFCGD
W
KYRHFA



















deleterious
DEP

DE









A11
preferred
A
1° Anchor
YFW
YFW
A
YFW
YFW
P
1° Anchor










VTLMISAGNCD
KRYH





















F











deleterious
DEP





A
G


A24
preferred
YFWRHK
1° Anchor

STC


YFW
YFW
1° Anchor


9-mer


YFWM






FLIW
















deleterious
DEG

DE
G
QNP
DERHKG
AQN


















A24
Preferred

1° Anchor

P
YFWP

P


1° Anchor


10-


YFWM







FLIW


mer



Deleterious


GDE
QN
RHK
DE
A
QN
DEA












POSITION




























9













or
C-




1
2
3
4
5
6
7
8
C-terminus
terminus





A3101
Preferred
RHK
1° Anchor
YFW
P

YFW
YFW
AP
1° Anchor





MVTALIS






RK



Deleterious
DEP

DE

ADE
DE
DE
DE


A3301
Preferred

1° Anchor
YFW



AYFW

1° Anchor





MVALFIST






RK



Deleterious
GP

DE


A6801
Preferred
YFWSTC
1° Anchor


YFWLIV

YFW
P
1° Anchor





AVTMSLI


M



RK



deleterious
GP

DEG

RHK


A


B0702
Preferred
RHKFWY
1° Anchor
RHK

RHK
RHK
RHK
PA
1° Anchor





P






LMFWYAI












V



deleterious
DEQNP

DEP
DE
DE
GDE
QN
DE


B3501
Preferred
FWYLIVM
1° Anchor
FWY



FWY

1° Anchor





P






LMFWYIV












A



deleterious
AGP



G
G


B51
Preferred
LIVMFWY
1° Anchor
FWY
STC
FWY

G
FWY
1° Anchor





P






LIVFWYA












M



deleterious
AGPDER



DE
G
DEQN
GDE




HKSTC

















B5301
preferred
LIVMFWY
1° Anchor
FWY
STC
FWY

LIVMFWYFWY
1° Anchor






P





IMFWYAL











V



















deleterious
AGPQN




G
RHKQN
DE




B5401
preferred
FWY
1° Anchor
FWYLIVM

LIVM

ALIVM
FWYA
1° Anchor





P





P
ATIVLMF












WY



deleterious
GPQNDE

GDESTC

RHKDE
DE
QNDGE
DE










TABLE IV (F):


Summary of HLA-supertypes


Overall phenotypic frequencies of HLA-supertypes in different ethnic populations








Specificity
Phenotypic frequency















Supertype
Position 2
C-Terminus
Caucasian
N.A. Black
Japanese
Chinese
Hispanic
Average





B7
P
AILMVFWY
43.2
55.1
57.1
43.0
49.3
49.5


A3
AILMVST
RK
37.5
42.1
45.8
52.7
43.1
44.2


A2
AILMVT
AILMVT
45.8
39.0
42.4
45.9
43.0
42.2


A24
YF (WIVLMT)
FI (YWLM)
23.9
38.9
58.6
40.1
38.3
40.0


B44
E (D)
FWYLIMVA
43.0
21.2
42.9
39.1
39.0
37.0


A1
TI (LVMS)
FWY
47.1
16.1
21.8
14.7
26.3
25.2


B27
RHK
FYL (WMI)
28.4
26.1
13.3
13.9
35.3
23.4


B62
QL (IVMP)
FWY (MIV)
12.6
4.8
36.5
25.4
11.1
18.1


B58
ATS
FWY (LIV)
10.0
25.1
1.6
9.0
5.9
10.3










TABLE IV (G):


Calculated population coverage afforded by different HLA-supertype combinations










Phenotypic frequency














HLA-supertypes
Caucasian
N.A Blacks
Japanese
Chinese
Hispanic
Average





A2, A3 and B7
83.0
86.1
87.5
88.4
86.3
86.2


A2, A3, B7, A24, B44
99.5
98.1
100.0
99.5
99.4
99.3


and A1
99.9
99.6
100.0
99.8
99.9
99.8


A2, A3, B7, A24,


B44, A1, B27, B62,


and B58





Motifs indicate the residues defining supertype specificites. The motifs incorporate residues determined on the basis of published data to be recognized by multiple alleles within the supertype. Residues within brackets are additional residues also predicted to be tolerated by multiple alleles within the supertype.













TABLE V







Frequently Occurring Motifs











avrg. %




Name
identity
Description
Potential Function





zf-C2H2
34%
Zinc finger, C2H2 type
Nucleic acid-binding protein functions as transcription factor,





nuclear location probable


cytochrome_b_N
68%
Cytochrome b(N-
membrane bound oxidase, generate superoxide




terminal)/b6/petB


Ig
19%
Immunoglobulin domain
domains are one hundred amino acids long and include a





conserved intradomain disulfide bond.


WD40
18%
WD domain, G-beta repeat
tandem repeats of about 40 residues, each containing a Trp-Asp





motif. Function in signal transduction and protein interaction


PDZ
23%
PDZ domain
may function in targeting signaling molecules to sub-membranous





sites


LRR
28%
Leucine Rich Repeat
short sequence motifs involved in protein-protein interactions


Pkinase
23%
Protein kinase domain
conserved catalytic core common to both serine/threonine and





tyrosine protein kinases containing an ATP binding site and a





catalytic site


PH
16%
PH domain
pleckstrin homology involved in intracellular signaling or as





constituents of the cytoskeleton


EGF
34%
EGF-like domain
30-40 amino-acid long found in the extracellular domain of





membrane-bound proteins or in secreted proteins


Rvt
49%
Reverse transcriptase




(RNA-dependent DNA




polymerase)


Ank
25%
Ank repeat
Cytoplasmic protein, associates integral membrane proteins to the





cytoskeleton


Oxidored_q1
32%
NADH-
membrane associated. Involved in proton translocation across the




Ubiquinone/plastoquinone
membrane




(complex I), various chains


Efhand
24%
EF hand
calcium-binding domain, consists of a 12 residue loop flanked on





both sides by a 12 residue alpha-helical domain


Rvp
79%
Retroviral aspartyl
Aspartyl or acid proteases, centered on a catalytic aspartyl residue




protease


Collagen
42%
Collagen triple helix repeat
extracellular structural proteins involved in formation of connective




(20 copies)
tissue. The sequence consists of the G-X-Y and the polypeptide





chains forms a triple helix.


Fn3
20%
Fibronectin type III domain
Located in the extracellular ligand-binding region of receptors and is





about 200 amino acid residues long with two pairs of cysteines





involved in disulfide bonds


7tm_1
19%
7 transmembrane receptor
seven hydrophobic transmembrane regions, with the N-terminus




(rhodopsin family)
located extracellularly while the C-terminus is cytoplasmic. Signal





through G proteins
















TABLE VI





Motifs and Post-translational Modifications


of 282P1G3







N-glycosylation site









87-90
NNSG
(SEQ ID NO: 54)


231-234
NDSS
(SEQ ID NO: 55)


315-318
NVSY
(SEQ ID NO: 56)


410-413
NHTA
(SEQ ID NO: 57)


492-495
NGTL
(SEQ ID NO: 58)


498-501
NRTT
(SEQ ID NO: 59)


529-532
NATK
(SEQ ID NO: 60)


578-581
NGTE
(SEQ ID NO: 61)


591-594
NLTI
(SEQ ID NO: 62)


596-599
NVTL
(SEQ ID NO: 63)


641-644
NRSV
(SEQ ID NO: 64)


657-660
NISE
(SEQ ID NO: 65)


783-786
NHTL
(SEQ ID NO: 66)


838-841
NSTL
(SEQ ID NO: 67)


961-964
NLTG
(SEQ ID NO: 68)


973-976
NDTY
(SEQ ID NO: 69)


985-988
NITT
(SEQ ID NO: 70)


1000-1003
NATT
(SEQ ID NO: 71)


1042-1045
NLTQ
(SEQ ID NO: 72)


1071-1074
NDSI
(SEQ ID NO: 73)


1213-1216
NGSS
(SEQ ID NO: 74)







Tyrosine sulfation site









817-831
TLYSGEDYPDTAPVI
(SEQ ID NO: 75)


1083-1097
GREYAGLYDDISTQG
(SEQ ID NO: 76)


1145-1159
KDETFGEYSDSDEKP
(SEQ ID NO: 77)


1176-1190
SADSLVEYGEGDHGL
(SEQ ID NO: 78)







cAMP- and cGMP-dependent protein kinase


phosphorylation site









684-687
KKTT
(SEQ ID NO: 79)







Pkinase C phosphorylation site








91-93
TFR


112-114
SNK


183-185
SQK


226-228
SLK


245-247
SIK


310-312
TLK


350-352
TKK


377-379
TIK


536-538
SPK


563-565
SLK


637-639
SER


643-645
SVR


766-768
TWK


785-787
TLR


1002-1004
TTK


1044-1046
TQK


1128-1130
SVK


1143-1145
SVK


1163-1165
SLR







Casein kinase II phosphorylation site









198-201
SRND
(SEQ ID NO: 80)


235-238
SSTE
(SEQ ID NO: 81)


260-263
SGSE
(SEQ ID NO: 82)


317-320
SYQD
(SEQ ID NO: 83)


385-388
SPVD
(SEQ ID NO: 84)


500-503
TTEE
(SEQ ID NO: 85)


501-504
TEED
(SEQ ID NO: 86)


554-557
SKCD
(SEQ ID NO: 87)


598-601
TLED
(SEQ ID NO: 88)


611-614
TALD
(SEQ ID NO: 89)


615-618
SAAD
(SEQ ID NO: 90)


623-626
TVLD
(SEQ ID NO: 91)


809-812
SGPD
(SEQ ID NO: 92)


820-823
SGED
(SEQ ID NO: 93)


870-873
SLLD
(SEQ ID NO: 94)


1027-1030
TLGE
(SEQ ID NO: 95)


1128-1131
SVKE
(SEQ ID NO: 96)


1143-1146
SVKD
(SEQ ID NO: 97)


1148-1151
TFGE
(SEQ ID NO: 98)


1153-1156
SDSD
(SEQ ID NO: 99)


1179-1182
SLVE
(SEQ ID NO: 100)







Tyrosine kinase phosphorylation site









480-487
KPL.EGRRY
(SEQ ID NO: 101)







N-myristoylation site









116-121
GIAMSE
(SEQ ID NO: 102)


240-245
GSKANS
(SEQ ID NO: 103)


261-266
GSESSI
(SEQ ID NO: 104)


322-327
GNYRCT
(SEQ ID NO: 105)


364-369
GILLCE
(SEQ ID NO: 106)


424-429
GTILAN
(SEQ ID NO: 107)


506-511
GSYSCW
(SEQ ID NO: 108)


579-584
GTEDGR
(SEQ ID NO: 109)


589-594
GANLTI
(SEQ ID NO: 110)


603-608
GIYCCS
(SEQ ID NO: 111)


651-656
GADHNS
(SEQ ID NO: 112)


888-893
GQRNSG
(SEQ ID NO: 113)


893-898
GMVPSL
(SEQ ID NO: 114)


960-965
GNLTGY
(SEQ ID NO: 115)


1040-1045
GVNLTQ
(SEQ ID NO: 116)


1101-1106
GLMCAI
(SEQ ID NO: 117)


1124-1129
GGKYSV
(SEQ ID NO: 118)


1162-1167
GSLRSL
(SEQ ID NO: 119)


1195-1200
GSFIGA
(SEQ ID NO: 120)


1199-1204
GAYAGS
(SEQ ID NO: 121)


1208-1213
GSVESN
(SEQ ID NO: 122)


1214-1219
GSSTAT
(SEQ ID NO: 123)







Amidation site









483-486
EGRR
(SEQ ID NO: 124)


682-685
QGKK
(SEQ ID NO: 125)
















TABLE VII





Search Peptides
















v.1 ORF: 272-3946



9-mers, 10-mers and 15-mers
(SEQ ID NO: 126)









MEPLLLGRGL IVYLMFLLLK FSKAIEIPSS VQQVPTIIKQ SKVQVAFPFD EYFQIECEAK
60



GNPEPTFSWT KDGNPFYFTD HRIIPSNNSG TFRIPNEGHI SHFQGKYRCF ASNKLGIAMS
120


EEIEFIVPSV PKLPKEKIDP LEVEEGDPIV LPCNPPKGLP PLHIYWMNIE LEHIEQDERV
180


YMSQKGDLYF ANVEEKDSRN DYCCFAAFPR LRTIVQKMPM KLTVNSLKHA NDSSSSTEIG
240


SKANSIKQRK PKLLLPPTES GSESSITILK GEILLLECFA EGLPTPQVDW NKIGGDLPKG
300


RETKENYGKT LKIENVSYQD KGNYRCTASN FLGTATHDFH VIVEEPPRWT KKPQSAVYST
360


GSNGILLCEA EGEPQPTIKW RVNGSPVDNH PFAGDVVFPR EISFTNLQPN HTAVYQCEAS
420


NVHGTILANA NIDVVDVRPL IQTKDGENYA TVVGYSAFLH CEFFASPEAV VSWQKVEEVK
480


PLEGRRYHIY ENGTLQINRT TEEDAGSYSC WVENAIGKTA VTANLDIRNA TKLRVSPKNP
540


RIPKLHMLEL HCESKCDSHL KHSLKLSWSK DGEAFEINGT EDGRIIIDGA NLTISNVTLE
600


DQGIYCCSAH TALDSAADIT QVTVLDVPDP PENLHLSERQ NRSVRLTWEA GADHNSNISE
660


YIVEFEGNKE EPGRWEELTR VQGKKTTVIL PLAPFVRYQF RVIAVNEVGR SQPSQPSDHH
720


ETPPAAPDRN PQNIRVQASQ PKEMIIKWEP LKSMEQNGPG LEYRVTWKPQ GAPVEWEEET
780


VTNHTLRVMT PAVYAPYDVK VQAINQLGSG PDPQSVTLYS GEDYPDTAPV IHGVDVINST
840


LVKVTWSTVP KDRVHGRLKG YQINWWKTKS LLDGRTHPKE VNILRFSGQR NSGMVPSLDA
900


FSEFHLTVLA YNSKGAGPES EPYIFQTPEG VPEQPTFLKV IKVDKDTATL SWGLPKKLNG
960


NLTGYLLQYQ IINDTYEIGE LNDINITTPS KPSWHLSNLN ATTKYKFYLR ACTSQGCGKP
1020


ITEESSTLGE GSKGIGKISG VNLTQKTHPI EVFEPGAEHI VRLMTKNWGD NDSIFQDVIE
1080


TRGREYAGLY DDISTQGWFI GLMCAIALLT LLLLTVCFVK RNRGGKYSVK EKEDLHPDPE
1140


IQSVKDETFG EYSDSDEKPL KGSLRSLNRD MQPTESADSL VEYGEGDHGL FSEDGSFIGA
1200


YAGSKEKGSV ESNGSSTATF PLRA
1224


 







v.2 ORF: 272-3787









9-mers




aa 125-141
FIVPSVPKFPKEKIDPL
(SEQ ID NO: 127)


aa 295-311
GDLPKGREAKENYGKTL
(SEQ ID NO: 128)


aa 1024-1040
ESSTLGEGKYAGLYDDI
(SEQ ID NO: 129)


 


10-mers


aa 124-142
EFIVPSVPKFPKEKIDPLE
(SEQ ID NO: 130)


aa 294-312
GGDLPKGREAKENYGKTLK
(SEQ ID NO: 131)


aa 1023-1041
EESSTLGEGKYAGLYDDIS
(SEQ ID NO: 132)


 


15-mers


aa 119-147
MSEEIEFIVPSVPKFPKEKIDPLEVEEGD
(SEQ ID NO: 133)


aa 289-317
DWNKIGGDLPKGREAKENYGKTLKIENVS
(SEQ ID NO: 134)


aa 1018-1046
GKPITEESSTLGEGKYAGLYDDISTQGWF
(SEQ ID NO: 135)


 







v.3 ORF: 272...2953 Frame +2









9-mers




aa 830-848
VIHGVDVINTTYVSN TTYVSNATGSPQ PSIFICSKEQ ELSYRNRNML AEDFIQKSTS CNYVEKSSTF
(SEQ ID NO: 136)



FKI


 


10-mers


aa 829-849
PVIHGVDVINTTYVSN TTYVSNATGSPQ PSIFICSKEQ ELSYRNRNML AEDFIQKSTS CNYVEKSSTF
(SEQ ID NO: 137)



FKI


 


15-mers


aa 824-854
YPDTAPVIHGVDVINTTYVSN TTYVSNATGSPQ PSIFICSKEQ ELSYRNRNML AEDFIQKSTS
(SEQ ID NO: 138)



CNYVEKSSTF FKI


 







v.4 ORF: 272...3625 Frame +2









9-mers




aa 816-832
VTLYSGEDLPEQPTFLK
(SEQ ID NO: 139)


 


10-mers


aa 815-833
SVTLYSGEDLPEQPTFLKV
(SEQ ID NO: 140)


 


15-mers


aa 810-838
GPDPQSVTLYSGEDLPEQPTFLKVIKVDK
(SEQ ID NO: 141)


 







v.5 ORF: 272...3898 Frame +2









9-mers




aa 219-235
PMKLTVNSSNSIKQRKP
(SEQ ID NO: 142)


 


10-mers


aa 218-236
MPMKLTVNSSNSIKQRKPK
(SEQ ID NO: 143)


 


15-mers


aa 213-241
TIVQKMPMKLTVNSSNSIKQRKPKLLLPP
(SEQ ID NO: 144)


 







v.6 ORF: 272...3823 Frame +2









9-mers




aa 121-137
EEIEFIVPKLEHIEQDE
(SEQ ID NO: 145)


 


10-mers


aa 122-139
SEEIEFIVPKLEHIEQDER
(SEQ ID NO: 146)


 


15-mers


aa 115-143
LGIAMSEEIEFIVPKLEHIEQDERVYMSQ
(SEQ ID NO: 147)


 







v.7 ORF: 272...3982 Frame +2









9-mers




aa 337-364
HDFHVIVEDNISHELFTLHPEPPRWTKK
(SEQ ID NO: 148)


 


10 mers


aa 336-365
THDFHVIVEDNISHELFTLHPEPPRWTKKP
(SEQ ID NO: 149)


 


15-mers


aa 331-370
FLGTATHDFHVIVEDNISHELFTLHPEPPRWTKKPQSAVY
(SEQ ID NO: 150)









Tables VIII-XXI:










TABLE VIII





Start
Subsequence
Score















V1-HLA-A1-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each start position is


specified, the length of peptide is 9 amino acids, and the end


position for each peptide is the start position plus eight.









500
TTEEDAGSY
112.500


919
ESEPYIFQT
67.500


173
HIEQDERVY
45.000


1078
VIETRGREY
45.000


371
EGEPQPTIK
45.000


931
VPEQPTFLK
22.500


524
NLDIRNATK
20.000


760
GLEYRVTWK
18.000


547
MLELHCESK
18.000


579
GTEDGRIII
11.250


871
LLDGRTHPK
10.000


343
VEEPPRWTK
9.000


1191
FSEDGSFIG
6.750


119
MSEEIEFIV
6.750


78
FTDHRIIPS
6.250


145
EGDPIVLPC
6.250


721
ETPPAAPDR
5.000


915
GAGPESEPY
5.000


396
VVFPREISF
5.000


168
NIELEHIEQ
4.500


598
TLEDQGIYC
4.500


917
GPESEPTIF
4.500


149
IVLPCNPPK
4.000


1154
DSDEKPLKG
3.750


434
VVDVRPLIQ
2.500


948
ATLSWGLPK
2.500


961
NLTGYLLQY
2.500


287
QVDWNKIGG
2.500


789
MTPAVYAPY
2.500


586
IIDGANLTI
2.500


810
GPDPQSVTL
2.500


236
STEIGSKAN
2.250


1021
ITEESSTLG
2.250










V1-HLA-A1-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each start position is


specified, the length of peptide is 9 amino acids, and the end


position for each peptide is the start position plus eight.









1183
YGEGDHGLF
2.250


62
NPEPTFSWT
2.250


416
QCEASNVHG
1.800


142
EVEEGDPIV
1.800


122
EIEFIVPSV
1.800


1175
ESADSLVEY
1.500


261
GSESSITIL
1.350


901
FSEFHLTVL
1.350


627
VPDPPENLH
1.250


1144
VKDETFGEY
1.250


1136
HPDPEIQSV
1.250


816
VTLYSGEDY
1.250


70
TKDGNPFYF
1.250


597
VTLEDQGIY
1.250


157
KGLPPLHIY
1.250


571
DGEAFEING
1.125


270
KGEILLLEC
1.125


978
IGELNDINI
1.125


1112
LLLTVCFVK
1.000


137
KIDPLEVEE
1.000


616
AADITQVTV
1.000


45
VAFPFDEYF
1.000


835
DVINSTLVK
1.000


279
FAEGLPTPQ
0.900


369
EAEGEPQPT
0.900


54
QIECEAKGN
0.900


342
IVEEPPRWT
0.900


192
NVEEKDSRN
0.900


753
SMEQNGPGL
0.900


511
WVENAIGKT
0.900


1056
WVENAIGKT
0.900


1056
GAEHIVRLM
0.900


551
HCESKCDSH
0.900


367
LCEAEGEPQ
0.900










V1-HLA-A1-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each start position is


specified, the length of peptide is 9 amino acids, and the end


position for each peptide is the start position plus eight.









1180
LVEYGEGDH
0.900


275
LLECFAEGL
0.900


24
AIEIPSSVQ
0.900


1209
SVESNGSST
0.900


738
ASQPKEMII
0.750


316
CSYQDKGNY
0.750


1152
YSDSDEKPL
0.750


199
RNDYCCFAA
0.625


1068
WGDNDSIFQ
0.625


44
QVAFPFDEY
0.500


99
HISHFQGKY
0.500


1000
NATTKYKFY
0.500


158
GLPPLHIYW
0.500


117
IAMSEEIEF
0.500


392
FAGDVVFPR
0.500


1176
SADSLVEYG
0.500


612
ALDSAADIT
0.500


651
GADHNSNIS
0.500


875
RTHPKEVNI
0.500


833
GVDVINSTL
0.500


202
YCCFAAFPR
0.500


897
SLDAFSEFH
0.500


906
LTVLAYNSK
0.500


986
ITTPSKPSW
0.500


555
KCDSHLKHS
0.500


893
GMVPSLDAF
0.500


957
KLNGNLTGY
0.500


689
ILPLAPFVR
0.500


853
RVHGRLKGY
0.500


13
YLMFLLLKF
0.500


929
EGVPEQPTF
0.500


1052
VFEPGAEHI
0.450


213
TIVQKMPMK
0.400










V1-HLA-A1-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each start position is


specified, the length of peptide is 9 amino acids, and the end


position for each peptide is the start position plus eight.









949
TLSWGLPKK
0.400










V2-HLA-A1-9mers-(SET 1-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 9 amino acids, and the end


position for each peptide is the start position plus eight.









1
FIVPSVPKF
2.000


3
VPSVPKFPK
0.250


5
SVPKFPKEK
0.020


4
PSVPKFPKE
0.003


2
IVPSVPKFP
0.001


6
VPKFPKEKI
0.000


9
FPKEKIDPL
0.000


7
PKFPKEKID
0.000


8
KFPKEKIDP
0.000










V2-HLA-A1-9mers-(SET 2)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 9 amino acids, and the end


position for each peptide is the start position plus eight.









9
AKENYGKTL
0.045


6
GREAKENYG
0.045


2
DLPKGREAK
0.020


5
KGREAKENY
0.013


1
GDLPKGREA
0.005


7
REAKENYGK
0.002


8
EAKENYGKT
0.001


3
LPKGREAKE
0.000










V2-HLA-A1-9mers-(SET2)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 9 amino acids, and the end


position for each peptide is the start position plus eight.









4
PKGREAKEN
0.000










V2-HLA-A1-9mers-(SET3)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 9 amino acids, and the end


position for each peptide is the start position plus eight.









2
SSTLGEGKY
0.750


5
LGEGKYAGL
0.450


1
ESSTLGEGK
0.300


3
STLGEGKYA
0.025


4
TLGEGKYAG
0.020


6
GEGKYAGLY
0.003


9
KYAGLYDDI
0.001


7
EGKYAGLYD
0.000


8
GKYAGLYDD
0.000










V3-HLA-A1-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each start position is


specified, the length of peptide is 9 amino acids, and the end


position for each peptide is the start position plus eight.









4
GVDVINTTY
25.000


36
EQELSYRNR
1.350


10
TTYVSNTTY
1.250


55
STSCNYVEK
1.000


46
MLAEDFIQK
1.000


47
LAEDFIQKS
0.900


60
YVEKSSTFF
0.900


34
SKEQELSYR
0.450










V3-HLA-A1-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each start position is


specified, the length of peptide is 9 amino acids, and the end


position for each peptide is the start position plus eight.









33
CSKEQELSY
0.375


23
TGSPQPSIF
0.250


25
SPQPSIFIC
0.125


24
GSPQPSIFI
0.075


22
ATGSPQPSI
0.050


27
QPSIFICSK
0.050


16
TTYVSNATG
0.050


13
VSNTTYVSN
0.030


15
NTTYVSNAT
0.025


48
AEDFIQKST
0.025


45
NMLAEDFIQ
0.025


9
NTTYVSNTT
0.025


1
VIHGVDVIN
0.020


7
VINTTYVSN
0.020


12
YVSNTTYVS
0.020


6
DVINTTYVS
0.020


19
VSNATGSPQ
0.015


56
TSCNYVEKS
0.015


29
SIFICSKEQ
0.010


21
NATGSPQPS
0.010


57
SCNYVEKSS
0.010


38
ELSTRNRNM
0.010


31
FICSKEQEL
0.010


52
IQKSTSCNY
0.007


61
VEKSSTFFK
0.005


59
NYVEKSSTF
0.005


43
NRNMLAEDF
0.005


54
KSTSCNYVE
0.003


3
HGVDVINTT
0.003


44
RNMLAEDFI
0.003


8
INTIYVSNT
0.003


58
CNYVEKSST
0.003


14
SNTTYVSNA
0.003


62
EKSSTFFKI
0.003










V3-HLA-A1-9mers-282-P1G3


Each peptide is a portion of SEQ ID NO: 7; each start position is


specified, the length of peptide is 9 amino acids, and the end


position for each peptide is the start position plus eight.









2
IHGVDVINT
0.003


39
LSYRNRNML
0.002


51
FIQKSTSCN
0.001


18
YVSNATGSP
0.001


32
ICSKEQELS
0.001


35
KEQELSYRN
0.001


26
PQPSIFICS
0.001


37
QELSYRNRN
0.001


20
SNATGSPQP
0.001


5
VDVINTTYV
0.001


49
EDFIQKSTS
0.001


11
TYVSNTTYV
0.001


50
DFIQKSTSC
0.001


53
QKSTSCNYV
0.001


17
TYVSNATGS
0.001


40
SYRNRNMLA
0.000


28
PSIFICSKE
0.000


42
RNRNMLAED
0.000


30
IFICSKEQE
0.000


41
YRNRNMLAE
0.000










V4-HLA-A1-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each start position is


specified, the length of peptide is 9 amino acids, and the end


position for each peptide is the start position plus eight.









9
LPEQPTFLK
22.500


7
EDLPEQPTF
0.100


4
YSGEDLPEQ
0.030


6
GEDLPEQPT
0.025


1
VTLTSGEDL
0.025


5
SGEDLPEQP
0.022










V4-HLA-A1-9mers-282-P1G3


Each peptide is a portion of SEQ ID NO: 9; each start position is


specified, the length of peptide is 9 amino acids, and the end


position for each peptide is the start position plus eight.









8
DLPEQPTFL
0.010


2
TLYSGEDLP
0.001


3
LYSGEDLPE
0.000










V5-HLA-A1-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each start position is


specified, the length of peptide is 9 amino acids, and the end


position for each peptide is the start position plus eight.









8
SSNSIKQRK
0.300


5
TVNSSNSIK
0.200


7
NSSNSIKQR
0.150


4
LTVNSSNSI
0.025


6
VNSSNSIKQ
0.013


3
KLTVNSSNS
0.010


2
MKLTVNSSN
0.001


9
SNSIKQRKP
0.000


1
PMKLTVNSS
0.000










V6-HLA-A1-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each start position is


specified, the length of peptide is 9 amino acids, and the end


position for each peptide is the start position plus eight.









2
EIEFIVPKL
1.800


5
FIVPKLEHI
0.100


9
KLIHIEQDE
0.090


1
EEIEFIVPK
0.020


4
EFIVPKLEH
0.003


7
VPKLEHIEQ
0.001










V6-HLA-A1-9mers-282-P1G3


Each peptide is a portion of SEQ ID NO: 13; each start position is


specified, the length of peptide is 9 amino acids, and the end


position for each peptide is the start position plus eight.









6
IVPKLEHIE
0.000


3
IEFIVPKLE
0.000


8
PKLEHIEQD
0.000










V7-HLA-A1-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each start position is


specified, the length of peptide is 9 amino acids, and the end


position for each peptide is the start position plus eight.









19
HPEPPRWTK
45.000


16
FTLHPEPPR
0.500


17
TLHPEPPRW
0.200


6
IVEDNISHE
0.090


5
VIVEDNISH
0.050


10
NISHELFTL
0.050


7
VEDNISHEL
0.025


12
SHELFTLHP
0.022


11
ISHELFTLH
0.015


9
DNISHELFT
0.013


20
PEPPRWTKK
0.010


4
HVIVEDNIS
0.010


8
EDNISHELF
0.005


14
ELFTLHPEP
0.002


2
DFHVIVEDN
0.001


18
LHPEPPRWT
0.001


3
FHVIVEDNI
0.001


1
HDFHVIVED
0.000


15
LFTLHPEPP
0.000


13
HELFTLHPE
0.000
















TABLE IX







V1-HLA-A1-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3;


each start position is specified, the length of


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









Start
Subsequence
Score












261
GSESSITILK
135.000


810
GPDPQSVTLY
62.500


62
NPEPTFSWTK
45.000


1152
YSDSDEKPLK
30.000


1136
HPDPEIQSVK
25.000


1028
LGEGSKGIGK
22.500


312
KIENVSYQDK
18.000


342
IVEEPPRWTK
18.000


738
ASQPKEMIIK
15.000


371
EGEPQPTIKW
11.250


406
NLQPNHTAVY
10.000


343
VEEPPRWTKK
9.000


170
ELEHIEQDER
9.000


658
ISEYIVEFEG
6.750


1191
FSEDGSFIGA
6.750


627
VPDPPENLHL
6.250


788
VMTPAVYAPY
5.000


688
VILPLAPFVR
5.000


137
KIDPLEVEEG
5.000


1056
GAEHIVRLMT
4.500


481
PLEGRRYHIY
4.500


236
STEIGSKANS
4.500


1149
FGEYSDSDEK
4.500


466
SPEAVVSWQK
4.500


475
KVEEVKPLEG
4.500


142
EVEEGDPIVL
4.500


901
FSEFHLTVLA
2.700


434
VVDVRPLIQT
2.500


897
SLDAFSEFHL
2.500


78
FTDHRIIPSN
2.500


145
EGDPIVLPCN
2.500


4
LLLGRGLIVY
2.500


199
RNDYCCFAAF
2.500


612
ALDSAADITQ
2.500


500
TTEEDAGSYS
2.250


747
KWEPLKSMEQ
2.250


270
KGEILLLECF
2.250


369
EAEGEPQPTI
1.800


279
FAEGLPTPQV
1.800


460
HCEFFASPEA
1.800


173
HIEQDERVYM
1.800


24
AIEIPSSVQQ
1.800


300
GRETKENYGK
1.800


598
TLEDQGIYCC
1.800


919
ESEPYIFQTP
1.350


636
LSERQNRSVR
1.350


1192
SEDGSFIGAY
1.250


499
RTTEEDAGSY
1.250


917
GPESEPYIFQ
1.125


1021
ITEESSTLGE
1.125


1183
YGEGDHGLFS
1.125


579
GTEDGRIIID
1.125


931
VPEQPTFLKV
1.125


930
GVPEQPTFLK
1.000


948
ATLSWGLPKK
1.000


1111
LLLLTVCFVK
1.000


212
RTIVQKMPMK
1.000


11
IVYLMFLLLK
1.000


126
IVPSVPKLPK
1.000


689
ILPLAPFVRY
1.000


30
SVQQVPTIIK
1.000


624
VLDVPDPPEN
1.000


947
TATLSWGLPK
1.000


1078
VIETRGREYA
0.900


511
WVENAIGKTA
0.900


1180
LVEYGEGDHG
0.900


416
QCEASNVHGT
0.900


547
MLELHCESKC
0.900


551
HCESKCDSHL
0.900


303
TKENYGKTLK
0.900


675
WEELTRVQGK
0.900


1209
SVESNGSSTA
0.900


996
LSNLNATTKY
0.750


1154
DSDEKPLKGS
0.750


1075
FQDVIETRGR
0.750


119
MSEEIEFIVP
0.675


824
YPDTAPVIHG
0.625


960
GNLTGYLLQY
0.625


431
NIDVVDVRPL
0.500


616
AADITQVTVL
0.500


586
IIDGANLTIS
0.500


847
STVPKDRVHG
0.500


395
DVVFPREISF
0.500


524
NLDIRNATKL
0.500


555
KCDSHLKHSL
0.500


833
GVDVINSTLV
0.500


686
TTVILPLAPF
0.500


596
NVTLEDQGIY
0.500


14
LMFLLLKFSK
0.500


315
NVSYQDKGNY
0.500


815
SVTLYSGEDY
0.500


478
EVKPLEGRRY
0.500


440
LIQTKDGENY
0.500


283
LPTPQVDWNK
0.500


116
GIAMSEEIEF
0.500


1077
DVIETRGREY
0.500


1109
LTLLLLTVCF
0.500


1134
DLHPDPEIQS
0.500


445
DGENYATVVG
0.450


669
KEEPGRWEEL
0.450










V2-HLA-A1-10mers-(SET 1)-282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score












3
IVPSVPKFPK
1.000


5
PSVPKFPKEK
0.300


1
EFIVPSVPKF
0.010


2
FIVPSVPKFP
0.010


6
SVPKFPKEKI
0.001


4
VPSVPKFPKE
0.001


8
PKFPKEKIDP
0.000


10
FPKEKIDPLE
0.000


9
KFPKEKIDPL
0.000


7
VPKFPKEKID
0.000










V2-HLA-A1-10mers-(SET 2)-282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score












5
LGEGKYAGLY
11.250


1
ESSTLGEGKY
0.750


3
STLGEGKYAG
0.050


4
TLGEGKYAGL
0.020


2
SSTLGEGKYA
0.015


8
GKYAGLYDDI
0.001


7
EGKYAGLYDD
0.000


6
GEGKYAGLYD
0.000










V2-HLA-A1-10mers-(SET 3)-282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score












6
LGEGKYAGLY
11.250


2
ESSTLGEGKY
0.750


4
STLGEGKYAG
0.050


5
TLGEGKYAGL
0.020


3
SSTLGEGKYA
0.015


1
EESSTLGEGK
0.010


10
KYAGLYDDIS
0.001


9
GKYAGLYDDI
0.001


8
EGKYAGLYDD
0.000


7
GEGKYAGLYD
0.000










V3-HLA-A1-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score












61
YVEKSSTFFK
9.000


10
NTTYVSNTTY
1.250


48
LAEDFIQKST
0.900


55
KSTSCNYVEK
0.600


46
NMLAEDFIQK
0.500


5
GVDVINTTYV
0.500


33
ICSKEQELSY
0.250


23
ATGSPQPSIF
0.250


37
EQELSYRNRN
0.135


26
SPQPSIFICS
0.125


4
HGVDVINTTY
0.125


24
TGSPQPSIFI
0.125


35
SKEQELSYRN
0.090


25
GSPQPSIFIC
0.075


52
FIQKSTSCNY
0.050


16
NTTYVSNATG
0.050


2
VIHGVDVINT
0.050


49
AEDFIQKSTS
0.025


56
STSCNYVEKS
0.025


11
TTYVSNTTYV
0.025


17
TTYVSNATGS
0.025


59
CNYVEKSSTF
0.025


13
YVSNTTYVSN
0.020


22
NATGSPQPSI
0.020


7
DVINTTYVSN
0.020


34
CSKEQELSYR
0.015


14
VSNTTYVSNA
0.015


57
TSCNYVEKSS
0.015


45
RNMLAEDFIQ
0.013


47
MLAEDFIQKS
0.010


32
FICSKEQELS
0.010


58
SCNYVEKSST
0.010


8
VINTTYVSNT
0.010


19
YVSNATGSPQ
0.010


39
ELSYRNRNML
0.010


40
LSYRNRNMLA
0.008


60
NYVEKSSTFF
0.005


36
KEQELSYRNR
0.005


20
VSNATGSPQP
0.003


27
PQPSIFICSK
0.003


15
SNTTYVSNAT
0.003


43
RNRNMLAEDF
0.003


9
INTTYVSNTT
0.003


21
SNATGSPQPS
0.003


1
PVIHGVDVIN
0.002


29
PSIFICSKEQ
0.002


12
TYVSNTTYVS
0.001


30
SIFICSKEQE
0.001


6
VDVINTTYVS
0.001


31
IFICSKEQEL
0.001


38
QELSYRNRNM
0.001


3
IHGVDVINTT
0.001


51
DFIQKSTSCN
0.001


50
EDFIQKSTSC
0.001


44
NRNMLAEDFI
0.001


62
VEKSSTFFKI
0.000


28
QPSIFICSKE
0.000


53
IQKSTSCNYV
0.000


54
QKSTSCNYVE
0.000


18
TYVSNATGSP
0.000


41
SYRNRNMLAE
0.000


42
YRNRNMLAED
0.000










V4-HLA-A1-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9;


each start position is specified, the length of


peptide is 10 amino acids, and the end position 


for each peptide is the start position plus nine.









Start
Subsequence
Score












10
LPEQPTFLKV
1.125


9
DLPEQPTFLK
1.000


7
GEDLPEQPTF
0.500


6
SGEDLPEQPT
0.225


1
SVTLYSGEDL
0.010


8
EDLPEQPTFL
0.005


3
TLYSGEDLPE
0.005


2
VTLYSGEDLP
0.003


5
YSGEDLPEQP
0.002


4
LYSGEDLPEQ
0.001










V5-HLA-A1-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score












5
LTVNSSNSIK
0.500


8
NSSNSIKQRK
0.300


10
SNSIKQRKPK
0.050


6
TVNSSNSIKQ
0.050


7
VNSSNSIKQR
0.025


4
KLTVNSSNSI
0.010


9
SSNSIKQRKP
0.002


3
MKLTVNSSNS
0.001


1
MPMKLTVNSS
0.000


2
PMKLTVNSSN
0.000
















TABLE X







V1-HLA-A0201-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3;


each start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Start
Subsequence
Score












1111
LLLLTVCFV
5534.148


688
VILPLAPFV
330.846


1108
LLTLLLLTV
271.948


9
GLIVYLMFL
270.234


17
LLLKFSKAI
249.365


118
AMSEEIEFI
191.488


1101
GLMCAIALL
181.794


4
LLLGRGLIV
179.368


16
FLLLKFSKA
160.655


426
ILANANIDV
118.238


923
YIFQTPEGV
79.757


406
NLQPNHTAV
69.552


10
LIVYLMFLL
66.613


1107
ALLTLLLLT
63.417


840
TLVKVTWST
55.890


1027
TLGEGSKGI
42.774


930
GVPEQPTFL
42.151


47
FPFDEYFQI
41.346


166
WMNIELEHI
39.062


836
VINSTLVKV
37.393


125
FIVPSVPKL
31.077


591
NLTISNVTL
21.362


11
IVYLMFLLL
19.320


544
KLHMLELHC
17.388


1166
SLNRDMQPT
17.140


23
KAIEIPSSV
13.862


103
FQGKYRCFA
12.744


267
TILKGEILL
10.868


1042
NLTQKTHPI
10.433


451
TVVGYSAFL
10.281


1001
ATTKYKFYL
9.465


967
LQYQIINDT
9.453


1102
LMCAIALLT
9.149


787
RVMTPAVYA
8.846


1073
SIFQDVIET
8.720


908
VLAYNSKGA
8.446


471
VSWQKVEEV
7.220


598
TLEDQGIYC
7.170


585
IIIDGANLT
7.142


335
ATHDFHVIV
6.171


680
RVQGKKTTV
6.086


780
TVTNHTLRV
6.086


333
GTATHDFHV
5.603


785
TLRVMTPAV
5.286


1092
DISTQGWFI
4.438


275
LLECFAEGL
4.328


1106
IALLTLLLL
4.292


14
LMFLLLKFS
4.282


458
FLHCEFFAS
3.778


619
ITQVTVLDV
3.777


174
IEQDERVYM
3.703


1033
KGIGKISGV
3.655


274
LLLECFAEG
3.651


774
VEWEEETVT
3.437


603
GIYCCSAHT
3.279


214
IVQKMPMKL
3.178


1105
AIALLTLLL
2.937


584
RIIIDGANL
2.937


268
ILKGEILLL
2.923


13
YLMFLLLKF
2.917


942
KVDKDTATL
2.617


1053
FEPGAEHIV
2.551


980
ELNDINITT
2.291


939
KVIKVDKDT
2.282


429
NANIDVVDV
2.222


589
GANLTISNV
2.222


611
TALDSAADI
2.198


976
YEIGELNDI
2.146


444
KDGENYATV
2.079


863
INWWKTKSL
1.968


950
LSWGLPKKL
1.968


83
IIPSNNSGT
1.742


26
EIPSSVQQV
1.650


916
AGPESEPYI
1.536


970
QIINDTYEI
1.435


819
YSGEDYPDT
1.376


280
AEGLPTPQV
1.352


1099
FIGLMCAIA
1.288


185
KGDLYFANV
1.208


374
PQPTIKWRV
1.164


991
KPSWHLSNL
1.123


988
TPSKPSWHL
1.046


272
EILLLECFA
1.043


846
WSTVPKDRV
1.023


753
SMEQNGPGL
0.987


203
CCFAAFPRL
0.980


586
IIDGANLTI
0.975


1066
KNWGDNDSI
0.969


252
KLLLPPTES
0.965


743
EMIIKWEPL
0.964


318
YQDKGNYRC
0.927


746
IKWEPLKSM
0.918


534
RVSPKNPRI
0.913


596
NVTLEDQGI
0.913


1100
IGLMCAIAL
0.877


210
RLRTIVQKM
0.868


736
VQASQPKEM
0.856


370
AEGEPQPTI
0.832


1214
GSSTATFPL
0.809


427
LANANIDVV
0.759










V2-HLA-A201-9mers-(SET 1)-282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Start
Subsequence
Score












1
FIVPSVPKF
2.000


3
VPSVPKFPK
0.250


5
SVPKFPKEK
0.020


4
PSVPKFPKE
0.003


2
IVPSVPKFP
0.001


6
VPKFPKEKI
0.000


9
FPKEKIDPL
0.000


7
PKFPKEKID
0.000


8
KFPKEKIDP
0.000










V2-HLA-A201-9mers-(SET 2)-282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Start
Subsequence
Score












1
GDLPKGREA
0.005


9
AKENYGKTL
0.002


2
DLPKGREAK
0.001


7
REAKENYGK
0.000


5
KGREAKENY
0.000


8
EAKENYGKT
0.000


3
LPKGREAKE
0.000


6
GREAKENYG
0.000


4
PKGREAKEN
0.000










V2-HLA-A201-9mers-(SET 3)-282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Start
Subsequence
Score












3
STLGEGKYA
1.404


4
TLGEGKYAG
0.306


5
LGEGKYAGL
0.023


9
KYAGLYDDI
0.004


6
GEGKYAGLY
0.000


8
GKYAGLYDD
0.000


2
SSTLGEGKY
0.000


1
ESSTLGEGK
0.000


7
EGKYAGLYD
0.000










V3-HLA-A201-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7;


each start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Start
Subsequence
Score





31
FICSKEQEL
13.512


5
VDVINTTYV
0.903


39
LSYRNRNML
0.759


44
RNMLAEDFI
0.679


53
QKSTSCNYV
0.531


24
GSPQPSIFI
0.375


46
MLAEDFIQK
0.197


8
INTTYVSNT
0.190


25
SPQPSIFIC
0.177


58
CNYVEKSST
0.156


22
ATGSPQPSI
0.145


9
NTTYVSNTT
0.104


15
NTTYVSNAT
0.104


45
NMLAEDFIQ
0.095


14
SNTTYVSNA
0.075


38
ELSYRNRNM
0.075


48
AEDFIQKST
0.058


11
TYVSNTTYV
0.053


51
FIQKSTSCN
0.047


7
VINTTYVSN
0.026


35
KEQELSYRN
0.021


2
IHGVDVINT
0.020


3
HGVDVINTT
0.016


12
YVSNTTYVS
0.012


62
EKSSTFFKI
0.012


60
YVEKSSTFF
0.011


29
SIFICSKEQ
0.008


1
VIHGVDVIN
0.007


37
QELSYRNRN
0.005


47
LAEDFIQKS
0.004


10
TTYVSNTTY
0.003


16
TTYVSNATG
0.003


4
GVDVINTTY
0.003


13
VSNTTYVSN
0.001


21
NATGSPQPS
0.001


27
QPSIFICSK
0.001


18
YVSNATGSP
0.001


61
VEKSSTFFK
0.001


56
TSCNYVEKS
0.001


57
SCNYVEKSS
0.000


52
IQKSTSCNY
0.000


32
ICSKEQELS
0.000


26
PQPSIFICS
0.000


55
STSCNYVEK
0.000


50
DFIQKSTSC
0.000


6
DVINTTYVS
0.000


23
TGSPQPSIF
0.000


19
VSNATGSPQ
0.000


54
KSTSCNYVE
0.000


20
SNATGSPQP
0.000


33
CSKEQELSY
0.000


40
SYRNRNMLA
0.000


59
NYVEKSSTF
0.000


49
EDFIQKSTS
0.000


41
YRNRNMLAE
0.000


42
RNRNMLAED
0.000


34
SKEQELSYR
0.000


17
TYVSNATGS
0.000


30
IFICSKEQE
0.000


43
NRNMLAEDF
0.000


36
EQELSYRNR
0.000


28
PSIFICSKE
0.000










V4-HLA-A0201-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9;


each start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Start
Subsequence
Score





8
DLPEQPTFL
36.129


1
VTLYSGEDL
0.914


6
GEDLPEQPT
0.058


2
TLYSGEDLP
0.023


4
YSGEDLPEQ
0.004


9
LPEQPTFLK
0.000


7
EDLPEQPTF
0.000


5
SGEDLPEQP
0.000


3
LYSGEDLPE
0.000










V5-HLA-A0201-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11;


each start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Start
Subsequence
Score





3
KLTVNSSNS
0.261


4
LTVNSSNSI
0.246


2
MKLTVNSSN
0.001


5
TVNSSNSIK
0.001


7
NSSNSIKQR
0.000


6
VNSSNSIKQ
0.000


8
SSNSIKQRK
0.000


1
PMKLTVNSS
0.000


9
SNSIKQRKP
0.000










V6-HLA-A0201-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13;


each start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Start
Subsequence
Score





5
FIVPKLEHI
7.437


2
EIEFIVPKL
0.032


9
KLEHIEQDE
0.003


3
IEFIVPKLE
0.002


6
IVPKLEHIE
0.001


1
EEIEFIVPK
0.001


8
PKLEHIEQD
0.000


7
VPKLEHIEQ
0.000


4
EFIVPKLEH
0.000










V7-HLA-A0201-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15;


each start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Start
Subsequence
Score





10
NISHELFTL
39.184


7
VEDNISHEL
0.282


17
TLHPEPPRW
0.075


5
VIVEDNISH
0.071


18
LHPEPPRWT
0.040


9
DNISHELFT
0.020


3
FHVIVEDNI
0.016


11
ISHELFTLH
0.006


14
ELFTLHPEP
0.004


16
FTLHPEPPR
0.004


6
IVEDNISHE
0.001


4
HVIVEDNIS
0.000


13
HELFTLHPE
0.000


20
PEPPRWTKK
0.000


15
LFTLHPEPP
0.000


1
HDFHVIVED
0.000


2
DFHVIVEDN
0.000


8
EDNISHELF
0.000


12
SHELFTLHP
0.000


19
HPEPPRWTK
0.000
















TABLE XI







V6-HLA-A1-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score





10
KLEHIEQDER
9.000


1
SEEIEFIVPK
1.800


3
EIEFIVPKLE
0.090


6
FIVPKLEHIE
0.010


7
IVPKLEHIEQ
0.005


4
IEFIVPKLEH
0.003


2
EEIEFIVPKL
0.001


5
EFIVPKLEHI
0.001


8
VPKLEHIEQD
0.000


9
PKLEHIEQDE
0.000










V7-HLA-A1-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score





20
HPEPPRWTKK
45.000


7
IVEDNISHEL
0.900


8
VEDNISHELF
0.250


18
TLHPEPPRWT
0.100


5
HVIVEDNISH
0.050


17
FTLHPEPPRW
0.050


10
DNISHELFTL
0.013


19
LHPEPPRWTK
0.010


16
LFTLHPEPPR
0.010


11
NISHELFTLH
0.010


12
ISHELFTLHP
0.007


1
THDFHVIVED
0.005


13
SHELFTLHPE
0.005


9
EDNISHELFT
0.003


15
ELFTLHPEPP
0.001


6
VIVEDNISHE
0.001


2
HDFHVIVEDN
0.001


4
FHVIVEDNIS
0.001


3
DFHVIVEDNI
0.001


14
HELFTLHPEP
0.000


21
PEPPRWTKKP
0.000










V1-HLA-A0201-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score





1110
TLLLLTVCFV
3255.381


274
LLLECFAEGL
1025.804


16
FLLLKFSKAI
674.752


1107
ALLTLLLLTV
591.888


118
AMSEEIEFIV
489.752


5
LLGRGLIVYL
459.398


9
GLIVYLMFLL
284.974


1189
GLFSEDGSFI
212.307


840
TLVKVTWSTV
118.238


132
KLPKEKIDPL
84.264


158
GLPPLHIYWM
62.845


1102
LMCAIALLTL
60.325


426
ILANANIDVV
54.634


957
KLNGNLTGYL
53.459


396
VVFPREISFT
51.883


897
SLDAFSEFHL
49.561


221
KLTVNSLKHA
39.992


150
VLPCNPPKGL
36.316


425
TILANANIDV
35.385


687
TVILPLAPFV
33.472


966
LLQYQIINDT
29.137


1101
GLMCAIALLT
27.572


267
TILKGEILLL
24.997


949
TLSWGLPKKL
21.362


792
AVYAPYDVKV
19.475


413
AVYQCEASNV
19.475


114
KLGIAMSEEI
17.892


13
YLMFLLLKFS
16.044


765
VTWKPQGAPV
13.630


1099
FIGLMCAIAL
13.512


470
VVSWQKVEEV
11.660


585
IIIDGANLTI
9.999


597
VTLEDQGIYC
9.787


693
APFVRYQFRV
9.743


36
TIIKQSKVQV
9.563


10
LIVYLMFLLL
9.488


1000
NATTKYKFYL
9.465


524
NLDIRNATKL
8.545


456
SAFLHCEFFA
8.144


1108
LLTLLLLTVC
7.964


341
VIVEEPPRWT
7.856


752
KSMEQNGPGL
7.404


859
KGYQINWWKT
6.947


541
RIPKLHMLEL
6.756


1105
AIALLTLLLL
6.756


8
RGLIVYLMFL
6.527


117
IAMSEEIEFI
5.649


25
IEIPSSVQQV
5.288


969
YQIINDTYEI
4.866


441
IQTKDGENYA
4.710


742
KEMIIKWEPL
4.481


332
LGTATHDFHV
4.477


615
SAADITQVTV
3.961


141
LEVEEGDPIV
3.865


480
KPLEGRRYHI
3.616


598
TLEDQGIYCC
2.998


1034
GIGKISGVNL
2.937


213
TIVQKMPMKL
2.937


862
QINWWKTKSL
2.937


356
AVYSTGSNGI
2.921


839
STLVKVTWST
2.872


953
GLPKKLNGNL
2.777


214
IVQKMPMKLT
2.550


461
CEFFASPEAV
2.452


833
GVDVINSTLV
2.434


3
PLLLGRGLIV
2.321


512
VENAIGKTAV
2.299


565
KLSWSKDGEA
2.260


1181
VEYGEGDHGL
2.260


987
TTPSKPSWHL
2.225


603
GIYCCSAHTA
2.186


61
GNPEPTFSWT
2.084


795
APYDVKVQAI
2.055


1100
IGLMCAIALL
2.017


218
MPMKLTVNSL
2.017


863
INWWKTKSLL
1.968


635
HLSERQNRSV
1.939


1172
QPTESADSLV
1.861


1171
MQPTESADSL
1.804


1008
YLRACTSQGC
1.737


405
TNLQPNHTAV
1.680


618
DITQVTVLDV
1.650


836
VINSTLVKVT
1.643


1043
LTQKTHPIEV
1.642


450
ATVVGYSAFL
1.632


907
TVLAYNSKGA
1.608


681
VQGKKTTVIL
1.510


334
TATHDFHVIV
1.505


1106
IALLTLLLLT
1.497


206
AAFPRLRTIV
1.465


452
VVGYSAFLHC
1.404


378
IKWRVNGSPV
1.363


181
YMSQKGDLYF
1.362


202
YCCFAAFPRL
1.219


171
LEHIEQDERV
1.127


1113
LLTVCFVKRN
1.107


835
DVINSTLVKV
1.050


934
QPTFLKVIKV
1.044


428
ANANIDVVDV
1.044


82
RIIPSNNSGT
1.025










V2-HLA-A0201-10mers-(SET 1)-282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score





6
SVPKFPKEKI
0.447


9
KFPKEKIDPL
0.059


2
FIVPSVPKFP
0.052


3
IVPSVPKFPK
0.013


4
VPSVPKFPKE
0.000


10
FPKEKIDPLE
0.000


1
EFIVPSVPKF
0.000


5
PSVPKFPKEK
0.000


7
VPKFPKEKID
0.000


8
PKFPKEKIDP
0.000










V2-HLA-A0201-10mers-(SET 2)-282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score





4
TLGEGKYAGL
131.379


2
SSTLGEGKYA
0.178


8
GKYAGLYDDI
0.034


3
STLGEGKYAG
0.004


6
GEGKYAGLYD
0.002


5
LGEGKYAGLY
0.000


1
ESSTLGEGKY
0.000


7
EGKYAGLYDD
0.000










V2-HLA-A0201-10mers-(SET 3)282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score





5
TLGEGKYAGL
131.379


3
SSTLGEGKYA
0.178


9
GKYAGLYDDI
0.034


4
STLGEGKYAG
0.004


7
GEGKYAGLYD
0.002


1
EESSTLGEGK
0.000


10
KYAGLYDDIS
0.000


6
LGEGKYAGLY
0.000


2
ESSTLGEGKY
0.000


8
EGKYAGLYDD
0.000










V3-HLA-A0201-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score





11
TTYVSNTTYV
17.002


5
GVDVINTTYV
13.389


47
MLAEDFIQKS
4.540


8
VINTTYVSNT
4.006


2
VIHGVDVINT
4.006


53
IQKSTSCNYV
2.308


39
ELSYRNRNML
1.602


24
TGSPQPSIFI
0.375


25
GSPQPSIFIC
0.177


40
LSYRNRNMLA
0.176


22
NATGSPQPSI
0.145


62
VEKSSTFFKI
0.133


14
VSNTTYVSNA
0.127


9
INTTYVSNTT
0.083


46
NMLAEDFIQK
0.076


38
QELSYRNRNM
0.071


15
SNTTYVSNAT
0.049


58
SCNYVEKSST
0.049


52
FIQKSTSCNY
0.047


48
LAEDFIQKST
0.046


13
YVSNTTYVSN
0.045


31
IFICSKEQEL
0.025


32
FICSKEQELS
0.023


3
IHGVDVINTT
0.020


61
YVEKSSTFFK
0.012


19
YVSNATGSPQ
0.006


44
NRNMLAEDFI
0.004


30
SIFICSKEQE
0.004


17
TTYVSNATGS
0.003


50
EDFIQKSTSC
0.002


59
CNYVEKSSTF
0.002


36
KEQELSYRNR
0.001


56
STSCNYVEKS
0.001


10
NTTYVSNTTY
0.001


16
NTTYVSNATG
0.001


26
SPQPSIFICS
0.001


45
RNMLAEDFIQ
0.001


33
ICSKEQELSY
0.001


7
DVINTTYVSN
0.001


49
AEDFIQKSTS
0.001


55
KSTSCNYVEK
0.001


57
TSCNYVEKSS
0.000


21
SNATGSPQPS
0.000


23
ATGSPQPSIF
0.000


27
PQPSIFICSK
0.000


60
NYVEKSSTFF
0.000


34
CSKEQELSYR
0.000


20
VSNATGSPQP
0.000


28
QPSIFICSKE
0.000


6
VDVINTTYVS
0.000


4
HGVDVINTTY
0.000


1
PVIHGVDVIN
0.000


37
EQELSYRNRN
0.000


42
YRNRNMLAED
0.000


43
RNRNMLAEDF
0.000


54
QKSTSCNYVE
0.000


35
SKEQELSYRN
0.000


12
TYVSNTTYVS
0.000


51
DFIQKSTSCN
0.000


29
PSIFICSKEQ
0.000


41
SYRNRNMLAE
0.000


18
TYVSNATGSP
0.000










V4-HLA-0201-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score





1
SVTLYSGEDL
0.916


10
LPEQPTFLKV
0.094


3
TLYSGEDLPE
0.048


8
EDLPEQPTFL
0.045


9
DLPEQPTFLK
0.027


6
SGEDLPEQPT
0.013


5
YSGEDLPEQP
0.001


2
VTLYSGEDLP
0.001


7
GEDLPEQPTF
0.001


4
LYSGEDLPEQ
0.000










V5-HLA-0201-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score





4
KLTVNSSNSI
36.515


1
MPMKLTVNSS
0.007


6
TVNSSNSIKQ
0.001


3
MKLTVNSSNS
0.001


7
VNSSNSIKQR
0.000


5
LTVNSSNSIK
0.000


10
SNSIKQRKPK
0.000


8
NSSNSIKQRK
0.000


2
PMKLTVNSSN
0.000


9
SSNSIKQRKP
0.000










V6-HLA-0201-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score





2
EEIEFIVPKL
0.294


4
IEFIVPKLEH
0.009


6
FIVPKLEHIE
0.004


7
IVPKLEHIEQ
0.002


10
KLEHIEQDER
0.002


5
EFIVPKLEHI
0.001


1
SEEIEFIVPK
0.000


3
EIEFIVPKLE
0.000


9
PKLEHIEQDE
0.000


8
VPKLEHIEQD
0.000










V7-HLA-0201-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start_position plus nine.









Start
Subsequence
Score





18
TLHPEPPRWT
8.197


7
IVEDNISHEL
0.834


10
DNISHELFTL
0.140


6
VIVEDNISHE
0.033


11
NISHELFTLH
0.019


17
FTLHPEPPRW
0.018


9
EDNISHELFT
0.004


12
ISHELFTLHP
0.003


15
ELFTLHPEPP
0.002


19
LHPEPPRWTK
0.001


8
VEDNISHELF
0.000


3
DFHVIVEDNI
0.000


5
HVIVEDNISH
0.000


4
FHVIVEDNIS
0.000


14
HELFTLHPEP
0.000


16
LFTLHPEPPR
0.000


2
HDFHVIVEDN
0.000


1
THDFHVIVED
0.000


21
PEPPRWTKKP
0.000


13
SHELFTLHPE
0.000


20
HPEPPRWTKK
0.000
















TABLE XII







V1-HLA-A3-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3;


each start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Start
Subsequence
Score





760
GLEYRVTWK
180.000


1112
LLLTVCFVK
135.000


961
NLTGYLLQY
54.000


937
FLKVIKVDK
30.000


949
TLSWGLPKK
30.000


871
LLDGRTHPK
30.000


957
KLNGNLTGY
27.000


9
GLIVYLMFL
24.300


893
GMVPSLDAF
20.250


524
NLDIRNATK
20.000


547
MLELHCESK
20.000


792
AVYAPYDVK
15.000


5
LLGRGLIVY
12.000


1113
LLTVCFVKR
12.000


689
ILPLAPFVR
12.000


998
NLNATTKYK
10.000


744
MIIKWEPLK
9.000


1189
GLFSEDGSF
9.000


13
YLMFLLLKF
9.000


948
ATLSWGLPK
9.000


843
KVTWSTVPK
6.000


296
DLPKGRETK
6.000


213
TIVQKMPMK
4.500


149
IVLPCNPPK
4.500


661
YIVEFEGNK
4.050


1101
GLMCAIALL
4.050


181
YMSQKGDLY
4.000


310
TLKIENVSY
4.000


396
VVFPREISF
3.000


1110
TLLLLTVCF
3.000


530
ATKLRVSPK
3.000


268
ILKGEILLL
2.700


677
ELTRVQGKK
2.700


331
FLGTATHDF
2.000


11
IVYLMFLLL
1.800


275
LLECFAEGL
1.800


857
RLKGYQINW
1.800


739
SQPKEMIIK
1.800


44
QVAFPFDEY
1.800


835
DVINSTLVK
1.800


31
VQQVPTIIK
1.800


158
GLPPLHIYW
1.800


1197
FIGAYAGSK
1.800


1002
TTKYKFYLR
1.800


906
LTVLAYNSK
1.500


1199
GAYAGSKEK
1.500


859
KGYQINWWK
1.350


118
AMSEEIEFI
1.350


17
LLLKFSKAI
1.350


436
DVRPLIQTK
1.350


657
NISEYIVEF
1.350


861
YQINWWKTK
1.350


221
KLTVNSLKH
1.200


544
KLHMLELHC
1.200


129
SVPKLPKEK
1.000


166
WMNIELEHI
0.900


931
VPEQPTFLK
0.900


4
LLLGRGLIV
0.900


1111
LLLLTVCFV
0.900


1150
GEYSDSDEK
0.900


867
KTKSLLDGR
0.900


282
GLPTPQVDW
0.900


210
RLRTIVQKM
0.900


242
KANSIKQRK
0.900


16
FLLLKFSKA
0.900


1094
STQGWFIGL
0.810


840
TLVKVTWST
0.675


687
TVILPLAPF
0.675


520
AVTANLDIR
0.600


163
HIYWMNIEL
0.600


591
NLTISNVTL
0.600


983
DINITTPSK
0.600


1108
LLTLLLLTV
0.600


1042
NLTQKTHPI
0.600


127
VPSVPKLPK
0.600


897
SLDAFSEFH
0.600


1118
FVKRNRGGK
0.600


74
NPFYFTDHR
0.600


340
HVIVEEPPR
0.600


536
SPKNPRIPK
0.600


753
SMEQNGPGL
0.600


882
NILRFSGQR
0.540


392
FAGDVVFPR
0.540


562
HSLKLSWSK
0.450


284
PTPQVDWNK
0.450


853
RVHGRLKGY
0.450


45
VAFPFDEYF
0.450


1027
TLGEGSKGI
0.450


1088
GLYDDISTQ
0.450


1107
ALLTLLLLT
0.450


125
FIVPSVPKL
0.405


10
LIVYLMFLL
0.405


451
TVVGYSAFL
0.405


343
VEEPPRWTK
0.405


702
VIAVNEVGR
0.400


426
ILANANIDV
0.400


598
TLEDQGIYC
0.400


161
PLHIYWMNI
0.360


99
HISHFQGKY
0.360


458
FLHCEFFAS
0.360










V2-HLA-A3-9mers-(SET 1)-282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Start
Subsequence
Score





5
SVPKFPKEK
3.000


1
FIVPSVPKF
1.350


3
VPSVPKFPK
0.900


9
FPKEKIDPL
0.013


6
VPKFPKEKI
0.009


2
IVPSVPKFP
0.002


8
KFPKEKIDP
0.000


4
PSVPKFPKE
0.000


7
PKFPKEKID
0.000










V2-HLA-A3-9mers-(SET 2)-282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Start
Subsequence
Score





2
DLPKGREAK
6.000


7
REAKENYGK
0.180


5
KGREAKENY
0.018


9
AKENYGKTL
0.001


3
LPKGREAKE
0.000


1
GDLPKGREA
0.000


8
EAKENYGKT
0.000


6
GREAKENYG
0.000


4
PKGREAKEN
0.000










V2-HLA-A3-9mers-(SET 3)-282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Start
Subsequence
Score





4
TLGEGKYAG
0.090


6
GEGKYAGLY
0.032


1
ESSTLGEGK
0.030


3
STLGEGKYA
0.011


2
SSTLGEGKY
0.006


9
KYAGLYDDI
0.003


8
GKYAGLYDD
0.001


5
LGEGKYAGL
0.001


7
EGKYAGLYD
0.000










V3-A3-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7;


each start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Start
Subsequence
Score





46
MLAEDFIQK
180.000


4
GVDVINTTY
1.800


10
TTYVSNTTY
1.000


55
STSCNYVEK
1.000


27
QPSIFICSK
0.900


60
YVEKSSTFF
0.200


61
VEKSSTFFK
0.180


52
IQKSTSCNY
0.120


45
NMLAEDFIQ
0.090


33
CSKEQELSY
0.060


31
FICSKEQEL
0.060


22
ATGSPQPSI
0.045


24
GSPQPSIFI
0.027


39
LSYRNRNML
0.015


25
SPQPSIFIC
0.013


12
YVSNTTYVS
0.012


15
NTTYVSNAT
0.007


9
NTTYVSNTT
0.007


34
SKEQELSYR
0.006


38
ELSYRNRNM
0.006


6
DVINTTYVS
0.005


29
SIFICSKEQ
0.005


16
TTYVSNATG
0.005


59
NYVEKSSTF
0.005


1
VIHGVDVIN
0.005


14
SNTTYVSNA
0.004


36
EQELSYRNR
0.004


23
TGSPQPSIF
0.003


43
NRNMLAEDF
0.002


51
FIQKSTSCN
0.002


7
VINTTYVSN
0.002


44
RNMLAEDFI
0.002


8
INTTYVSNT
0.002


56
TSCNYVEKS
0.002


47
LAEDFIQKS
0.002


62
EKSSTFFKI
0.002


26
PQPSIFICS
0.001


58
CNYVEKSST
0.001


54
KSTSCNYVE
0.001


35
KEQELSYRN
0.001


21
NATGSPQPS
0.001


18
YVSNATGSP
0.001


2
IHGVDVINT
0.001


32
ICSKEQELS
0.000


40
SYRNRNMLA
0.000


3
HGVDVINTT
0.000


5
VDVINTTYV
0.000


11
TYVSNTTYV
0.000


57
SCNYVEKSS
0.000


37
QELSYRNRN
0.000


48
AEDFIQKST
0.000


53
QKSTSCNYV
0.000


19
VSNATGSPQ
0.000


13
VSNTTYVSN
0.000


50
DFIQKSTSC
0.000


42
RNRNMLAED
0.000


17
TYVSNATGS
0.000


41
YRNRNMLAE
0.000


49
EDFIQKSTS
0.000


20
SNATGSPQP
0.000


30
IFICSKEQE
0.000


28
PSIFICSKE
0.000










V4-HLA-A3-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9;


each start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Start
Subsequence
Score





9
LPEQPTFLK
0.900


8
DLPEQPTFL
0.270


2
TLYSGEDLP
0.100


1
VTLYSGEDL
0.045


7
EDLPEQPTF
0.001


6
GEDLPEQPT
0.001


4
YSGEDLPEQ
0.000


3
LYSGEDLPE
0.000


5
SGEDLPEQP
0.000










V5-HLA-A3-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11;


each start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Start
Subsequence
Score





5
TVNSSNSIK
2.000


8
SSNSIKQRK
0.150


3
KLTVNSSNS
0.120


4
LTVNSSNSI
0.045


7
NSSNSIKQR
0.015


1
PMKLTVNSS
0.012


6
VNSSNSIKQ
0.000


2
MKLTVNSSN
0.000


9
SNSIKQRKP
0.000










V6-HLA-A3-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13;


each start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Start
Subsequence
Score





5
FIVPKLEHI
0.203


1
EEIEFIVPK
0.182


9
KLEHIEQDE
0.090


2
EIEFIVPKL
0.081


6
IVPKLEHIE
0.002


7
VPKLEHIEQ
0.000


4
EFIVPKLEH
0.000


3
IEFIVPKLE
0.000


8
PKLEHIEQD
0.000










V7-HLA-A3-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15;


each start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Start
Subsequence
Score





19
HPEPPRWTK
1.350


16
FTLHPEPPR
0.450


17
TLHPEPPRW
0.300


10
NISHELFTL
0.270


5
VIVEDNISH
0.090


14
ELFTLHPEP
0.030


20
PEPPRWTKK
0.009


4
HVIVEDNIS
0.006


11
ISHELFTLH
0.005


6
IVEDNISHE
0.003


7
VEDNISHEL
0.003


3
FHVIVEDNI
0.001


8
EDNISHELF
0.001


1
HDFHVIVED
0.000


9
DNISHELFT
0.000


13
HELTLHPE
0.000


12
SHELFTLHP
0.000


2
DFHVIVEDN
0.000


18
LHPEPPRWT
0.000


15
LFTLHPEPP
0.000
















TABLE XIII







V1-HLA-A3-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score





14
LMFLLLKFSK
300.000


1111
LLLLTVCFVK
135.000


11
IVYLMFLLLK
90.000


187
DLYFANVEEK
90.000


546
HMLELHCESK
45.000


930
GVPEQPTFLK
40.500


870
SLLDGRTHPK
30.000


743
EMIIKWEPLK
27.000


4
LLLGRGLIVY
27.000


995
HLSNLNATTK
20.000


905
HLTVLAYNSK
20.000


532
KLRVSPKNPR
18.000


1112
LLLTVCFVKR
18.000


689
ILPLAPFVRY
18.000


342
IVEEPPRWTK
13.500


1037
KISGVNLTQK
13.500


9
GLIVYLMFLL
12.150


788
VMTPAVYAPY
9.000


1189
GLFSEDGSFI
9.000


312
KIENVSYQDK
6.000


30
SVQQVPTIIK
6.000


406
NLQPNHTAVY
6.000


126
IVPSVPKLPK
6.000


998
NLNATTKYKF
6.000


1073
SIFQDVIETR
4.500


274
LLLECFAEGL
4.050


158
GLPPLHIYWM
4.050


633
NLHLSERQNR
4.000


181
YMSQKGDLYF
4.000


785
TLRVMTPAVY
4.000


33
QVPTIIKQSK
3.000


219
PMKLTVNSLK
3.000


62
NPEPTFSWTK
2.700


132
KLPKEKIDPL
2.700


688
VILPLAPFVR
2.700


212
RTIVQKMPMK
2.250


948
ATLSWGLPKK
2.250


509
SCWVENAIGK
2.000


733
NIRVQASQPK
2.000


1102
LMCAIALLTL
1.800


11001
ATTKYKFYLR
1.800


897
SLDAFSEFHL
1.800


114
KLGIAMSEEI
1.800


1101
GLMCAIALLT
1.350


118
AMSEEIEFIV
1.350


691
PLAPFVRYQF
1.350


16
FLLLKFSKAI
1.350


283
LPTPQVDWNK
1.350


18
LLKFSKAIEI
1.200


170
ELEHIEQDER
1.200


848
TVPKDRVHGR
1.200


116
GIAMSEEIEF
1.200


947
TATLSWGLPK
1.200


105
GKYRCFASNK
0.900


967
LQYQIINDTY
0.900


5
LLGRGLIVYL
0.900


488
HIYENGTLQI
0.900


466
SPEAVVSWQK
0.900


598
TLEDQGIYCC
0.900


261
GSESSITILK
0.900


1107
ALLTLLLLTV
0.900


292
KIGGDLPKGR
0.900


1110
TLLLLTVCFV
0.900


309
KTLKIENVSY
0.900


957
KLNGNLTGYL
0.810


43
VQVAFPFDEY
0.810


471
VSWQKVEEVK
0.750


844
VTWSTVPKDR
0.750


481
PLEGRRYHIY
0.600


524
NLDIRNATKL
0.600


44
QVAFPFDEYF
0.600


529
NATKLRVSPK
0.600


701
RVIAVNEVGR
0.600


238
EIGSKANSIK
0.600


810
GPDPQSVTLY
0.540


10
LIVYLMFLLL
0.540


953
GLPKKLNGNL
0.540


738
ASQPKEMIIK
0.450


857
RLKGYQINWW
0.450


221
KLTVNSLKHA
0.450


123
IEFIVPSVPK
0.450


1088
GLYDDISTQG
0.450


150
VLPCNPPKGL
0.450


1136
HPDPEIQSVK
0.450


440
LIQTKDGENY
0.400


815
SVTLYSGEDY
0.400


559
HLKHSLKLSW
0.400


902
SEFHLTVLAY
0.360


1143
SVKDETFGEY
0.360


686
TTVILPLAPF
0.338


960
GNLTGYLLQY
0.324


148
PIVLPCNPPK
0.300


1108
LLTLLLLTVC
0.300


356
AVYSTGSNGI
0.300


426
ILANANIDVV
0.300


817
TLYSGEDYPD
0.300


535
VSPKNPRIPK
0.300


69
WTKDGNPFYF
0.300


840
TLVKVTWSTV
0.300


603
GIYCCSAHTA
0.300










V2-HLA-A3-10mers-(SET 1)-282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score





3
IVPSVPKFPK
9.000


6
SVPKFPKEKI
0.090


5
PSVPKFPKEK
0.034


2
FIVPSVPKFP
0.003


9
KFPKEKIDPL
0.003


1
EFIVPSVPKF
0.003


4
VPSVPKFPKE
0.001


10
FPKEKIDPLE
0.000


7
VPKFPKEKID
0.000


8
PKFPKEKIDP
0.000










V2-HLA-A3-10mers-(SET 2)-282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score





4
TLGEGKYAGL
0.900


8
GKYAGLYDDI
0.009


3
STLGEGKYAG
0.007


5
LGEGKYAGLY
0.005


1
ESSTLGEGKY
0.002


2
SSTLGEGKYA
0.001


6
GEGKYAGLYD
0.000


7
EGKYAGLYDD
0.000










V2-HLA-A3-10mers-(SET 3)-282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score





5
TLGEGKYAGL
0.900


1
EESSTLGEGK
0.018


9
GKYAGLYDDI
0.009


4
STLGEGKYAG
0.007


6
LGEGKYAGLY
0.005


2
ESSTLGEGKY
0.002


10
KYAGLYDDIS
0.001


3
SSTLGEGKYA
0.001


7
GEGKYAGLYD
0.000


8
GEGKYAGLYD
0.000










V3-HLA-A3-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score





46
NMLAEDFIQK
180.000


61
YVEKSSTFFK
6.000


52
FIQKSTSCNY
0.400


55
KSTSCNYVEK
0.300


47
MLAEDFIQKS
0.270


27
PQPSIFICSK
0.270


10
NTTYVSNTTY
0.200


39
ELSYRNRNML
0.180


23
ATGSPQPSIF
0.100


8
VINTTYVSNT
0.090


2
VIHGVDVINT
0.090


33
ICSKEQELSY
0.080


5
GVDVINTTYV
0.060


11
TTYVSNTTYV
0.050


34
CSKEQELSYR
0.045


59
CNYVEKSSTF
0.020


56
STSCNYVEKS
0.018


62
VEKSSTFFKI
0.016


25
GSPQPSIFIC
0.013


22
NATGSPQPSI
0.013


30
SIFICSKEQE
0.010


17
TTYVSNATGS
0.010


40
LSYRNRNMLA
0.010


4
HGVDVINTTY
0.009


14
VSNTTYVSNA
0.009


53
IQKSTSCNYV
0.006


26
SPQPSIFICS
0.005


36
KEQELSYRNR
0.005


60
NYVEKSSTFF
0.005


32
FICSKEQELS
0.004


43
RNRNMLAEDF
0.004


24
TGSPQPSIFI
0.003


19
YVSNATGSPQ
0.002


13
YVSNTTYVSN
0.002


16
NTTYVSNATG
0.001


58
SCNYVEKSST
0.001


31
IFICSKEQEL
0.001


7
DVINTTYVSN
0.001


48
LAEDFIQKST
0.001


44
NRNMLAEDFI
0.001


37
EQELSYRNRN
0.001


1
PVIHGVDVIN
0.000


28
QPSIFICSKE
0.000


15
SNTTYVSNAT
0.000


9
INTTYVSNTT
0.000


50
EDFIQKSTSC
0.000


3
IHGVDVINTT
0.000


45
RNMLAEDFIQ
0.000


12
TYVSNTTYVS
0.000


6
VDVINTTYVS
0.000


57
TSCNYVEKSS
0.000


49
AEDFIQKSTS
0.000


20
VSNATGSPQP
0.000


38
QELSYRNRNM
0.000


21
SNATGSPQPS
0.000


35
SKEQELSYRN
0.000


54
QKSTSCNYVE
0.000


41
SYRNRNMLAE
0.000


42
YRNRNMLAED
0.000


18
TYVSNATGSP
0.000


51
DFIQKSTSCN
0.000


29
PSIFICSKEQ
0.000










V4-HLA-A3-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score





9
DLPEQPTFLK
40.500


3
TLYSGEDLPE
0.200


1
SVTLYSGEDL
0.060


7
GEDLPEQPTF
0.018


10
LPEQPTFLKV
0.012


2
VTLYSGEDLP
0.002


8
EDLPEQPTFL
0.000


6
SGEDLPEQPT
0.000


5
YSGEDLPEQP
0.000


4
LYSGEDLPEQ
0.000










V5-HLA-A3-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score





4
KLTVNSSNSI
1.800


5
LTVNSSNSIK
1.500


8
NSSNSIKQRK
0.150


10
SNSIKQRKPK
0.020


7
VNSSNSIKQR
0.006


6
TVNSSNSIKQ
0.004


2
PMKLTVNSSN
0.003


1
MPMKLTVNSS
0.002


3
MKLTVNSSNS
0.000


9
SSNSIKQRKP
0.000










V6-HLA-A3-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score





10
KLEHIEQDER
12.000


1
SEEIEFIVPK
0.270


4
IEFIVPKLEH
0.009


2
EEIEFIVPKL
0.005


6
FIVPKLEHIE
0.005


7
IVPKLEHIEQ
0.004


3
EIEFIVPKLE
0.000


5
EFIVPKLEHI
0.000


8
VPKLEHIEQD
0.000


9
PKLEHIEQDE
0.000










V7-HLA-A3-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15;


each start position is specified, the length of


peptide is 10 amino acids, and the end position for


each peptide is the start position plus nine.









Start
Subsequence
Score





20
HPEPPRWTKK
0.300


19
LHPEPPRWTK
0.135


7
IVEDNISHEL
0.090


11
NISHELFTLH
0.060


5
HVIVEDNISH
0.060


15
ELFTLHPEPP
0.030


18
TLHPEPPRWT
0.022


17
FTLHPEPPRW
0.015


6
VIVEDNISHE
0.007


16
LFTLHPEPPR
0.006


8
VEDNISHELF
0.006


10
DNISHELFTL
0.002


12
ISHELFTLHP
0.001


2
HDFHVIVEDN
0.000


3
DFHVIVEDNI
0.000


14
HELFTLHPEP
0.000


4
FHVIVEDNIS
0.000


9
EDNISHELFT
0.000


1
THDFHVIVED
0.000


13
SHELFTLHPE
0.000


21
PEPPRWTKKP
0.000
















TABLE XIV







V1-HLA-A1101-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3;


each start position is specified, the length of


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









Start
Subsequence
Score





843
KVTWSTVPK
6.000


792
AVYAPYDVK
4.000


149
IVLPCNPPK
3.000


948
ATLSWGLPK
3.000


1118
FVKRNRGGK
2.000


835
DVINSTLVK
1.800


1112
LLLTVCFVK
1.800


906
LTVLAYNSK
1.500


739
SQPKEMIIK
1.200


760
GLEYRVTWK
1.200


12
VYLMFLLLK
1.200


106
KYRCFASNK
1.200


31
VQQVPTIIK
1.200


129
SVPKLPKEK
1.000


530
ATKLRVSPK
1.000


15
MFLLLKFSK
0.900


188
LYFANVEEK
0.800


1199
GAYAGSKEK
0.600


436
DVRPLIQTK
0.600


340
HVIVEEPPR
0.600


744
MIIKWEPLK
0.600


931
VPEQPTFLK
0.600


242
KANSIKQRK
0.600


661
YIVEFEGNK
0.600


867
KTKSLLDGR
0.600


213
TIVQKMPMK
0.600


861
YQINWWKTK
0.450


127
VPSVPKLPK
0.400


536
SPKNPRIPK
0.400


871
LLDGRTHPK
0.400


937
FLKVIKVDK
0.400


547
MLELHCESK
0.400


524
NLDIRNATK
0.400


520
AVTANLDIR
0.400


1002
TTKYKFYLR
0.400


949
TLSWGLPKK
0.400


1197
FIGAYAGSK
0.400


1150
GEYSDSDEK
0.360


301
RETKENYGK
0.360


1029
GEGSKGIGK
0.360


859
KGYQINWWK
0.240


689
ILPLAPFVR
0.240


34
VPTIIKQSK
0.200


52
YFQIECEAK
0.200


934
QPTFLKVIK
0.200


1011
ACTSQGCGK
0.200


998
NLNATTKYK
0.200


284
PTPQVDWNK
0.209


304
KENYGKTLK
0.180


787
RVMTPAVYA
0.120


983
DINITTPSK
0.120


262
SESSITILK
0.120


478
EVKPLEGRR
0.120


392
FAGDVVFPR
0.120


343
VEEPPRWTK
0.120


296
DLPKGRETK
0.120


882
NILRFSGQR
0.120


202
YCCFAAFPR
0.120


677
ELTRVQGKK
0.120


98
GHISHFQGK
0.090


333
GTATHDFHV
0.090


212
RTIVQKMPM
0.090


779
ETVTNHTLR
0.090


124
EFIVPSVPK
0.090


702
VIAVNEVGR
0.080


74
NPFYFTDHR
0.080


11
IVYLMFLLL
0.080


877
HPKEVNILR
0.080


1113
LLTVCFVKR
0.080


396
VVFPREISF
0.080


693
APFVRYQFR
0.080


317
SYQDKGNYR
0.080


562
HSLKLSWSK
0.060


291
NKIGGDLPK
0.060


510
CWVENAIGK
0.060


680
RVQGKKTTV
0.060


764
RVTWKPQGA
0.060


942
KVDKDTATL
0.060


721
ETPPAAPDR
0.060


534
RVSPKNPRI
0.060


930
GVPEQPTFL
0.060


833
GVDVINSTL
0.060


313
IENVSYQDK
0.060


452
VVGYSAFLH
0.060


579
GTEDGRIII
0.060


424
GTILANANI
0.045


1115
TVCFVKRNR
0.040


780
TVTNHTLRV
0.040


214
IVQKMPMKL
0.040


849
VPKDRVHGR
0.040


1074
IFQDVIETR
0.040


9
GLIVYLMFL
0.036


247
KQRKPKLLL
0.036


734
IRVQASQPK
0.030


220
MKLTVNSLK
0.030


1124
GGKYSVKEK
0.030


685
KTTVILPLA
0.030


687
TVILPLAPF
0.030


1001
ATTKYKFYL
0.030


875
RTHPKEVNI
0.030










V2-HLA-A1101-9mers-(SET 1)-282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









Start
Subsequence
Score





5
SVPKFPKEK
1.000


3
VPSVPKFPK
0.600


1
FIVPSVPKF
0.006


6
VPKFPKEKI
0.002


9
FPKEKIDPL
0.002


8
KFPKEKIDP
0.001


2
IVPSVPKFP
0.001


4
PSVPKFPKE
0.000


7
PKFPKEKID
0.000










V2-HLA-A1101-9mers-(SET 2)-282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









Start
Subsequence
Score





7
REAKENYGK
0.360


2
DLPKGREAK
0.120


5
KGREAKENY
0.001


3
LPKGREAKE
0.000


9
AKENYGKTL
0.000


1
GDLPKGREA
0.000


6
GREAKENYG
0.000


8
EAKENYGKT
0.000


4
PKGREAKEN
0.000










V2-HLA-A1101-9mers-(SET 3)-282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









Start
Subsequence
Score





9
KYAGLYDDI
0.012


3
STLGEGKYA
0.007


1
ESSTLGEGK
0.006


6
GEGKYAGLY
0.002


4
TLGEGKYAG
0.001


8
GKYAGLYDD
0.000


5
LGEGKYAGL
0.000


2
SSTLGEGKY
0.000


7
EGKYAGLYD
0.000










V3-HLA-A1101-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7;


each start position is specified, the length of


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









Start
Subsequence
Score





55
STSCNYVEK
1.000


46
MLAEDFIQK
0.800


27
QPSIFICSK
0.200


61
VEKSSTFFK
0.180


4
GVDVINTTY
0.060


60
YVEKSSTFF
0.020


10
TTYVSNTTY
0.020


22
ATGSPQPSI
0.010


40
SYRNRNMLA
0.008


52
IQKSTSCNY
0.006


59
NYVEKSSTF
0.006


11
TYVSNTTYV
0.006


12
YVSNTTYVS
0.004


31
FICSKEQEL
0.004


34
SKEQELSYR
0.004


36
EQELSYRNR
0.004


44
RNMLAEDFI
0.002


18
YVSNATGSP
0.002


16
TTYVSNATG
0.002


45
NMLAEDFIQ
0.002


6
DVINTTYVS
0.002


24
GSPQPSIFI
0.001


9
NTTYVSNTT
0.001


15
NTTYVSNAT
0.001


25
SPQPSIFIC
0.001


17
TYVSNATGS
0.001


33
CSKEQELSY
0.000


39
LSYRNRNML
0.000


51
FIQKSTSCN
0.000


14
SNTTYVSNA
0.000


7
VINTTYVSN
0.000


1
VIHGVDVIN
0.000


29
SIFICSKEQ
0.000


35
KEQELSYRN
0.000


30
IFICSKEQE
0.000


5
VDVINTTYV
0.000


47
LAEDFIQKS
0.000


43
NRNMLAEDF
0.000


32
ICSKEQELS
0.000


23
TGSPQPSIF
0.000


53
QKSTSCNYV
0.000


21
NATGSPQPS
0.000


62
EKSSTFFKI
0.000


26
PQPSIFICS
0.000


42
RNRNMLAED
0.000


54
KSTSCNYVE
0.000


38
ELSYRNRNM
0.000


57
SCNYVEKSS
0.000


37
QELSYRNRN
0.000


50
DFIQKSTSC
0.000


58
CNYVEKSST
0.000


20
SNATGSPQP
0.000


2
IHGVDVINT
0.000


8
INTTYVSNT
0.000


41
YRNRNMLAE
0.000


3
HGVDVINTT
0.000


48
AEDFIQKST
0.000


13
VSNTTYVSN
0.000


19
VSNATGSPQ
0.000


56
TSCNYVEKS
0.000


49
EDFIQKSTS
0.000


28
PSIFICSKE
0.000










V4-HLA-A1101-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9;


each start position is specified, the length of


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









Start
Subsequence
Score





9
LPEQPTFLK
0.600


1
VTLYSGEDL
0.015


8
DLPEQPTFL
0.001


3
LYSGEDLPE
0.001


2
TLYSGEDLP
0.001


6
GEDLPEQPT
0.000


7
EDLPEQPTF
0.000


4
YSGEDLPEQ
0.000


5
SGEDLPEQP
0.000










V5-HLA-A1101-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11;


each start position is specified, the length of


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









Start
Subsequence
Score





5
TVNSSNSIK
2.000


8
SSNSIKQRK
0.020


4
LTVNSSNSI
0.015


7
NSSNSIKQR
0.002


3
KLTVNSSNS
0.001


6
VNSSNSIKQ
0.000


1
PMKLTVNSS
0.000


2
MKLTVNSSN
0.000


9
SNSIKQRKP
0.000










V6-HLA-A1101-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13;


each start position is specified, the length of


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









Start
Subsequence
Score





1
EEIEFIVPK
0.027


5
FIVPKLEHI
0.006


6
IVPKLEHIE
0.002


4
EFIVPKLEH
0.002


9
KLEHIEQDE
0.001


2
EIEFIVPKL
0.001


7
VPKLEHIEQ
0.000


3
IEFIVPKLE
0.000


8
PKLEHIEQD
0.000










V7-HLA-A1101-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15;


each start position is specified, the length of


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









Start
Subsequence
Score





19
HPEPPRWTK
0.400


16
FTLHPEPPR
0.300


5
VIVEDNISH
0.012


10
NISHELFTL
0.012


20
PEPPRWTKK
0.006


17
TLHPEPPRW
0.004


4
HVIVEDNIS
0.003


6
IVEDNISHE
0.002


7
VEDNISHEL
0.001


3
FHVIVEDNI
0.000


14
ELFTLHPEP
0.000


11
ISHELFTLH
0.000


15
LFTLHPEPP
0.000


13
HELFTLHPE
0.000


2
DFHVIVEDN
0.000


8
EDNISHELF
0.000


1
HDFHVIVED
0.000


12
SHELFTLHP
0.000


9
DNISHELFT
0.000


18
LHPEPPRWL
0.000
















TABLE XV







V1-HLA-A1101-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3;


each start position is specified, the length of


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









Start
Subsequence
Score





930
GVPEQPTFLK
18.000


11
IVYLMFLLLK
8.000


212
RTIVQKMPMK
4.500


342
IVEEPPRWTK
4.000


126
IVPSVPKLPK
4.000


30
SVQQVPTIIK
4.000


14
LMFLLLKFSK
2.400


33
QVPTIIKQSK
2.000


1111
LLLLTVCFVK
1.800


701
RVIAVNEVGR
1.800


948
ATLSWGLPKK
1.500


1037
KISGVNLTQK
1.200


312
KIENVSYQDK
1.200


509
SCWVENAIGK
0.800


1010
RACTSQGCGK
0.600


870
SLLDGRTHPK
0.600


860
GYQINWWKTK
0.600


546
HMLELHCESK
0.600


1001
ATTKYKFYLR
0.400


995
HLSNLNATTK
0.400


733
NIRVQASQPK
0.400


848
TVPKDRVHGR
0.400


947
TATLSWGLPK
0.400


283
LPTPQVDWNK
0.400


466
SPEAVVSWQK
0.400


905
HLTVLAYNSK
0.400


62
NPEPTFSWTK
0.400


688
VILPLAPFVR
0.360


1196
SFIGAYAGSK
0.300


936
TFLKVIKVDK
0.300


1117
CFVKRNRGGK
0.300


51
EYFQIECEAK
0.240


532
KLRVSPKNPR
0.240


187
DLYFANVEEK
0.240


1136
HPDPEIQSVK
0.200


208
FPRLRTIVQK
0.200


529
NATKLRVSPK
0.200


844
VTWSTVPKDR
0.200


933
EQPTFLKVIK
0.180


660
EYIVEFEGNK
0.180


743
EMIIKWEPLK
0.180


1073
SIFQDVIETR
0.160


1121
RNRGGKYSVK
0.120


105
GKYRCFASNK
0.120


561
KHSLKLSWSK
0.120


300
GRETKENYGK
0.120


261
GSESSITILK
0.120


123
IEFIVPSVPK
0.120


292
KIGGDLPKGR
0.120


238
EIGSKANSIK
0.120


1112
LLLTVCFVKR
0.120


1057
AEHIVRLMTK
0.120


179
RVYMSQKGDL
0.120


295
GDLPKGRETK
0.090


451
TVVGYSAFLH
0.090


939
KVIKVDKDTA
0.090


290
WNKIGGDLPK
0.080


633
NLHLSERQNR
0.080


201
DYCCFAAFPR
0.072


523
ANLDIRNATK
0.060


834
VDVINSTLVK
0.060


148
PIVLPCNPPK
0.060


519
TAVTANLDIR
0.060


680
RVQGKKTTVI
0.060


552
CESKCDSHLK
0.060


370
AEGEPQPTIK
0.060


381
RVNGSPVDNH
0.060


833
GVDVINSTLV
0.060


518
KTAVTANLDI
0.060


343
VEEPPRWTKK
0.060


90
GTFRIPNEGH
0.060


675
WEELTRVQGK
0.060


9
GLIVYLMFLL
0.054


309
KTLKIENVSY
0.045


692
LAPFVRYQFR
0.040


99
HISHFQGKYR
0.040


535
VSPKNPRIPK
0.040


858
LKGYQINWWK
0.040


738
ASQPKEMIIK
0.040


471
VSWQKVEEVK
0.040


356
AVYSTGSNGI
0.040


429
NANIDVVDVR
0.040


219
PMKLTVNSLK
0.040


84
IPSNNSGTFR
0.040


792
AVYAPYDVKV
0.040


1028
LGEGSKGIGK
0.040


190
FANVEEKDSR
0.040


726
APDRNPQNIR
0.040


413
AVYQCEASNV
0.040


639
RQNRSVRLTW
0.036


755
EQNGPGLEYR
0.036


842
VKVTWSTVPK
0.030


435
VDVRPLIQTK
0.030


982
NDINITTPSK
0.030


791
PAVYAPYDVK
0.030


997
SNLNATTKYK
0.030


1123
RGGKYSVKEK
0.030


340
HVIVEEPPRW
0.030


735
RVQASQPKEM
0.030


499
RTTEEDAGSY
0.030










V2-HLA-A1101-10mers-(SET 1)-282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









Start
Subsequence
Score





3
IVPSVPKFPK
6.000


6
SVPKFPKEKI
0.020


9
KFPKEKIDPL
0.006


5
PSVPKFPKEK
0.002


1
EFIVPSVPKF
0.001


2
FIVPSVPKFP
0.000


10
FPKEKIDPLE
0.000


4
VPSVPKFPKE
0.000


7
VPKFPKEKID
0.000


8
PKFPKEKIDP
0.000










V2-HLA-A1101-10mers-(SET 2)-282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









Start
Subsequence
Score





4
TLGEGKYAGL
0.004


3
STLGEGKYAG
0.003


8
GKYAGLYDDI
0.001


6
GEGKYAGLYD
0.000


5
LGEGKYAGLY
0.000


2
SSTLGEGKYA
0.000


1
ESSTLGEGKY
0.000


7
EGKYAGLYDD
0.000










V2-HLA-A1101-10mers-(SET 3)-282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









Start
Subsequence
Score





1
EESSTLGEGK
0.018


5
TLGEGKYAGL
0.004


4
STLGEGKYAG
0.003


9
GKYAGLYDDI
0.001


10
KYAGLYDDIS
0.001


7
GEGKYAGLYD
0.000


6
LGEGKYAGLY
0.000


3
SSTLGEGKYA
0.000


2
ESSTLGEGKY
0.000


8
EGKYAGLYDD
0.000










V3-HLA-A1101-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7;


each start position is specified, the length of


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









Start
Subsequence
Score





61
YVEKSSTFFK
6.000


46
NMLAEDFIQK
1.200


55
KSTSCNYVEK
0.060


27
PQPSIFICSK
0.060


5
GVDVINTTYV
0.060


11
TTYVSNTTYV
0.020


10
NTTYVSNTTY
0.010


23
ATGSPQPSIF
0.010


60
NYVEKSSTFF
0.006


53
IQKSTSCNYV
0.006


33
ICSKEQELSY
0.004


34
CSKEQELSYR
0.004


52
FIQKSTSCNY
0.004


36
KEQELSYRNR
0.004


31
IFICSKEQEL
0.003


22
NATGSPQPSI
0.002


17
TTYVSNATGS
0.002


19
YVSNATGSPQ
0.002


13
YVSNTTYVSN
0.002


62
VEKSSTFFKI
0.002


12
TYVSNTTYVS
0.001


43
RNRNMLAEDF
0.001


39
ELSYRNRNML
0.001


56
STSCNYVEKS
0.001


16
NTTYVSNATG
0.001


7
DVINTTYVSN
0.001


30
SIFICSKEQE
0.001


41
SYRNRNMLAE
0.001


2
VIHGVDVINT
0.001


59
CNYVEKSSTF
0.001


40
LSYRNRNMLA
0.001


45
RNMLAEDFIQ
0.001


18
TYVSNATGSP
0.001


47
MLAEDFIQKS
0.000


24
TGSPQPSIFI
0.000


8
VINTTYVSNT
0.000


32
FICSKEQELS
0.000


26
SPQPSIFICS
0.000


4
HGVDVINTTY
0.000


1
PVIHGVDVIN
0.000


44
NRNMLAEDFI
0.000


28
QPSIFICSKE
0.000


58
SCNYVEKSST
0.000


14
VSNTTYVSNA
0.000


25
GSPQPSIFIC
0.000


37
EQELSYRNRN
0.000


48
LAEDFIQKST
0.000


51
DFIQKSTSCN
0.000


38
QELSYRNRNM
0.000


6
VDVINTTYVS
0.000


49
AEDFIQKSTS
0.000


54
QKSTSCNYVE
0.000


9
INTTYVSNTT
0.000


21
SNATGSPQPS
0.000


35
SKEQELSYRN
0.000


15
SNTTYVSNAT
0.000


20
VSNATGSPQP
0.000


3
IHGVDVINTT
0.000


42
YRNRNMLAED
0.000


50
EDFIQKSTSC
0.000


57
TSCNYVEKSS
0.000


29
PSIFICSKEQ
0.000










V4-HLA-A1101-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9;


each start position is specified, the length of


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









Start
Subsequence
Score





9
DLPEQPTFLK
0.360


1
SVTLYSGEDL
0.020


10
LPEQPTFLKV
0.004


7
GEDLPEQPTF
0.002


3
TLYSGEDLPE
0.002


2
VTLYSGEDLP
0.002


4
LYSGEDLPEQ
0.000


8
EDLPEQPTFL
0.000


6
SGEDLPEQPT
0.000


5
YSGEDLPEQP
0.000










V5-HLA-A1101-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11;


each start position is specified, the length of


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









Start
Subsequence
Score





5
LTVNSSNSIK
1.500


8
NSSNSIKQRK
0.020


10
SNSIKQRKPK
0.020


4
KLTVNSSNSI
0.012


6
TVNSSNSIKQ
0.004


7
VNSSNSIKQR
0.004


1
MPMKLTVNSS
0.000


2
PMKLTVNSSN
0.000


3
MKLTVNSSNS
0.000


9
SSNSIKQRKP
0.000










V6-HLA-A1101-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13;


each start position is specified, the length of


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









Start
Subsequence
Score





10
KLEHIEQDER
0.240


1
SEEIEFIVPK
0.060


7
IVPKLEHIEQ
0.004


4
IEFIVPKLEH
0.002


5
EFIVPKLEHI
0.001


6
FIVPKLEHIE
0.001


2
EEIEFIVPKL
0.000


8
VPKLEHIEQD
0.000


3
EIEFIVPKLE
0.000


9
PKLEHIEQDE
0.000










V7-HLA-A1101-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15;


each start position is specified, the length of


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









Start
Subsequence
Score





20
HPEPPRWTKK
0.200


5
HVIVEDNISH
0.060


16
LFTLHPEPPR
0.040


19
LHPEPPRWTK
0.040


7
IVEDNISHEL
0.020


17
FTLHPEPPRW
0.015


11
NISHELFTLH
0.004


6
VIVEDNISHE
0.001


8
VEDNISHELF
0.001


3
DFHVIVEDNI
0.001


10
DNISHELFTL
0.001


15
ELFTLHPEPP
0.000


14
HELFTLHPEP
0.000


18
TLHPEPPRWT
0.000


12
ISHELFTLHP
0.000


2
HDFHVIVEDN
0.000


4
FHVIVEDNIS
0.000


13
SHELFTLHPE
0.000


1
THDFHVIVED
0.000


9
EDNISHELFT
0.000


21
PEPPRWTKKP
0.000
















TABLE XVI







V1-HLA-A24-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3;


each start position is specified, the length of


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









Start
Subsequence
Score





180
VYMSQKGDL
300.000


1182
EYGEGDHGL
240.000


323
NYRCTASNF
100.000


823
DYPDTAPVI
90.000


964
GYLLQYQII
90.000


489
IYENGTLQI
75.000


1085
EYAGLYDDI
60.000


357
VYSTGSNGI
60.000


76
FYFTDHRII
50.000


102
HFQGKYRCF
15.000


697
RYQFRVIAV
15.000


584
RIIIDGANL
12.000


486
RYHIYENGT
12.000


968
QYQIINDTY
10.500


1004
KYKFYLRAC
10.000


1052
VFEPGAEHI
9.000


1098
WFIGLMCAI
9.000


660
EYIVEFEGN
9.000


8
RGLIVYLMF
8.400


289
DWNKIGGDL
8.400


860
GYQINWWKT
8.250


991
KPSWHLSNL
8.000


942
KVDKDTATL
8.000


247
KQRKPKLLL
8.000


890
RNSGMVPSL
8.000


125
FIVPSVPKL
7.920


51
EYFQIECEA
7.700


414
VYQCEASNV
7.500


10
LIVYLMFLL
7.200


2
EPLLLGRGL
7.200


1094
STQGWFIGL
7.200


1104
CAIALLTLL
7.200


626
DVPDPPENL
7.200


930
GVPEQPTFL
7.200


448
NYATVVGYS
7.000


793
VYAPYDVKV
6.600


214
IVQKMPMKL
6.600


1100
IGLMCAIAL
6.000


451
TVVGYSAFL
6.000


1101
GLMCAIALL
6.000


1190
LFSEDGSFI
6.000


9
GLIVYLMEL
6.000


154
NPPKGLPPL
6.000


796
PYDVKVQAI
6.000


419
ASNVHGTIL
6.000


959
NGNLTGYLL
6.000


275
LLECFAEGL
6.000


261
GSESSITIL
6.000


267
TILKGEILL
6.000


753
SMEQNGPGL
6.000


901
FSEFHLTVL
6.000


743
EMIIKWEPL
6.000


266
ITILKGEIL
6.000


1127
YSVKEKEDL
6.000


1106
IALLTLLLL
6.000


833
GVDVINSTL
5.600


39
KQSKVQVAF
5.600


950
LSWGLPKKL
5.280


507
SYSCWVENA
5.000


109
CFASNKLGI
5.000


604
IYCCSAHTA
5.000


1172
QPTESADSL
4.800


946
DTATLSWGL
4.800


958
LNGNLTGYL
4.800


133
LPKEKIDPL
4.800


11
IVYLMFLLL
4.800


203
CCFAAFPRL
4.800


6
LGRGLIVYL
4.800


810
GPDPQSVTL
4.800


1105
AIALLTLLL
4.800


954
LPKKLNGNL
4.800


245
SIKQRKPKL
4.400


163
HIYWMNIEL
4.400


542
IPKLHMLEL
4.400


692
LAPFVRYQF
4.200


359
STGSNGILL
4.000


358
YSTGSNGIL
4.000


1103
MCAIALLTL
4.000


1152
YSDSDEKPL
4.900


1001
ATTKYKFYL
4.000


864
NWWKTKSLL
4.000


151
LPCNPPKGL
4.000


1035
IGKISGVNL
4.000


591
NLTISNVTL
4.000


682
QGKKTTVIL
4.000


1214
GSSTATFPL
4.000


863
INWWKTKSL
4.000


268
ILKGEILLL
4.000


605
YCCSAHTAL
4.000


988
TPSKPSWHL
4.000


13
YLMFLLLKF
3.960


893
GMVPSLDAF
3.600


1110
TLLLLTVCF
3.600


929
EGVPEQPTF
3.600


117
IAMSEEIEF
3.300


384
GSPVDNHPF
3.000


1183
YGEGDHGLF
3.000


450
ATVVGYSAF
3.000


687
TVILPLAPF
3.000


917
GPESEPYIF
3.000










V2-HLA-A24-9mers-(SET 1)-282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









Start
Subsequence
Score





9
FPKEKIDPL
4.800


1
FIVPSVPKF
3.960


6
VPKFPKEKI
1.100


8
KFPKEKIPD
0.150


2
IVPSVPKFP
0.021


5
SVPKFPKEK
0.017


3
VPSVPKFPK
0.010


4
PSVPKFPKE
0.002


7
PKFPKEKID
0.000










V2-HLA-A24-9mers-(SET 2)-282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









Start
Subsequence
Score





9
AKENYGKTL
0.600


5
KGREAKENY
0.240


8
EAKENYGKT
0.132


1
GDLPKGREA
0.020


2
DLPKGREAK
0.015


3
LPKGREAKE
0.011


7
REAKENYGK
0.002


6
GREAKENYG
0.002


4
PKGREAKEN
0.001










V2-HLA-A24-9mers-(SET 3)-282P1G3


Each peptide is a portion of SEQ ID NO: 5;


each start position is specified, the length of


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









Start
Subsequence
Score





9
KYAGLYDDI
120.000


5
LGEGKYAGL
6.000


3
STLGEGKYA
0.150


2
SSTLGEGKY
0.110


1
ESSTLGEGK
0.012


4
TLGEGKYAG
0.012


6
GEGKYAGLY
0.010


7
EGKYAGLYD
0.010


8
GKYAGLYDD
0.001










V3-HLA-A24-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7;


each start position is specified, the length of


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









Start
Subsequence
Score





59
NYVEKSSTF
180.000


17
TYVSNATGS
7.500


11
TYVSNTTYV
7.500


31
FICSKEQEL
5.280


40
SYRNRNMLA
5.000


39
LSYRNRNML
4.800


60
YVEKSSTFF
3.000


44
RNMLAEDFI
3.000


23
TGSPQPSIF
2.400


24
GSPQPSIFI
1.500


22
ATGSPQPSI
1.000


50
DFIQKSTSC
0.750


38
ELSYRNRNM
0.500


43
NRNMLAEDF
0.360


3
HGVDVINTT
0.302


47
LAEDFIQKS
0.238


57
SCNYVEKSS
0.210


25
SPQPSIFIC
0.180


15
NTTYVSNAT
0.168


9
NTTYVSNTT
0.168


51
FIQKSTSCN
0.150


13
VSNTTYVSN
0.150


6
DVINTTYVS
0.150


7
VINTTYVSN
0.150


1
VIHGVDVIN
0.140


4
GVDVINTTY
0.140


62
EKSSTFFKI
0.132


33
CSKEQELSY
0.120


21
NATGSPQPS
0.120


56
TSCNYVEKS
0.110


10
TTYVSNTTY
0.100


14
SNTTYVSNA
0.100


32
ICSKEQELS
0.100


8
INTTYVSNT
0.100


12
YVSNTTYVS
0.100


52
IQKSTSCNY
0.100


58
CNYVEKSST
0.100


30
IFICSKEQE
0.075


35
KEQELSYRN
0.043


26
PQPSIFICS
0.025


42
RNRNMLAED
0.022


54
KSTSCNYVE
0.020


37
QELSYRNRN
0.018


19
VSNATGSPQ
0.015


36
EQELSYRNR
0.015


45
NMLAEDFIQ
0.015


5
VDVINTTYV
0.015


46
MLAEDFIQK
0.014


48
AEDFIQKST
0.014


53
QKSTSCNYV
0.012


55
STSCNYVEK
0.011


29
SIFICSKEQ
0.011


2
IHGVDVINT
0.010


16
TTYVSNATG
0.010


20
SNATGSPQP
0.010


27
QPSIFICSK
0.010


18
YVSNATGSP
0.010


49
EDFIQKSTS
0.010


28
PSIFICSKE
0.002


34
SKEQELSYR
0.002


41
YRNRNMLAE
0.002


61
VEKSSTFFK
0.001










V4-HLA-A24-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9;


each start position is specified, the length of


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









Start
Subsequence
Score





8
DLPEQPTFL
7.200


1
VTLYSGEDL
6.000


3
LYSGEDLPE
0.500


7
EDLPEQPTF
0.360


5
SGEDLPEQP
0.022


9
LPEQPTFLK
0.015


4
YSGEDLPEQ
0.013


6
GEDLPEQPT
0.012


2
TLYSGEDLP
0.010










V5-HLA-A24-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11;


each start position is specified, the length of


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









Start
Subsequence
Score





4
LTVNSSNSI
1.800


3
KLTVNSSNS
0.200


8
SSNSIKQRK
0.025


2
MKLTVNSSN
0.021


5
TVNSSNSIK
0.015


1
PMKLTVNSS
0.012


6
VNSSNSIKQ
0.011


9
SNSIKQRKP
0.011


7
NSSNSIKQR
0.010










V6-HLA-A24-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13;


each start position is specified, the length of


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









Start
Subsequence
Score





2
EIEFIVPKL
9.240


5
FIVPKLEHI
1.800


4
EFIVPKLEH
0.083


9
KLEHIEQDE
0.050


6
IVPKLEHIE
0.018


7
VPKLEHIEQ
0.011


1
EEIEFIVPK
0.002


3
IEFIVPKLE
0.001


8
PKLEHIEQD
0.000










V7-HLA-A24-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15;


each start position is specified, the length of


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









Start
Subsequence
Score





10
NISHELFTL
4.000


2
DFHVIVEDN
0.700


7
VEDNISHEL
0.616


8
EDNISHELF
0.300


3
FHVIVEDNI
0.210


4
HVIVEDNIS
0.180


9
DNISHELFT
0.150


17
TLHPEPPRW
0.120


15
LFTLHPEPP
0.050


18
LHPEPPRWT
0.018


6
IVEDNISHE
0.018


5
VIVEDNISH
0.018


19
HPEPPRWTK
0.018


11
ISHELFTLH
0.017


16
FTLHPEPPR
0.015


14
ELFTLHPEP
0.013


7
HDFHVIVED
0.002


13
HELFTLHPE
0.002


12
SHELFTLHP
0.002


20
PEPPRWTKK
0.000


















TABLE XVII





Start
Subsequence
Score















V1-HLA-A24-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









106
KYRCFASNKL
528.000


1126
KYSVKEKEDL
400.000


486
RYHIYENGTL
400.000


323
NYRCTASNFL
240.000


1151
EYSDSDEKPL
240.000


357
VYSTGSNGIL
200.000


604
IYCCSAHTAL
200.000


12
VYLMFLLLKF
198.000


454
GYSAFLHCEF
132.000


1182
EYGEGDHGLF
120.000


975
TYEIGELNDI
90.000


507
SYSCWVENAI
84.000


124
EFIVPSVPKL
33.000


900
AFSEFHLTVL
24.000


697
RYQFRVIAVN
21.000


330
NFLGTATHDF
15.000


752
KSMEQNGPGL
14.400


957
KLNGNLTGYL
14.400


541
RIPKLHMLEL
13.200


1019
KPITEESSTL
12.000


1158
KPLKGSLRSL
12.000


132
KLPKEKIDPL
12.000


8
RGLIVYLMFL
12.000


1004
KYKFYLRACT
12.000


875
RTHPKEVNIL
11.520


832
HGVDVINSTL
10.080


555
KCDSHLKHSL
9.600


489
IYENGTLQIN
9.000


317
SYQDKGNYRC
9.000


179
RVYMSQKGDL
8.000


180
VYMSQKGDLY
7.500


964
GYLLQYQIIN
7.500


46
AFPFDEYFQI
7.500


1089
LYDDISTQGW
7.200


153
CNPPKGLPPL
7.200


9
GLIVYLMFLL
7.200


929
EGVPEQPTFL
7.200


218
MPMKLTVNSL
7.200


953
GLPKKLNGNL
7.200


274
LLLECFAEGL
7.200


142
EVEEGDPIVL
7.200


10
LIVYLMFLLL
7.200


809
SGPDPQSVTL
7.200


270
KGEILLLECF
7.200


1104
CAIALLTLLL
7.200


448
NYATVVGYSA
7.000


849
VPKDRVHGRL
6.720


213
TIVQKMPMKL
6.600


306
NYGKTLKIEN
6.600


244
NSIKQRKPKL
6.600


1100
IGLMCAIALL
6.000


551
HCESKCDSHL
6.000


590
ANLTISNVTL
6.000


266
ITILKGEILL
6.000


267
TILKGEILLL
6.000


150
VLPCNPPKGL
6.000


987
TTPSKPSWHL
6.000


862
QINWWKTKSL
6.000


1171
MQPTESADSL
6.000


450
ATVVGYSAFL
6.000


398
FPREISFTNL
5.760


516
IGKTAVTANL
5.600


949
TLSWGLPKKL
5.280


1200
AYAGSKEKGS
5.000


818
LYSGEDYPDT
5.000


885
RFSGQRNSGM
5.000


91
TFRIPNEGHI
5.000


1085
EYAGLYDDIS
5.000


260
SGSESSITIL
4.800


798
DVKVQAINQL
4.800


627
VPDPPENLHL
4.800


1093
ISTQGWFIGL
4.800


302
ETKENYGKTL
4.800


202
YCCFAAFPRL
4.800


5
LLGRGLIVYL
4.800


1103
MCAIALLTLL
4.800


199
RNDYCCFAAF
4.800


536
SPKNPRIPKL
4.400


557
DSHLKHSLKL
4.400


972
INDTYEIGEL
4.400


524
NLDIRNATKL
4.400


681
VQGKKTTVIL
4.000


245
SIKQRKPKLL
4.000


539
NPRIPKLHML
4.000


1034
GIGKISGVNL
4.000


1105
AIALLTLLLL
4.000


431
NIDVVDVRPL
4.000


863
INWWKTKSLL
4.000


1099
FIGLMCAIAL
4.000


1102
LMCAIALLTL
4.000


1213
NGSSTATFPL
4.000


1054
EPGAEHIVRL
4.000


958
LNGNLTGYLL
4.000


897
SLDAFSEFHL
4.000


1066
KNWGDNDSIF
4.000


418
EASNVHGTIL
4.000


1000
NATTKYKFYL
4.000


358
YSTGSNGILL
4.000


1080
ETRGREYAGL
4.000


616
AADITQVTVL
4.000










V2-HLA-A24-10mers-(SET 1)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









9
KFPKEKIDPL
60.000


1
EFIVPSVPKF
16.500


6
SVPKFPKEKI
1.650


2
FIVPSVPKFP
0.025


10
FPKEKIDPLE
0.017


3
IVPSVPKFPK
0.015


4
VPSVPKFPKE
0.013


7
VPKFPKEKID
0.010


5
PSVPKFPKEK
0.002


8
PKFPKEKIDP
0.000










V2-HLA-A24-10mers-(SET 2)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









4
TLGEGKYAGL
4.800


5
LGEGKYAGLY
0.150


8
GKYAGLYDDI
0.120


1
ESSTLGEGKY
0.110


2
SSTLGEGKYA
0.100


3
STLGEGKYAG
0.015


7
EGKYAGLYDD
0.010


6
GEGKYAGLYD
0.001










V2-HLA-A24-10mers-(SET 3)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









10
KYAGLYDDIS
10.000


5
TLGEGKYAGL
4.800


6
LGEGKYAGLY
0.150


9
GKYAGLYDDI
0.120


2
ESSTLGEGKY
0.110


3
SSTLGEGKYA
0.100


4
STLGEGKYAG
0.015


8
EGKYAGLYDD
0.010


1
EESSTLGEGK
0.001


7
GEGKYAGLYD
0.001










V3-HLA-A24-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









60
NYVEKSSTFF
180.000


31
IFICSKEQEL
39.600


12
TYVSNTTYVS
7.500


43
RNRNMLAEDF
4.800


39
ELSYRNRNML
4.800


23
ATGSPQPSIF
2.000


59
CNYVEKSSTF
2.000


24
TGSPQPSIFI
1.200


22
NATGSPQPSI
1.000


51
DFIQKSTSCN
0.750


18
TYVSNATGSP
0.750


41
SYRNRNMLAE
0.500


26
SPQPSIFICS
0.302


4
HGVDVINTTY
0.252


48
LAEDFIQKST
0.252


37
EQELSYRNRN
0.180


15
SNTTYVSNAT
0.168


9
INTTYVSNTT
0.168


47
MLAEDFIQKS
0.158


25
GSPQPSIFIC
0.150


8
VINTTYVSNT
0.150


14
VSNTTYVSNA
0.150


7
DVINTTYVSN
0.150


44
NRNMLAEDFI
0.150


52
FIQKSTSCNY
0.150


58
SCNYVEKSST
0.150


57
TSCNYVEKSS
0.140


62
VEKSSTFFKI
0.132


53
IQKSTSCNYV
0.120


21
SNATGSPQPS
0.120


56
STSCNYVEKS
0.110


17
TTYVSNATGS
0.100


10
NTTYVSNTTY
0.100


2
VIHGVDVINT
0.100


5
GVDVINTTYV
0.100


11
TTYVSNTTYV
0.100


32
FICSKEQELS
0.100


33
ICSKEQELSY
0.100


13
YVSNTTYVSN
0.100


40
LSYRNRNMLA
0.100


38
QELSYRNRNM
0.075


45
RNMLAEDFIQ
0.030


55
KSTSCNYVEK
0.022


1
PVIHGVDVIN
0.021


35
SKEQELSYRN
0.018


46
NMLAEDFIQK
0.018


3
IHGVDVINTT
0.017


28
QPSIFICSKE
0.015


20
VSNATGSPQP
0.015


6
VDVINTTYVS
0.015


61
YVEKSSTFFK
0.015


34
CSKEQELSYR
0.012


50
EDFIQKSTSC
0.010


49
AEDFIQKSTS
0.010


16
NTTYVSNATG
0.010


19
YVSNATGSPQ
0.010


30
SIFICSKEQE
0.010


36
KEQELSYRNR
0.004


29
PSIFICSKEQ
0.002


42
YRNRNMLAED
0.002


27
PQPSIFICSK
0.002


54
QKSTSCNYVE
0.001










V4-HLA-A24-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each start


position is specified, the length of peptide is 10 amino acids, and


the end position for each peptide is the start position plus nine.









1
SVTLYSGEDL
4.000


8
EDLPEQPTFL
0.720


4
LYSGEDLPEQ
0.550


6
SGEDLPEQPT
0.216


7
GEDLPEQPTF
0.200


10
LPEQPTFLKV
0.198


9
DLPEQPTFLK
0.018


2
VTLYSGEDLP
0.015


5
YSGEDLPEQP
0.014


3
TLYSGEDLPE
0.010










V5-HLA-A24-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









4
KLTVNSSNSI
2.400


1
MPMKLTVNSS
0.180


8
NSSNSIKQRK
0.017


6
TVNSSNSIKQ
0.017


9
SSNSIKQRKP
0.017


3
MKLTVNSSNS
0.015


5
LTVNSSNSIK
0.015


2
PMKLTVNSSN
0.014


7
VNSSNSIKQR
0.010


10
SNSIKQRKPK
0.010










V6-HLA-A24-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









5
EFIVPKLEHI
7.500


2
EEIEFIVPKL
1.109


10
KLEHIEQDER
0.033


6
FIVPKLEHIE
0.022


3
EIEFIVPKLE
0.021


7
IVPKLEHIEQ
0.017


8
VPKLEHIEQD
0.010


1
SEEIEFIVPK
0.002


4
IEFIVPKLEH
0.001


9
PKLEHIEQDE
0.000










V7-HLA-A24-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









7
IVEDNISHEL
11.088


3
DFHVIVEDNI
7.000


10
DNISHELFTL
6.000


8
VEDNISHELF
0.200


17
FTLHPEPPRW
0.150


18
TLHPEPPRWT
0.120


16
LFTLHPEPPR
0.050


20
HPEPPRWTKK
0.020


4
FHVIVEDNIS
0.018


6
VIVEDNISHE
0.018


5
HVIVEDNISH
0.015


9
EDNISHELFT
0.015


11
NISHELFTLH
0.014


2
HDFHVIVEDN
0.014


12
ISHELFTLHP
0.012


15
ELFTLHPEPP
0.010


14
HELFTLHPEP
0.002


19
LHPEPPRWTK
0.002


1
THDFHVIVED
0.002


13
SHELFTLHPE
0.002


21
PEPPRWTKKP
0.000


















TABLE XVIII





Start
Subsequence
Score















V1-HLA-B7-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each start


position is specified, the length of peptide is 9 amino acids, and


the end position for each peptide is the start position plus eight.









539
NPRIPKLHM
300.000


151
LPCNPPKGL
120.000


988
TPSKPSWHL
120.000


2
EPLLLGRGL
80.000


133
LPKEKIDPL
80.000


991
KPSWHLSNL
80.000


1172
QPTESADSL
80.000


542
IPKLHMLEL
80.000


954
LPKKLNGNL
80.000


154
NPPKGLPPL
80.000


247
KQRKPKLLL
60.000


6
LGRGLIVYL
40.000


626
DVPDPPENL
30.000


810
GPDPQSVTL
24.000


695
FVRYQFRVI
20.000


11
IVYLMFLLL
20.000


214
IVQKMPMKL
20.000


930
GVPEQPTFL
20.000


451
TVVGYSAFL
20.000


159
LPPLHIYWM
20.000


1106
IALLTLLLL
12.000


1101
GLMCAIALL
12.000


1001
ATTKYKFYL
12.000


130
VPKLPKEKI
12.000


828
APVIHGVDV
12.000


1105
AIALLTLLL
12.000


1104
CAIALLTLL
12.000


419
ASNVHGTIL
12.000


1163
SLRSLNRDM
10.000


210
RLRTIVQKM
10.000


285
TPQVDWNKI
8.000


47
FPFDEYFQI
8.000


726
APDRNPQNI
7.200


833
GVDVINSTL
6.000


772
APVEWEEET
6.000


942
KVDKDTATL
6.000


795
APYDVKVQA
6.000


1100
IGLMCAIAL
4.000


398
FPREISFTN
4.000


863
INWWKTKSL
4.000


267
TILKGEILL
4.000


9
GLIVYLMFL
4.000


946
DTATLSWGL
4.000


591
NLTISNVTL
4.000


10
LIVYLMFLL
4.000


268
ILKGEILLL
4.000


1127
YSVKEKEDL
4.000


950
LSWGLPKKL
4.000


266
ITILKGEIL
4.000


1214
GSSTATFPL
4.000


203
CCFAAFPRL
4.000


1103
MCAIALLTL
4.000


959
NGNLTGYLL
4.000


358
YSTGSNGIL
4.000


605
YCCSAHTAL
4.000


743
EMIIKWEPL
4.000


584
RIIIDGANL
4.000


125
FIVPSVPKL
4.000


163
HIYWMNIEL
4.000


890
RNSGMVPSL
4.000


1094
STQGWFIGL
4.000


359
STGSNGILL
4.000


1035
IGKISGVNL
4.000


682
QGKKTTVIL
4.000


245
SIKQRKPKL
4.000


958
LNGNLTGYL
4.000


855
HGRLKGYQI
4.000


206
AAFPRLRTI
3.600


730
NPQNIRVQA
3.000


111
ASNKLGIAM
3.000


433
DVVDVRPLI
3.000


787
RVMTPAVYA
2.250


250
KPKLLLPPT
2.000


758
GPGLEYRVT
2.000


352
KPQSAVYST
2.000


208
FPRLRTIVQ
2.000


596
NVTLEDQGI
2.000


534
RVSPKNPRI
2.000


30
SVQQVPTII
2.000


785
TLRVMTPAV
2.000


385
SPVDNHPFA
2.000


671
EPGRWEELT
2.000


873
DGRTHPKEV
2.000


1019
KPITEESST
2.000


1121
RNRGGKYSV
2.000


737
QASQPKEMI
1.800


753
SMEQNGPGL
1.200


334
TATHDFHVI
1.200


916
AGPESEPYI
1.200


118
AMSEEIEFI
1.200


519
TAVTANLDI
1.200


180
VYMSQKGDL
1.200


261
GSESSITIL
1.200


738
ASQPKEMII
1.200


650
AGADHNSNI
1.200


611
TALDSAADI
1.200


901
FSEFHLTVL
1.200


1152
YSDSDEKPL
1.200


218
MPMKLTVNS
1.200


418
EASNVHGTI
1.200










V2-HLA-B7-9mers-(SET 1)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









9
FPKEKIDPL
80.000


6
VPKFPKEKI
12.000


3
VPSVPKFPK
0.300


5
SVPKFPKEK
0.050


2
IVPSVPKFP
0.050


1
FIVPSVPKF
0.020


4
PSVPKFPKE
0.001


8
KFPKEKIDP
0.001


7
PKFPKEKID
0.000










V2-HLA-B7-9mers-(SET 2)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









9
AKENYGKTL
0.360


8
EAKENYGKT
0.300


5
KGREAKENY
0.200


3
LPKGREAKE
0.200


2
DLPKGREAK
0.015


1
GDLPKGREA
0.010


7
REAKENYGK
0.001


6
GREAKENYG
0.000


4
PKGREAKEN
0.000










V2-HLA-B7-9mers-(SET 3)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









5
LGEGKYAGL
1.200


3
STLGEGKYA
0.100


9
KYAGLYDDI
0.040


2
SSTLGEGKY
0.020


7
EGKYAGLYD
0.010


1
ESSTLGEGK
0.010


4
TLGEGKYAG
0.010


6
GEGKYAGLY
0.002


8
GKYAGLYDD
0.001










V3-HLA-B7-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









39
LSYRNRNML
6.000


31
FICSKEQEL
4.000


25
SPQPSIFIC
2.000


22
ATGSPQPSI
1.800


44
RNMLAEDFI
1.200


38
ELSYRNRNM
1.000


24
GSPQPSIFI
0.600


27
QPSIFICSK
0.200


8
INTTYVSNT
0.100


15
NTTYVSNAT
0.100


3
HGVDVINTT
0.100


12
YVSNTTYVS
0.100


58
CNYVEKSST
0.100


42
RNRNMLAED
0.100


6
DVINTTYVS
0.100


40
SYRNRNMLA
0.100


14
SNTTYVSNA
0.100


9
NTTYVSNTT
0.100


21
NATGSPQPS
0.060


18
YVSNATGSP
0.050


62
EKSSTFFKI
0.040


60
YVEKSSTFF
0.030


4
GVDVINTTY
0.030


10
TTYVSNTTY
0.020


32
ICSKEQELS
0.020


56
TSCNYVEKS
0.020


5
VDVINTTYV
0.020


13
VSNTTYVSN
0.020


51
FIQKSTSCN
0.020


53
QKSTSCNYV
0.020


33
CSKEQELSY
0.020


1
VIHGVDVIN
0.020


57
SCNYVEKSS
0.020


23
TGSPQPSIF
0.020


7
VINTTYVSN
0.020


52
IQKSTSCNY
0.020


11
TYVSNTTYV
0.020


47
LAEDFIQKS
0.018


16
TTYVSNATG
0.010


2
IHGVDVINT
0.010


46
MLAEDFIQK
0.010


45
NMLAEDFIQ
0.010


19
VSNATGSPQ
0.010


20
SNATGSPQP
0.010


54
KSTSCNYVE
0.010


50
DFIQKSTSC
0.010


29
SIFICSKEQ
0.010


55
STSCNYVEK
0.010


48
AEDFIQKST
0.009


37
QELSYRNRN
0.003


36
EQELSYRNR
0.003


59
NYVEKSSTF
0.002


26
PQPSIFICS
0.002


43
NRNMLAEDF
0.002


35
KEQELSYRN
0.002


17
TYVSNATGS
0.002


49
EDFIQKSTS
0.002


61
VEKSSTFFK
0.001


28
PSIFICSKE
0.001


41
YRNRNMLAE
0.001


30
IFICSKEQE
0.001


34
SKEQELSYR
0.000










V4-HLA-B7-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









1
VTLYSGEDL
4.000


8
DLPEQPTFL
4.000


9
LPEQPTFLK
0.090


4
YSGEDLPEQ
0.010


2
TLYSGEDLP
0.010


6
GEDLPEQPT
0.004


5
SGEDLPEQP
0.003


7
EDLPEQPTF
0.002


3
LYSGEDLPE
0.001










V5-HLA-B7-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









4
LTVNSSNSI
0.400


5
TVNSSNSIK
0.050


3
KLTVNSSNS
0.020


8
SSNSIKQRK
0.010


6
VNSSNSIKQ
0.010


7
NSSNSIKQR
0.010


9
SNSIKQRKP
0.010


2
MKLTVNSSN
0.002


1
PMKLTVNSS
0.002










V6-HLA-B7-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









2
EIEFIVPKL
1.200


5
FIVPKLEHI
0.400


7
VPKLEHIEQ
0.200


6
IVPKLEHIE
0.050


9
KLEHIEQDE
0.003


4
EFIVPKLEH
0.002


3
IEFIVPKLE
0.001


1
EEIEFIVPK
0.001


8
PKLEHIEQD
0.000










V7-HLA-B7-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









10
NISHELFTL
4.000


19
HPEPPRWTK
0.135


7
VEDNISHEL
0.120


4
HVIVEDNIS
0.100


9
DNISHELFT
0.100


3
FHVIVEDNI
0.040


17
TLHPEPPRW
0.020


16
FTLHPEPPR
0.015


6
IVEDNISHE
0.015


18
LHPEPPRWT
0.015


11
ISHELFTLH
0.010


14
ELFTLHPEP
0.010


5
VIVEDNISH
0.010


8
EDNISHELF
0.002


2
DFHVIVEDN
0.002


13
HELFTLHPE
0.001


1
HDFHVIVED
0.001


15
LFTLHPEPP
0.001


12
SHELFTLHP
0.000


20
PEPPRWTKK
0.000


















TABLE XIX





Start
Subsequence
Score















V1-HLA-B7-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









398
FPREISFTNL
800.000


539
NPRIPKLHML
800.000


218
MPMKLTVNSL
240.000


1054
EPGAEHIVRL
80.000


1158
KPLKGSLRSL
80.000


849
VPKDRVHGRL
80.000


536
SPKNPRIPKL
80.000


1019
KPITEESSTL
80.000


1080
ETRGREYAGL
40.000


828
APVIHGVDVI
24.000


627
VPDPPENLHL
24.000


795
APYDVKVQAI
24.000


798
DVKVQAINQL
20.000


179
RVYMSQKGDL
20.000


1105
AIALLTLLLL
12.000


693
APFVRYQFRV
12.000


752
KSMEQNGPGL
12.000


450
ATVVGYSAFL
12.000


772
APVEWEEETV
12.000


1000
NATTKYKFYL
12.000


480
KPLEGRRYHI
12.000


590
ANLTISNVTL
12.000


418
EASNVHGTIL
12.000


2
EPLLLGRGLI
12.000


1104
CAIALLTLLL
12.000


616
AADITQVTVL
10.800


6
LGRGLIVYLM
10.000


74
NPFYFTDHRI
8.000


695
FVRYQFRVIA
7.500


356
AVYSTGSNGI
6.000


150
VLPCNPPKGL
6.000


142
EVEEGDPIVL
6.000


987
TTPSKPSWHL
6.000


643
SVRLTWEAGA
5.000


735
RVQASQPKEM
5.000


780
TVTNHTLRVM
5.000


863
INWWKTKSLL
4.000


790
TPAVYAPYDV
4.000


1034
GIGKISGVNL
4.000


266
ITILKGEILL
4.000


1103
MCAIALLTLL
4.000


9
GLIVYLMFLL
4.000


953
GLPKKLNGNL
4.000


323
NYRCTASNFL
4.000


106
KYRCFASNKL
4.000


862
QINWWKTKSL
4.000


274
LLLECFAEGL
4.000


541
RIPKLHMLEL
4.000


260
SGSESSITIL
4.000


949
TLSWGLPKKL
4.000


213
TIVQKMPMKL
4.000


557
DSHLKHSLKL
4.000


957
KLNGNLTGYL
4.000


1102
LMCAIALLTL
4.000


1093
ISTQGWFIGL
4.000


934
QPTFLKVIKV
4.000


132
KLPKEKIDPL
4.000


5
LLGRGLIVYL
4.000


8
RGLIVYLMFL
4.000


832
HGVDVINSTL
4.000


1099
FIGLMCAIAL
4.000


267
TILKGEILLL
4.000


929
EGVPEQPTFL
4.000


516
IGKTAVTANL
4.000


202
YCCFAAFPRL
4.000


473
WQKVEEVKPL
4.000


244
NSIKQRKPKL
4.000


373
EPQPTIKWRV
4.000


1171
MQPTESADSL
4.000


302
ETKENYGKTL
4.000


681
VQGKKTTVIL
4.000


1213
NGSSTATFPL
4.000


1100
IGLMCAIALL
4.000


809
SGPDPQSVTL
4.000


958
LNGNLTGYLL
4.000


358
YSTGSNGILL
4.000


637
SERQNRSVRL
4.000


153
CNPPKGLPPL
4.000


875
RTHPKEVNIL
4.000


1172
QPTESADSLV
4.000


265
SITILKGEIL
4.000


245
SIKQRKPKLL
4.000


34
VPTIIKQSKV
4.000


10
LIVYLMFLLL
4.000


725
AAPDRNPQNI
3.600


117
IAMSEEIEFI
3.600


110
FASNKLGIAM
3.000


792
AVYAPYDVKV
3.000


413
AVYQCEASNV
3.000


129
SVPKLPKEKI
3.000


206
AAFPRLRTIV
2.700


1048
HPIEVFEPGA
2.000


680
RVQGKKTTVI
2.000


27
IPSSVQQVPT
2.000


526
DIRNATKLRV
2.000


408
QPNHTAVYQC
2.000


1051
EVFEPGAEHI
2.000


954
LPKKLNGNLT
2.000


208
FPRLRTIVQK
2.000


538
KNPRIPKLHM
1.500










V2-HLA-B7-10mers-(SET 1)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









6
SVPKFPKEKI
3.000


9
KFPKEKIDPL
0.400


7
VPKFPKEKID
0.200


4
VPSVPKFPKE
0.200


10
FPKEKIDPLE
0.200


3
IVPSVPKFPK
0.075


2
FIVPSVPKFP
0.010


1
EFIVPSVPKF
0.002


5
PSVPKFPKEK
0.001


8
PKFPKEKIDP
0.000










V2-HLA-B7-10mers-(SET 2)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









4
TLGEGKYAGL
4.000


2
SSTLGEGKYA
0.100


8
GKYAGLYDDI
0.040


1
ESSTLGEGKY
0.020


3
STLGEGKYAG
0.010


7
EGKYAGLYDD
0.010


5
LGEGKYAGLY
0.006


6
GEGKYAGLYD
0.001










V2-HLA-B7-10mers-(SET 3)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









5
TLGEGKYAGL
4.000


3
SSTLGEGKYA
0.100


9
GKYAGLYDDI
0.040


2
ESSTLGEGKY
0.020


4
STLGEGKYAG
0.010


8
EGKYAGLYDD
0.010


6
LGEGKYAGLY
0.006


10
KYAGLYDDIS
0.002


7
GEGKYAGLYD
0.001


1
EESSTLGEGK
0.001










V3-HLA-B7-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









39
ELSYRNRNML
6.000


22
NATGSPQPSI
1.800


24
TGSPQPSIFI
0.600


31
IFICSKEQEL
0.400


26
SPQPSIFICS
0.400


5
GVDVINTTYV
0.300


53
IQKSTSCNYV
0.200


28
QPSIFICSKE
0.200


11
TTYVSNTTYV
0.200


43
RNRNMLAEDF
0.200


8
VINTTYVSNT
0.100


15
SNTTYVSNAT
0.100


58
SCNYVEKSST
0.100


38
QELSYRNRNM
0.100


9
INTTYVSNTT
0.100


14
VSNTTYVSNA
0.100


7
DVINTTYVSN
0.100


2
VIHGVDVINT
0.100


25
GSPQPSIFIC
0.100


40
LSYRNRNMLA
0.100


13
YVSNTTYVSN
0.100


48
LAEDFIQKST
0.090


23
ATGSPQPSIF
0.060


19
YVSNATGSPQ
0.050


62
VEKSSTFFKI
0.040


44
NRNMLAEDFI
0.040


45
RNMLAEDFIQ
0.030


17
TTYVSNATGS
0.020


47
MLAEDFIQKS
0.020


10
NTTYVSNTTY
0.200


32
FICSKEQELS
0.020


59
CNYVEKSSTF
0.020


21
SNATGSPQPS
0.020


52
FIQKSTSCNY
0.020


56
STSCNYVEKS
0.020


4
HGVDVINTTY
0.020


57
TSCNYVEKSS
0.020


33
ICSKEQELSY
0.020


61
YVEKSSTFFK
0.015


16
NTTYVSNATG
0.010


1
PVIHGVDVIN
0.010


20
VSNATGSPQP
0.010


41
SYRNRNMLAE
0.010


34
CSKEQELSYR
0.010


30
SIFICSKEQE
0.010


50
EDFIQKSTSC
0.010


55
KSTSCNYVEK
0.010


3
IHGVDVINTT
0.010


46
NMLAEDFIQK
0.010


37
EQELSYRNRN
0.009


60
NYVEKSSTFF
0.002


51
DFIQKSTSCN
0.002


12
TYVSNTTYVS
0.002


6
VDVINTTYVS
0.002


49
AEDFIQKSTS
0.002


36
KEQELSYRNR
0.001


29
PSIFICSKEQ
0.001


54
QKSTSCNYVE
0.001


27
PQPSIFICSK
0.001


42
YRNRNMLAED
0.001


18
TYVSNATGSP
0.001


35
SKEQELSYRN
0.001










V4-HLA-B7-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each start


position is specified, the length of peptide is 10 amino acids,


and the end position for each peptide is the start position plus nine.









1
SVTLYSGEDL
20.000


10
LPEQPTFLKV
1.200


8
EDLPEQPTFL
0.400


6
SGEDLPEQPT
0.045


9
DLPEQPTFLK
0.015


3
TLYSGEDLPE
0.010


5
YSGEDLPEQP
0.010


2
VTLYSGEDLP
0.010


4
LYSGEDLPEQ
0.001


7
GEDLPEQPTF
0.001










V5-HLA-B7-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each start


position is specified, the length of peptide is 10 amino acids,


and the end position for each peptide is the start position plus nine.









1
MPMKLTVNSS
1.200


4
KLTVNSSNSI
0.400


6
TVNSSNSIKQ
0.050


10
SNSIKQRKPK
0.015


7
VNSSNSIKQR
0.010


8
NSSNSIKQRK
0.010


9
SSNSIKQRKP
0.010


5
LTVNSSNSIK
0.010


2
PMKLTVNSSN
0.002


3
MKLTVNSSNS
0.002










V6-HLA-B7-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each start


position is specified, the length of peptide is 10 amino acids,


and the end position for each peptide is the start position plus nine.









2
EEIEFIVPKL
0.400


8
VPKLEHIEQD
0.200


7
IVPKLEHIEQ
0.050


5
EFIVPKLEHI
0.040


6
FIVPKLEHIE
0.010


10
KLEHIEQDER
0.003


3
EIEFIVPKLE
0.003


4
IEFIVPKLEH
0.002


1
SEEIEFIVPK
0.000


9
PKLEHIEQDE
0.000










V7-HLA-B7-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









7
IVEDNISHEL
6.000


10
DNISHELFTL
4.000


18
TLHPEPPRWT
0.150


20
HPEPPRWTKK
0.060


5
HVIVEDNISH
0.050


3
DFHVIVEDNI
0.040


17
FTLHPEPPRW
0.020


6
VIVEDNISHE
0.010


15
ELFTLHPEPP
0.010


11
NISHELFTLH
0.010


12
ISHELFTLHP
0.010


9
EDNISHELFT
0.010


19
LHPEPPRWTK
0.002


2
HDFHVIVEDN
0.002


4
FHVIVEDNIS
0.002


16
LFTLHPEPPR
0.002


14
HELFTLHPEP
0.001


8
VEDNISHELF
0.001


13
SHELFTLHPE
0.000


1
THDFHVIVED
0.000


21
PEPPRWTKKP
0.000


















TABLE XX





Start
Subsequence
Score















V1-HLA-B3501-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









539
NPRIPKLHM
120.000


133
LPKEKIDPL
120.000


740
QPKEMIIKW
60.000


542
IPKLHMLEL
60.000


954
LPKKLNGNL
60.000


991
KPSWHLSNL
40.000


159
LPPLHIYWM
40.000


690
LPLAPFVRY
40.000


1172
QPTESADSL
40.000


130
VPKLPKEKI
24.000


299
KGRETKENY
24.000


1082
RGREYAGLY
24.000


47
FPFDEYFQI
24.000


197
DSRNDYCCF
22.500


151
LPCNPPKGL
20.000


1175
ESADSLVEY
20.000


390
HPFAGDVVF
20.000


2
EPLLLGRGL
20.000


988
TPSKPSWHL
20.000


154
NPPKGLPPL
20.000


768
KPQGAPVEW
20.000


84
IPSNNSGTF
20.000


316
VSYQDKGNY
15.000


285
TPQVDWNKI
12.000


398
FPREISFTN
12.000


250
KPKLLLPPT
12.000


69
WTKDGNPFY
12.000


210
RLRTIVQKM
12.000


111
ASNKLGIAM
10.000


886
FSGQRNSGM
10.000


917
GPESEPYIF
9.000


915
GAGPESEPY
9.000


310
TLKIENVSY
9.000


1127
YSVKEKEDL
7.500


384
GSPVDNHPF
7.500


1000
NATTKYKFY
6.000


1019
KPITEESST
6.000


810
GPDPQSVTL
6.000


597
VTLEDQGIY
6.000


1163
SLRSLNRDM
6.000


247
KQRKPKLLL
6.000


1214
GSSTATFPL
5.000


950
LSWGLPKKL
5.000


455
YSAFLHCEF
5.000


465
ASPEAVVSW
5.000


182
MSQKGDLYF
5.000


358
YSTGSNGIL
5.000


419
ASNVHGTIL
5.000


667
GNKEEPGRW
4.500


117
IAMSEEIEF
4.500


268
ILKGEILLL
4.500


1158
KPLKGSLRS
4.000


385
SPVDNHPFA
4.000


853
RVHGRLKGY
4.000


957
KLNGNLTGY
4.000


828
APVIHGVDV
4.000


352
KPQSAVYST
4.000


795
APYDVKVQA
4.000


157
KGLPPLHIY
4.000


772
APVEWEEET
4.000


629
DPPENLHLS
4.000


212
RTIVQKMPM
4.000


1104
CAIALLTLL
3.000


682
QGKKTTVIL
3.000


692
LAPFVRYQF
3.000


45
VAFPFDEYF
3.000


441
IQTKDGENY
3.000


1106
IALLTLLLL
3.000


6
LGRGLIVYL
3.000


1035
IGKISGVNL
3.000


857
RLKGYQINW
3.000


456
SAFLHCEFF
3.000


758
GPGLEYRVT
3.000


584
RIIIDGANL
3.000


245
SIKQRKPKL
3.000


838
NSTLVKVTW
2.500


726
APDRNPQNI
2.400


611
ITALDSAADI
2.400


23
KAIEIPSSV
2.400


1152
YSDSDEKPL
2.250


5
LLGRGLIVY
2.000


997
SNLNATTKY
2.000


738
ASQPKEMII
2.000


722
TPPAAPDRN
2.000


181
YMSQKGDLY
2.000


657
NISEYIVEF
2.000


812
DPQSVTLYS
2.000


890
RNSGMVPSL
2.000


626
DVPDPPENL
2.000


39
KQSKVQVAF
2.000


44
QVAFPFDEY
2.000


730
NPQNIRVQA
2.000


283
LPTPQVDWN
2.000


508
YSCWVENAI
2.000


8
RGLIVYLMF
2.000


99
HISHFQGKY
2.000


736
VQASQPKEM
2.000


447
ENYATVVGY
2.000


755
EQNGPGLEY
2.000


1013
TSQGCGKPI
2.000










V2-HLA-B3501-9mers-(SET 1)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 9 amino acids, and the end


position for each peptide is the start position plus eight.









9
FPKEKIDPL
120.000


6
VPKFPKEKI
24.000


1
FIVPSVPKF
1.000


3
VPSVPKFPK
0.200


5
SVPKFPKEK
0.010


2
IVPSVPKFP
0.010


4
PSVPKFPKE
0.005


8
KFPKEKIDP
0.003


7
PKFPKEKID
0.000










V2-B3501-9mers-(SET 2)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start


position is specified, the length of peptide is 9 amino acids, and


the end position for each peptide is the start position plus eight.









5
KGREAKENY
24.000


8
EAKENYGKT
1.800


3
LPKGREAKE
0.600


9
AKENYGKTL
0.030


1
GDLPKGREA
0.010


2
DLPKGREAK
0.010


7
REAKENYGK
0.003


4
PKGREAKEN
0.002


6
GREAKENYG
0.000










V2-B3501-9mers-(SET 3)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









2
SSTLGEGKY
10.000


5
LGEGKYAGL
0.300


6
GEGKYAGLY
0.200


3
STLGEGKYA
0.150


9
KYAGLYDDI
0.080


1
ESSTLGEGK
0.050


7
EGKYAGLYD
0.030


4
TLGEGKYAG
0.020


8
GKYAGLYDD
0.001










V3-B3501-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each start position is


specified, the length of peptide is 9 amino acids, and the end


position for each peptide is the start position plus eight.









33
CSKEQELSY
60.00


52
IQKSTSCNY
6.000


39
LSYRNRNML
5.000


10
TTYVSNTTY
2.000


24
GSPQPSIFI
2.000


25
SPQPSIFIC
2.000


38
ELSYRNRNM
2.000


31
FICSKEQEL
1.000


23
TGSPQPSIF
1.000


44
RNMLAEDFI
0.800


4
GVDVINTTY
0.600


56
TSCNYVEKS
0.500


13
VSNTTYVSN
0.500


22
ATGSPQPSI
0.400


60
YVEKSSTFF
0.300


21
NATGSPQPS
0.300


59
NYVEKSSTF
0.200


3
HGVDVINTT
0.200


27
QPSIFICSK
0.200


47
LAEDFIQKS
0.180


32
ICSKEQELS
0.150


58
CNYVEKSST
0.150


9
NTTYVSNTT
0.100


7
VINTTYVSN
0.100


54
KSTSCNYVE
0.100


15
NTTYVSNAT
0.100


8
INTTYVSNT
0.100


12
YVSNTTYVS
0.100


51
FIQKSTSCN
0.100


43
NRNMLAEDF
0.100


57
SCNYVEKSS
0.100


1
VIHGVDVIN
0.100


14
SNTTYVSNA
0.100


6
DVINTTYVS
0.100


42
RNRNMLAED
0.060


19
VSNATGSPQ
0.050


35
KEQELSYRN
0.040


62
EKSSTFFKI
0.040


46
MLAEDFIQK
0.030


40
SYRNRNMLA
0.030


5
VDVINTTYV
0.020


11
TYVSNTTYV
0.020


53
QKSTSCNYV
0.020


2
IHGVDVINT
0.015


45
NMLAEDFIQ
0.015


26
PQPSIFICS
0.010


49
EDFIQKSTS
0.010


16
TTYVSNATG
0.010


37
QELSYRNRN
0.010


55
STSCNYVEK
0.010


17
TYVSNATGS
0.010


29
SIFICSKEQ
0.010


18
YVSNATGSP
0.010


20
SNATGSPQP
0.010


50
DFIQKSTSC
0.010


28
PSIFICSKE
0.005


61
VEKSSTFFK
0.003


48
AEDFIQKST
0.003


36
EQELSYRNR
0.003


30
IFICSKEQE
0.001


41
YRNRNMLAE
0.001


34
SKEQELSYR
0.000










V4-HLA-B3501-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each start position is


specified, the length of peptide is 9 amino acids, and the end


position for each peptide is the start position plus eight.









8
DLPEQPTFL
2.000


1
VTLYSGEDL
1.000


7
EDLPEQPTF
0.150


4
YSGEDLPEQ
0.150


9
LPEQPTFLK
0.060


2
TLYSGEDLP
0.010


5
SGEDLPEQP
0.006


6
GEDLPEQPT
0.003


3
LYSGEDLPE
0.002










V5-HLA-B3501 -9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each start position is


specified, the length of peptide is 9 amino acids, and the end


position for each peptide is the start position plus eight.









4
LTVNSSNSI
0.400


3
KLTVNSSNS
0.200


8
SSNSIKQRK
0.050


7
NSSNSIKQR
0.050


1
PMKLTVNSS
0.030


9
SNSIKQRKP
0.010


6
VNSSNSIKQ
0.010


2
MKLTVNSSN
0.010


5
TVNSSNSIK
0.010










V6-HLA-B3501-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each start position is


specified, the length of peptide is 9 amino acids, and the end


position for each peptide is the start position plus eight.









7
VPKLEHIEQ
0.900


5
FIVPKLEHI
0.400


2
EIEFIVPKL
0.300


6
IVPKLEHIE
0.010


9
KLEHIEQDE
0.006


1
EEIEFIVPK
0.002


4
EFIVPKLEH
0.001


3
IEFIVPKLE
0.001


8
PKLEHIEQD
0.000










V7-HLA-B3501-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each start position is


specified, the length of peptide is 9 amino acids, and the end


position for each peptide is the start position plus eight.









10
NISHELFTL
1.500


17
TLHPEPPRW
0.750


4
HVIVEDNIS
0.150


11
ISHELFTLH
0.100


8
EDNISHELF
0.100


9
DNISHELFT
0.100


19
HPEPPRWTK
0.060


3
FHVIVEDNI
0.040


5
VIVEDNISH
0.030


7
VEDNISHEL
0.030


18
LHPEPPRWT
0.020


2
DFHVIVEDN
0.010


16
FTLHPEPPR
0.010


14
ELFTLHPEP
0.010


6
IVEDNISHE
0.006


13
HELFTLHPE
0.001


1
HDFHVIVED
0.001


15
LFTLHPEPP
0.001


12
SHELFTLHP
0.000


20
PEPPRWTKK
0.000


















TABLE XXI





Start
Subsequence
Score















V1-HLA-B35-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









398
FPREISFTNL
120.000


849
VPKDRVHGRL
120.000


877
HPKEVNILRF
120.000


539
NPRIPKLHML
60.000


1019
KPITEESSTL
60.000


536
SPKNPRIPKL
60.000


1158
KPLKGSLRSL
40.000


94
IPNEGHISHF
40.000


480
KPLEGRRYHI
32.000


218
MPMKLTVNSL
20.000


1054
EPGAEHIVRL
20.000


895
VPSLDAFSEF
20.000


752
KSMEQNGPGL
20.000


568
WSKDGEAFEI
18.000


795
APYDVKVQAI
16.000


40
QSKVQVAFPF
15.000


1143
SVKDETFGEY
12.000


810
GPDPQSVTLY
12.000


499
RTTEEDAGSY
12.000


772
APVEWEEETV
12.000


996
LSNLNATTKY
10.000


758
GPGLEYRVTW
10.000


1162
GSLRSLNRDM
10.000


297
LPKGRETKEN
9.000


58
EAKGNPEPTF
9.000


478
EVKPLEGRRY
9.000


627
VPDPPENLHL
9.000


828
APVIHGVDVI
8.000


74
NPFYFTDHRI
8.000


1172
QPTESADSLV
8.000


2
EPLLLGRGLI
8.000


566
LSWSKDGEAF
7.500


67
FSWTKDGNPF
7.500


110
FASNKLGIAM
6.000


309
KTLKIENVSY
6.000


349
WTKKPQSAVY
6.000


69
WTKDGNPFYF
6.000


785
TLRVMTPAVY
6.000


6
LGRGLIVYLM
6.000


302
ETKENYGKTL
6.000


954
LPKKLNGNLT
6.000


1118
FVKRNRGGKY
6.000


745
IIKWEPLKSM
6.000


914
KGAGPESEPY
6.000


1121
ESNGSSTATF
5.000


244
NSIKQRKPKL
5.000


455
YSAFLHCEFF
5.000


557
DSHLKHSLKL
5.000


358
YSTGSNGILL
5.000


1093
ISTQGWFIGL
5.000


473
WQKVEEVKPL
4.500


1080
ETRGREYAGL
4.500


934
QPTFLKVIKV
4.000


735
RVQASQPKEM
4.000


1077
DVIETRGREY
4.000


991
KPSWHLSNLN
4.000


693
APFVRYQFRV
4.000


373
EPQPTIKWRV
4.000


34
VPTIIKQSKV
4.000


1048
HPIEVFEPGA
4.000


538
KNPRIPKLHM
4.000


790
TPAVYAPYDV
4.000


440
LIQTKDGENY
3.000


315
NVSYQDKGNY
3.000


449
YATVVGYSAF
3.000


916
AGPESEPYIF
3.000


516
IGKTAVTANL
3.000


1203
GSKEKGSVES
3.000


798
DVKVQAINQL
3.000


1000
NATTKYKFYL
3.000


1044
TQKTHPIEVF
3.000


418
EASNVHGTIL
3.000


596
NVTLEDQGIY
3.000


875
RTHPKEVNIL
3.000


857
RLKGYQINWW
3.000


1104
CAIALLTLLL
3.000


245
SIKQRKPKLL
3.000


725
AAPDRNPQNI
2.400


155
PPKGLPPLHI
2.400


21
FSKAIEIPSS
2.250


274
LLLECFAEGL
2.000


159
LPPLHIYWMN
2.000


957
KLNGNLTGYL
2.000


132
KLPKEKIDPL
2.000


43
VQVAFPFDEY
2.000


29
SSVQQVPTII
2.000


264
SSITILKGEI
2.000


988
TPSKPSWHLS
2.000


260
SGSESSITIL
2.000


689
ILPLAPFVRY
2.000


158
GLPPLHIYWM
2.000


780
TVTNHTLRVM
2.000


4
LLLGRGLIVY
2.000


960
GNLTGYLLQY
2.000


967
LQYQIINDTY
2.000


255
LPPTESGSES
2.000


730
NPQNIRVQAS
2.000


788
VMTPAVYAPY
2.000


8
RGLIVYLMFL
2.000


406
NLQPNHTAVY
2.000










V2-HLA-B3501-10mers-(SET 1)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









10
FPKEKIDPLE
1.200


7
VPKFPKEKID
0.600


6
SVPKFPKEKI
0.400


4
VPSVPKFPKE
0.200


9
KFPKEKIDPL
0.200


1
EFIVPSVPKF
0.100


3
IVPSVPKFPK
0.010


2
FIVPSVPKFP
0.010


5
PSVPKFPKEK
0.005


8
PKFPKEKIDP
0.000










V2-HLA-B3501-10mers-(SET 2)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









1
ESSTLGEGKY
10.000


4
TLGEGKYAGL
2.000


2
SSTLGEGKYA
0.750


5
LGEGKYAGLY
0.600


8
GKYAGLYDDI
0.040


7
EGKYAGLYDD
0.030


3
STLGEGKYAG
0.010


6
GEGKYAGLYD
0.001










V2-HLA-B3501-10mers-(SET 3)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









2
ESSTLGEGKY
10.000


5
TLGEGKYAGL
2.000


3
SSTLGEGKYA
0.750


6
LGEGKYAGLY
0.600


9
GKYAGLYDDI
0.040


8
EGKYAGLYDD
0.030


10
KYAGLYDDIS
0.020


4
STLGEGKYAG
0.010


7
GEGKYAGLYD
0.001


1
EESSTLGEGK
0.001










V3-HLA-B3501-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









43
RNRNMLAEDF
6.000


4
HGVDVINTTY
4.000


52
FIQKSTSCNY
2.000


26
SPQPSIFICS
2.000


33
ICSKEQELSY
2.000


10
NTTYVSNTTY
2.000


22
NATGSPQPSI
1.200


39
ELSYRNRNML
1.000


59
CNYVEKSSTF
1.000


23
ATGSPQPSIF
1.000


53
IQKSTSCNYV
0.600


14
VSNTTYVSNA
0.500


40
LSYRNRNMLA
0.500


57
TSCNYVEKSS
0.500


25
GSPQPSIFIC
0.500


34
CSKEQELSYR
0.450


24
TGSPQPSIFI
0.400


60
NYVEKSSTFF
0.200


28
QPSIFICSKE
0.200


11
TTYVSNTTYV
0.200


47
MLAEDFIQKS
0.200


38
QELSYRNRNM
0.200


48
LAEDFIQKST
0.180


32
FICSKEQELS
0.150


58
SCNYVEKSST
0.150


2
VIHGVDVINT
0.150


62
VEKSSTFFKI
0.120


9
INTTYVSNTT
0.100


31
IFICSKEQEL
0.100


7
DVINTTYVSN
0.100


13
YVSNTTYVSN
0.100


17
TTYVSNATGS
0.100


21
SNATGSPQPS
0.100


55
KSTSCNYVEK
0.100


56
STSCNYVEKS
0.100


8
VINTTYVSNT
0.100


15
SNTTYVSNAT
0.100


5
GVDVINTTYV
0.060


20
VSNATGSPQP
0.050


44
NRNMLAEDFI
0.040


45
RNMLAEDFIQ
0.030


37
EQELSYRNRN
0.030


46
NMLAEDFIQK
0.015


19
YVSNATGSPQ
0.010


51
DFIQKSTSCN
0.010


1
PVIHGVDVIN
0.010


30
SIFICSKEQE
0.010


16
NTTYVSNATG
0.010


6
VDVINTTYVS
0.010


12
TYVSNTTYVS
0.010


3
IHGVDVINTT
0.010


50
EDFIQKSTSC
0.010


29
PSIFICSKEQ
0.005


36
KEQELSYRNR
0.004


41
SYRNRNMLAE
0.003


49
AEDFIQKSTS
0.003


35
SKEQELSYRN
0.003


61
YVEKSSTFFK
0.003


54
QKSTSCNYVE
0.001


42
YRNRNMLAED
0.001


27
PQPSIFICSK
0.001


18
TYVSNATGSP
0.001










V4-HLA-B35-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each start


position is specified, the length of peptide is 10 amino acids,


and the end position for each peptide is the start position plus nine.









10
LPEQPTFLKV
1.200


1
SVTLYSGEDL
1.000


8
EDLPEQPTFL
0.100


5
YSGEDLPEQP
0.100


6
SGEDLPEQPT
0.060


7
GEDLPEQPTF
0.045


9
DLPEQPTFLK
0.020


3
TLYSGEDLPE
0.015


2
VTLYSGEDLP
0.010


4
LYSGEDLPEQ
0.002










V5-HLA-B35-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each start


position is specified, the length of peptide is 10 amino acids,


and the end position for each peptide is the start position plus nine.









1
MPMKLTVNSS
2.000


4
KLTVNSSNSI
0.800


8
NSSNSIKQRK
0.050


9
SSNSIKQRKP
0.050


2
PMKLTVNSSN
0.030


6
TVNSSNSIKQ
0.010


10
SNSIKQRKPK
0.010


3
MKLTVNSSNS
0.010


7
VNSSNSIKQR
0.010


5
LTVNSSNSIK
0.010










V6-HLA-B35-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each start


position is specified, the length of peptide is 10 amino acids,


and the end position for each peptide is the start position plus nine.









8
VPKLEHIEQD
0.600


2
EEIEFIVPKL
0.200


5
EFIVPKLEHI
0.040


7
IVPKLEHIEQ
0.015


6
FIVPKLEHIE
0.010


10
KLEHIEQDER
0.009


3
EIEFIVPKLE
0.003


4
IEFIVPKLEH
0.001


1
SEEIEFIVPK
0.000


9
PKLEHIEQDE
0.000










V7-HLA-B35-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each start


position is specified, the length of peptide is 10 amino acids,


and the end position for each peptide is the start position plus nine.









10
DNISHELFTL
1.500


17
FTLHPEPPRW
0.750


7
IVEDNISHEL
0.600


18
TLHPEPPRWT
0.100


12
ISHELFTLHP
0.100


20
HPEPPRWTKK
0.060


3
DFHVIVEDNI
0.040


8
VEDNISHELF
0.030


6
VIVEDNISHE
0.020


4
FHVIVEDNIS
0.015


5
HVIVEDNISH
0.015


15
ELFTLHPEPP
0.010


11
NISHELFTLH
0.010


2
HDFHVIVEDN
0.010


9
EDNISHELFT
0.010


19
LHPEPPRWTK
0.002


16
LFTLHPEPPR
0.001


14
HELFTLHPEP
0.001


13
SHELFTLHPE
0.000


1
THDFHVIVED
0.000


21
PEPPRWTKKP
0.000









Tables XXII-XLIX:










TABLE XXII





Pos
123456789
score















HLA-V1-A1-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each start


position is specified, the length of peptide is 9 amino acids,


and the end position for each peptide is the start position plus eight.









500
TTEEDAGSY
31


1144
VKDETFGEY
29


1078
VIETRGREY
27


173
HIEQDERVY
26


69
WTKDGNPFY
24


755
EQNGPGLEY
24


961
NLTGYLLQY
24


5
LLGRGLIVY
23


579
GTEDGRIII
23


789
MTPAVYAPY
23


816
VTLYSGEDY
23


350
TKKPQSAVY
22


597
VTLEDQGIY
22


903
EFHLTVLAY
22


1154
DSDEKPLKG
22


78
FTDHRIIPS
21


145
EGDPIVLPC
21


120
SEEIEFIVP
20


157
KGLPPLHIY
20


181
YMSQKGDLY
20


236
STEIGSKAN
20


316
VSYQDKGNY
20


1192
SEDGSFIGA
20


44
QVAFPFDEY
19


690
LPLAPFVRY
19


915
GAGPESEPY
19


919
ESEPYIFQT
19


997
SNLNATTKY
19


1119
VKRNRGGKY
19


1175
ESADSLVEY
19


257
PTESGSESS
18


489
IYENGTLQI
18


586
IIDGANLTI
18


598
TLEDQGIYC
18


627
VPDPPENLH
18


811
PDPQSVTLY
18


975
TYEIGELND
18


1021
ITEESSTLG
18


1082
RGREYAGLY
18


1173
PTESADSLV
18


62
NPEPTFSWT
17


99
HISHFQGKY
17


143
VEEGDPIVL
17


310
TLKIENVSY
17


343
VEEPPRWTK
17


434
VVDVRPLIQ
17


476
VEEVKPLEG
17


479
VKPLEGRRY
17


636
LSERQNRSV
17


669
KEEPGRWEE
17


957
KLNGNLTGY
17


1052
VFEPGAEHI
17


1083
GREYAGLYD
17


1129
VKEKEDLHP
17


1191
FSEDGSFIG
17


194
EEKDSRNDY
16


270
KGEILLLEC
16


336
THDFHVIVE
16


371
EGEPQPTIK
16


393
AGDVVFPRE
16


407
LQPNHTAVY
16


441
IQTKDGENY
16


447
ENYATVVGY
16


630
PPENLHLSE
16


786
LRVMTPAVY
16


810
GPDPQSVTL
16


853
RVHGRLKGY
16


878
PKEVNILRF
16


901
FSEFHLTVL
16


944
DKDTATLSW
16


968
QYQIINDTY
16


1022
TEESSTLGE
16


1094
STQGWFIGL
16


1152
YSDSDEKPL
16


49
FDEYFQIEC
15


299
KGRETKENY
15


318
YQDKGNYRC
15


326
CTASNFLGT
15


359
STGSNGILL
15


466
SPEAVVSWQ
15


482
LEGRRYHIY
15


580
TEDGRIIID
15


653
DHNSNISEY
15


658
ISEYIVEFE
15


747
KWEPLKSME
15


932
PEQPTFLKV
15


972
INDTYEIGE
15


1000
NATTKYKFY
15


1183
YGEGDHGLF
15


1193
EDGSFIGAY
15










HLA-V2-(SET1)-A1-9mers-(SET1)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start


position is specified, the length of peptide is 9 amino acids, and


the end position for each peptide is the start position plus eight.









4
PSVPKFPKE
13


1
FIVPSVPKF
8










HLA-V2-(SET2)-A1-9mers-(SET2)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start


position is specified, the length of peptide is 9 amino acids, and


the end position for each peptide is the start position plus eight.









5
KGREAKENY
15


9
AKENYGKTL
13


6
GREAKENYG
10










HLA-V2-(SET3)-A1-9mers-(SET3)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start


position is specified, the length of peptide is 9 amino acids, and


the end position for each peptide is the start position plus eight.









2
SSTLGEGKY
25


6
GEGKYAGLY
18










HLA-V3-A1-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each start


position is specified, the length of peptide is 9 amino acids, and


the end position for each peptide is the start position plus eight.









33
CSKEQELSY
27


4
GVDVINTTY
26


10
TTYVSNTTY
22


52
IQKSTSCNY
15










HLA-V4-A1-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each start


position is specified, the length of peptide is 9 amino acids, and


the end position for each peptide is the start position plus eight.









9
LPEQPTFLK
13


5
SGEDLPEQP
12


6
GEDLPEQPT
11


1
VTLYSGEDL
8


3
LYSGEDLPE
7


4
YSGEDLPEQ
6










HLA-V5-A1-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each start


position is specified, the length of peptide is 9 amino acids, and


the end position for each peptide is the start position plus eight.









6
VNSSNSIKQ
7


4
LTVNSSNSI
6


8
SSNSIKQRK
6


7
NSSNSIKQR
4


9
SNSIKQRKP
4


2
MKLTVNSSN
3










HLA-V6-A1-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each start


position is specified, the length of peptide is 9 amino acids, and


the end position for each peptide is the start position plus eight.









2
EIEFIVPKL
13


9
KLEHIEQDE
11


4
EFIVPKLEH
7










HLA-V7-A1-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each start


position is specified, the length of peptide is 9 amino acids, and


the end position for each peptide is the start position plus eight.









12
SHELFTLHP
18


19
HPEPPRWTK
16


7
VEDNISHEL
11


6
IVEDNISHE
10


11
ISHELFTLH
9


16
FTLHPEPPR
8
















TABLE XXIII







V1-HLA-A0201-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each start


position is specified, the length of peptide is 9 amino acids, and


the end position for each peptide is the start position plus eight.


Pos









1108
123456789
score












9
LLTLLLLTV
29


125
GLIVYLMFL
28


268
FIVPSVPKL
28


836
ILKGEILLL
28


1101
VINSTLVKV
28


1111
GLMCAIALL
28


4
LLLLTVCFV
28


1105
LLLGRGLIV
27


688
AIALLTLLL
26


118
VILPLAPFV
25


426
AMSEEIEFI
24


785
ILANANIDV
24


1107
TLRVMTPAV
24


1159
ALLTLLLLT
24


17
PLKGSLRSL
24


206
LLLKFSKAI
23


275
AAFPRLRTI
23


406
LLECFAEGL
23


586
NLQPNHTAV
23


591
IIDGANLTI
23


826
NLTISNVTL
23


970
DTAPVIHGV
23


1106
QIINDTYEI
23


245
IALLTLLLL
23


267
SIKQRKPKL
22


584
TILKGEILL
22


923
RIIIDGANL
22


1027
YIFQTPEGV
22


6
TLGEGSKGI
22


23
LGRGLIVYL
21


37
KAIEIPSSV
21


163
IIKQSKVQV
21


166
HIYWMNIEL
21


219
WMNIELEHI
21


253
PMKLTVNSL
21


427
LLLPPTESG
21


429
LANANIDVV
21


619
NANIDVVDV
21


753
ITQVTVLDV
21


942
SMEQNGPGL
21


1033
KVDKDTATL
21


1042
KGIGKISGV
21


1073
NLTQKTHPI
21


1104
SIFQDVIET
21


10
CAIALLTLL
21


13
LIVYLMFLL
20


16
YLMFLLLKF
20


122
FLLLKFSKA
20


210
EIEFIVPSV
20


265
RLRTIVQKM
20


274
SITILKGEI
20


335
LLLECFAEG
20


585
ATHDFHVIV
20


589
IIIDGANLT
20


616
GANLTISNV
20


793
AADITQVTV
20


3
VYAPYDVKV
20


26
PLLLGRGLI
19


266
EIPSSVQQV
19


451
ITILKGEIL
19


471
TVVGYSAFL
19


657
VSWQKVEEV
19


680
NISEYIVEF
19


890
RVQGKKTTV
19


935
RNSGMVPSL
19


949
PTFLKVIKV
19


957
TLSWGLPKK
19


976
KLNGNLTGY
19


995
YEIGELNDI
19


1088
HLSNLNATT
19


1094
GLYDDISTQ
19


1103
STQGWFIGL
19


5
MCAIALLTL
19


11
LLGRGLIVY
18


133
IVYLMFLLL
18


214
LPKEKIDPL
18


292
IVQKMPMKL
18


515
KIGGDLPKG
18


617
AIGKTAVTA
18


673
ADITQVTVL
18


700
GRWEELTRV
18


743
FRVIAVNEV
18


840
EMIIKWEPL
18


900
TLVKVTWST
18


953
AFSEFHLTV
18


1020
GLPKKLNGN
18


1136
PITEESSTL
18


29
HPDPEIQSV
18


154
SSVQQVPTI
17


238
NPPKGLPPL
17


333
EIGSKANSI
17


424
GTATHDFHV
17


444
GTILANANI
17


481
KDGENYATV
17


514
PLEGRRYHI
17


537
NAIGKTAVT
17


540
PKNPRIPKL
17


558
PRIPKLHML
17


611
SHLKHSLKL
17


810
TALDSAADI
17


829
GPDPQSVTL
17


833
PVIHGVDVI
17


841
GVDVINSTL
17


863
LVKVTWSTV
17


870
INWWKTKSL
17


871
SLLDGRTHP
17


875
LLDGRTHPK
17


930
RTHPKEVNI
17


946
GVPEQPTFL
17


950
DTATLSWGL
17


961
LSWGLPKKL
17


1055
NLTGYLLQY
17


1110
PGAEHIVRL
17


1121
TLLLLTVCF
17


1163
RNRGGKYSV
17


1166
SLRSLNRDM
17


35
SLNRDMQPT
17


137
PTIIKQSKV
16


161
KIDPLEVEE
16


216
PLHIYWMNI
16


252
QKMPMKLTV
16


280
KLLLPPTES
16


359
AEGLPTPQV
16


366
STGSNGILL
16


370
LLCEAEGEP
16


432
AEGEPQPTI
16


463
IDVVDVRPL
16


525
FFASPEAVV
16


534
LDIRNATKL
16


603
RVSPKNPRI
16


608
GIYCCSAHT
16


612
SAHTALDSA
16


691
ALDSAADIT
16


780
PLAPFVRYQ
16


788
TVTNHTLRV
16


799
VMTPAVYAP
16


893
VKVQAINQL
16


908
GMVPSLDAF
16


1001
VLAYNSKGA
16


1179
ATTKYKFYL
16


14
SLVEYGEGD
16


82
LMFLLLKFS
15


83
RIIPSNNSG
15


86
IIPSNNSGT
15


142
SNNSGTFRI
15


254
EVEEGDPIV
15


273
LLPPTESGS
15


308
ILLLECFAE
15


327
GKTLKIENV
15


349
TASNFLGTA
15


364
WTKKPQSAV
15


365
GILLCEAEG
15


474
ILLCEAEGE
15


487
QKVEEVKPL
15


546
YHIYENGTL
15


579
HMLELHCES
15


614
GTEDGRIII
15


615
DSAADITQV
15


626
SAADITQVT
15


684
DVPDPPENL
15


746
KKTTVILPL
15


808
IKWEPLKSM
15


876
GSGPDPQSV
15


926
THPKEVNIL
15


958
QTPEGVPEQ
15


965
LNGNLTGYL
15


966
YLLQYQIIN
15


980
LLQYQIIND
15


991
ELNDINITT
15


1092
KPSWHLSNL
15


1099
DISTQGWFI
15


1100
FIGLMCAIA
15


1102
IGLMCAIAL
15


1113
LMCAIALLT
15


19
LLTVCFVKR
15


30
LKFSKAIEI
14


107
SVQQVPTII
14


110
YRCFASNKL
14


115
FASNKLGIA
14


150
LGIAMSEEI
14


158
VLPCNPPKG
14


185
GLPPLHIYW
14


217
KGDLYFANV
14


260
KMPMKLTVN
14


261
SGSESSITI
14


282
GSESSITIL
14


305
GLPTPQVDW
14


331
ENYGKTLKI
14


489
FLGTATHDF
14


504
IYENGTLQI
14


517
DAGSYSCWV
14


524
GKTAVTANL
14


527
NLDIRNATK
14


542
IRNATKLRV
14


556
IPKLHMLEL
14


569
CDSHLKHSL
14


697
SKDGEAFEI
14


702
RYQFRVIAV
14


757
VIAVNEVGR
14


781
NGPGLEYRV
14


822
VTNHTLRVM
14


828
EDYPDTAPV
14


940
APVIHGVDV
14


973
VIKVDKDTA
14


1008
NDTYEIGEL
14


1036
YLRACTSQG
14


1037
GKISGVNLT
14


1053
KISGVNLTQ
14


1066
FEPGAEHIV
14


1098
KNWGDNDSI
14


1112
WFIGLMCAI
14


1189
LLLTVCFVK
14


1202
GLFSEDGSF
14


1216
AGSKEKGSV
14










V2-(SET1)-HLA-A0201-9mers-(SET1)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start


position is specified, the length of peptide is 9 amino acids, and


the end position for each peptide is the start position plus eight.









Pos
123456789
score












1
FIVPSVPKF
18


9
FPKEKIDPL
17


6
VPKFPKEKI
10


5
SVPKFPKEK
8










V2-(SET2)-HLA-A0201-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start


position is specified, the length of peptide is 9 amino acids, and


the end position for each peptide is the start position plus eight.









Pos
123456789
score












9
AKENYGKTL
13


1
GDLPKGREA
12


2
DLPKGREAK
12


8
EAKENYGKT
8


3
LPKGREAKE
7










V2-(SET3)-HLA-A0201-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start


position is specified, the length of peptide is 9 amino acids, and


the end position for each peptide is the start position plus eight.









Pos
123456789
score












5
LGEGKYAGL
17


4
TLGEGKYAG
16


9
KYAGLYDDI
14


3
STLGEGKYA
13


8
GKYAGLYDD
9










V3-HLA-A0201-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each start


position is specified, the length of peptide is 9 amino acids, and


the end position for each peptide is the start position plus eight.









Pos
123456789
score












31
FICSKEQEL
22


22
ATGSPQPSI
18


7
VINTTYVSN
14


39
LSYRNRNML
14


46
MLAEDFIQK
14


1
VIHGVDVIN
13


5
VDVINTTYV
13


47
LAEDFIQKS
13


3
HGVDVINTT
12


11
TYVSNTTYV
12


29
SIFICSKEQ
12


51
FIQKSTSCN
11


53
QKSTSCNYV
11


2
IHGVDVINT
10


8
INTTYVSNT
10


14
SNTTYVSNA
10


18
YVSNATGSP
10


24
GSPQPSIFI
10


38
ELSYRNRNM
10


45
NMLAEDFIQ
10


55
STSCNYVEK
10










V4-HLA-A0201-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each start


position is specified, the length of peptide is 9 amino acids, and


the end position for each peptide is the start position plus eight.









Pos
123456789
score












8
DLPEQPTFL
21


1
VTLYSGEDL
16


2
TLYSGEDLP
13


4
YSGEDLPEQ
11










V5-HLA-A0201-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each start


position is specified, the length of peptide is 9 amino acids, and


the end position for each peptide is the start position plus eight.









Pos
123456789
score












4
LTVNSSNSI
17


1
PMKLTVNSS
11


3
KLTVNSSNS
10










V6-HLA-A0201-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each start


position is specified, the length of peptide is 9 amino acids, and


the end position for each peptide is the start position plus eight.









Pos
123456789
score












5
FIVPKLEHI
24


2
EIEFIVPKL
20










V7-HLA-A0201-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each start


position is specified, the length of peptide is 9 amino acids, and


the end position for each peptide is the start position plus eight.









Pos
123456789
score












10
NISHELFTL
24


7
VEDNISHEL
14


5
VIVEDNISH
13


17
TLHPEPPRW
13


14
ELFTLHPEP
12


















TABLE XXIV





Pos
123456789
score















V1-HLA-A0203-9mers-282P1G3


NoResultsFound.


V2-(SET1)HLA-A0203-9mers-282P1G3


NoResultsFound.


V2-(SET2)-HLA-A0203-9mers-282P1G3


NoResultsFound.


V2-(SET3)-HLA-A0203-9mers-282P1G3


NoResultsFound.


V3-HLA-A0203-9mers-282P1G3


NoResultsFound.


V4-HLA-A0203-9mers-(SET2)-282P1G3


NoResultsFound.


V5-HLA-A0203-9mers-(SET2)-282P1G3


NoResultsFound.


V6-HLA-A0203-9mers-(SET2)-282P1G3


NoResultsFound.


V7-HLA-A0203-9mers-(SET2)-282P1G3


NoResultsFound.


















TABLE XXV





Pos
123456789
score















V1-HLA-A3-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3;


each start position is specified, the length of peptide


is 9 amino acids, and the end position for


each peptide is the start position plus eight.









792
AVYAPYDVK
33


835
DVINSTLVK
31


436
DVRPLIQTK
30


524
NLDIRNATK
30


149
IVLPCNPPK
27


296
DLPKGRETK
27


843
KVTWSTVPK
27


1112
LLLTVCFVK
27


1118
FVKRNRGGK
26


1197
FIGAYAGSK
26


5
LLGRGLIVY
25


677
ELTRVQGKK
25


937
FLKVIKVDK
25


310
TLKIENVSY
24


701
RVIAVNEVG
24


760
GLEYRVTWK
24


853
RVHGRLKGY
24


871
LLDGRTHPK
24


961
NLTGYLLQY
24


356
AVYSTGSNG
23


547
MLELHCESK
23


4
LLLGRGLIV
22


106
KYRCFASNK
22


129
SVPKLPKEK
22


209
PRLRTIVQK
22


413
AVYQCEASN
22


515
AIGKTAVTA
22


584
RIIIDGANL
22


680
RVQGKKTTV
22


689
ILPLAPFVR
22


949
TLSWGLPKK
22


957
KLNGNLTGY
22


998
NLNATTKYK
22


1088
GLYDDISTQ
22


381
RVNGSPVDN
21


396
VVFPREISF
21


687
TVILPLAPF
21


744
MIIKWEPLK
21


787
RVMTPAVYA
21


882
NILRFSGQR
21


948
ATLSWGLPK
21


983
DINITTPSK
21


1008
YLRACTSQG
21


1037
KISGVNLTQ
21


1051
EVFEPGAEH
21


1110
TLLLLTVCF
21


11
VYLMFLLL
20


82
RIIPSNNSG
20


213
TIVQKMPMK
20


221
KLTVNSLKH
20


291
NKIGGDLPK
20


530
ATKLRVSPK
20


661
YIVEFEGNK
20


704
AVNEVGRSQ
20


733
NIRVQASQP
20


942
KVDKDTATL
20


1058
EHIVRLMTK
20


1122
NRGGKYSVK
20


24
AIEIPSSVQ
19


44
QVAFPFDEY
19


177
DERVYMSQK
19


478
EVKPLEGRR
19


520
AVTANLDIR
19


585
IIIDGANLT
19


586
IIDGANLTI
19


591
NLTISNVTL
19


645
RLTWEAGAD
19


829
PVIHGVDVI
19


851
KDRVHGRLK
19


996
LSNLNATTK
19


1040
GVNLTQKTH
19


1078
VIETRGREY
19


1209
SVESNGSST
19


37
IIKQSKVQV
18


137
KIDPLEVEE
18


173
HIEQDERVY
18


179
RVYMSQKGD
18


187
DLYFANVEE
18


252
KLLLPPTES
18


268
ILKGEILLL
18


343
VEEPPRWTK
18


534
RVSPKNPRI
18


841
LVKVTWSTV
18


859
KGYQINWWK
18


861
YQINWWKTK
18


870
SLLDGRTHP
18


883
ILRFSGQRN
18


995
HLSNLNATT
18


1082
RGREYAGLY
18


1107
ALLTLLLLT
18


1108
LLTLLLLTV
18


1113
LLTVCFVKR
18


1128
SVKEKEDLH
18


1137
PDPEIQSVK
18


1199
GAYAGSKEK
18


3
PLLLGRGLI
17


13
YLMFLLLKF
17


16
FLLLKFSKA
17


114
KLGIAMSEE
17


124
EFIVPSVPK
17


210
RLRTIVQKM
17


253
LLLPPTESG
17


340
HVIVEEPPR
17


350
TKKPQSAVY
17


365
ILLCEAEGE
17


452
VVGYSAFLH
17


469
AVVSWQKVE
17


488
HIYENGTLQ
17


494
TLQINRTTE
17


532
KLRVSPKNP
17


657
NISEYIVEF
17


695
FVRYQFRVI
17


702
VIAVNEVGR
17


803
AINQLGSGP
17


833
GVDVINSTL
17


857
RLKGYQINW
17


869
KSLLDGRTH
17


897
SLDAFSEFH
17


907
TVLAYNSKG
17


939
KVIKVDKDT
17


1006
KFYLRACTS
17


1025
SSTLGEGSK
17


1180
LVEYGEGDH
17


1189
GLFSEDGSF
17


8
RGLIVYLMF
16


93
RIPNEGHIS
16


220
MKLTVNSLK
16


254
LLPPTESGS
16


273
ILLLECFAE
16


274
LLLECFAEG
16


304
KENYGKTLK
16


312
KIENVSYQD
16


344
EEPPRWTKK
16


390
HPFAGDVVF
16


421
NVHGTILAN
16


430
ANIDVVDVR
16


434
VVDVRPLIQ
16


447
ENYATVVGY
16


472
SWQKVEEVK
16


526
DIRNATKLR
16


544
KLHMLELHC
16


563
SLKLSWSKD
16


612
ALDSAADIT
16


621
QVTVLDVPD
16


637
SERQNRSVR
16


643
SVRLTWEAG
16


688
VILPLAPFV
16


735
RVQASQPKE
16


764
RVTWKPQGA
16


795
APYDVKVQA
16


817
TLYSGEDYP
16


934
QPTFLKVIK
16


1011
ACTSQGCGK
16


1020
PITEESSTL
16


1038
ISGVNLTQK
16


1060
IVRLMTKNW
16


1077
DVIETRGRE
16


1105
AIALLTLLL
16


1119
VKRNRGGKY
16







V2-(SET1)HLA-A3-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each


start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









5
SVPKFPKEK
22


1
FIVPSVPKF
15


2
IVPSVPKFP
14


3
VPSVPKFPK
10







V2-(SET2)-HLA-A3-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each


start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









2
DLPKGREAK
24


7
REAKENYGK
15


5
KGREAKENY
13


9
AKENYGKTL
11







V2-(SET3)-HLA-A3-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each


start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









4
TLGEGKYAG
14


1
ESSTLGEGK
13


2
SSTLGEGKY
11


6
GEGKYAGLY
10


3
STLGEGKYA
8


8
GKYAGLYDD
8


7
EGKYAGLYD
7


5
LGEGKYAGL
6


9
KYAGLYDDI
6







V3-HLA-A3-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each


start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









46
MLAEDFIQK
25


4
GVDVINTTY
21


6
DVINTTYVS
18


1
VIHGVDVIN
15


10
TTYVSNTTY
15


12
YVSNTTYVS
15


27
QPSIFICSK
15


55
STSCNYVEK
15


60
YVEKSSTFF
15


7
VINTTYVSN
14


18
YVSNATGSP
14


33
CSKEQELSY
13


59
NYVEKSSTF
13


38
ELSYRNRNM
12







V4-HLA-A3-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each


start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









2
TLYSGEDLP
16


7
EDLPEQPTF
13


8
DLPEQPTFL
13


9
LPEQPTFLK
10







V5-HLA-A3-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each


start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









5
TVNSSNSIK
23


3
KLTVNSSNS
16


8
SSNSIKQRK
11







V6-HLA-A3-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each


start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









1
EEIEFIVPK
18


9
KLEHIEQDE
13


5
FIVPKLEHI
12


6
IVPKLEHIE
12


4
EFIVPKLEH
10


2
EIEFIVPKL
8







V7-HLA-A3-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each


start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









19
HPEPPRWTK
18


6
IVEDNISHE
16


20
PEPPRWTKK
16


5
VIVEDNISH
15


17
TLHPEPPRW
14


4
HVIVEDNIS
13


10
NISHELFTL
12


14
ELFTLHPEP
10


11
ISHELFTLH
8


779
ETVTNHTLR
23


835
DVINSTLVK
23


1147
ETFGEYSDS
23


125
FIVPSVPKL
22


142
EVEEGDPIV
22


266
ITILKGEIL
22


395
DVVFPREIS
22


450
ATVVGYSAF
22


755
EQNGPGLEY
22


833
GVDVINSTL
22


880
EVNILRFSG
22


1091
DDISTQGWF
22


122
EIEFIVPSV
21


194
EEKDSRNDY
21


359
STGSNGILL
21


597
VTLEDQGIY
21


707
EVGRSQPSQ
21


826
DTAPVIHGV
21


974
DTYEIGELN
21


2
EPLLLGRGL
20


26
EIPSSVQQV
20


44
QVAFPFDEY
20


121
EEIEFIVPS
20


289
DWNKIGGDL
20


573
EAFEINGTE
20


778
EETVTNHTL
20


930
GVPEQPTFL
20


1156
DEKPLKGSL
20


1182
EYGEGDHGL
20


11
IVYLMFLLL
19


69
WTKDGNPFY
19


136
EKIDPLEVE
19


214
IVQKMPMKL
19


540
PRIPKLHML
19


721
ETPPAAPDR
19


798
DVKVQAINQ
19


816
VTLYSGEDY
19


935
PTFLKVIKV
19


942
KVDKDTATL
19


1058
EHIVRLMTK
19


1080
ETRGREYAG
19


145
EGDPIVLPC
18


474
QKVEEVKPL
18


477
EEVKPLEGR
18


592
LTISNVTLE
18


638
ERQNRSVRL
18


657
NISEYIVEF
18


670
EEPGRWEEL
18


789
MTPAVYAPY
18


829
PVIHGVDVI
18


893
GMVPSLDAF
18


1094
STQGWFIGL
18


10
LIVYLMFLL
17


33
QVPTIIKQS
17


175
EQDERVYMS
17


238
EIGSKANSI
17


271
GEILLLECF
17


581
EDGRIIIDG
17


584
RIIIDGANL
17


799
VKVQAINQL
17


961
NLTGYLLQY
17


977
EIGELNDIN
17


1001
ATTKYKFYL
17


1020
PITEESSTL
17


1104
CAIALLTLL
17


9
GLIVYLMEL
16


51
EYFQIECEA
16


99
HISHFQGKY
16


172
EHIEQDERV
16


197
DSRNDYCCF
16


222
LTVNSLKHA
16


263
ESSITILKG
16


277
ECFAEGLPT
16


314
ENVSYQDKG
16


401
EISFTNLQP
16


421
NVHGTILAN
16


432
IDVVDVRPL
16


511
WVENAIGKT
16


520
AVTANLDIR
16


617
ADITQVTVL
16


622
VTVLDVPDP
16


686
TTVILPLAP
16


919
ESEPYIFQT
16


1023
EESSTLGEG
16


1030
EGSKGIGKI
16


1114
LTVCFVKRN
16


1209
SVESNGSST
16


5
LLGRGLIVY
15


13
YLMFLLLKF
15


35
PTIIKQSKV
15


133
LPKEKIDPL
15


144
EEGDPIVLP
15


157
KGLPPLHIY
15


178
ERVYMSQKG
15


272
EILLLECFA
15


373
EPQPTIKWR
15


462
EFFASPEAV
15


468
EAVVSWQKV
15


469
AVVSWQKVE
15


629
DPPENLHLS
15


685
KTTVILPLA
15


847
STVPKDRVH
15


957
KLNGNLTGY
15


980
ELNDINITT
15


1078
VIETRGREY
15


1140
EIQSVKDET
15


1185
EGDHGLFSE
15


1189
GLFSEDGSF
15


90
GTFRIPNEG
14


124
EFIVPSVPK
14


173
HIEQDERVY
14


192
NVEEKDSRN
14


223
TVNSLKHAN
14


245
SIKQRKPKL
14


267
TILKGEILL
14


338
DFHVIVEEP
14


340
HVIVEEPPR
14


487
YHIYENGTL
14


525
LDIRNATKL
14


537
PKNPRIPKL
14


576
EINGTEDGR
14


582
DGRIIIDGA
14


660
EYIVEFEGN
14


676
EELTRVQGK
14


684
KKTTVILPL
14


701
RVIAVNEVG
14


704
AVNEVGRSQ
14


786
LRVMTPAVY
14


811
PDPQSVTLY
14


839
STLVKVTWS
14


844
VTWSTVPKD
14


852
DRVHGRLKG
14


867
KTKSLLDGR
14


878
PKEVNILRF
14


926
QTPEGVPEQ
14


933
EQPTFLKVI
14


939
KVIKVDKDT
14


973
NDTYEIGEL
14


1055
PGAEHIVRL
14


1073
SIFQDVIET
14


1103
MCAIALLTL
14


1105
AIALLTLLL
14


1128
SVKEKEDLH
14


6
LGRGLIVYL
13


41
SKVQVAFPF
13


45
VAFPFDEYF
13


65
PTFSWTKDG
13


78
FTDHRIIPS
13


95
PNEGHISHF
13


147
DPIVLPCNP
13


163
HIYWMNIEL
13


219
PMKLTVNSL
13


261
GSESSITIL
13


268
ILKGEILLL
13


305
ENYGKTLKI
13


356
AVYSTGSNG
13


456
SAFLHCEFF
13


530
ATKLRVSPK
13


614
DSAADITQV
13


618
DITQVTVLD
13


619
ITQVTVLDV
13


677
ELTRVQGKK
13


775
EWEEETVTN
13


780
TVTNHTLRV
13


815
SVTLYSGED
13


876
THPKEVNIL
13


890
RNSGMVPSL
13


899
DAFSEFHLT
13


906
LTVLAYNSK
13


907
TVLAYNSKG
13


962
LTGYLLQYQ
13


968
QYQIINDTY
13


983
DINITTPSK
13


991
KPSWHLSNL
13


1045
QKTHPIEVF
13


1082
RGREYAGLY
13


1092
DISTQGWFI
13


1106
IALLTLLLL
13


1109
LTLLLLTVC
13


1119
VKRNRGGKY
13


1144
VKDETFGEY
13


1154
DSDEKPLKG
13


1157
EKPLKGSLR
13


1159
PLKGSLRSL
13


















TABLE XXVI





Pos
123456789
score















V1-HLA-A26-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each


start position is specified, the length of peptide


is 9 amino acids, and the end position


for each peptide is the start position plus eight.









626
DVPDPPENL
27


687
TVILPLAPF
27


1051
EVFEPGAEH
27


1193
EDGSFIGAY
27


903
EFHLTVLAY
26


396
VVFPREISF
25


436
DVRPLIQTK
25


653
DHNSNISEY
25


946
DTATLSWGL
25


1077
DVIETRGRE
25


1175
ESADSLVEY
25


447
ENYATVVGY
24


853
RVHGRLKGY
24


929
EGVPEQPTF
24


302
ETKENYGKT
23


433
DVVDVRPLI
23


451
TVVGYSAFL
23


478
EVKPLEGRR
23


500
TTEEDAGSY
23


743
EMIIKWEPL
23







V2-(SET1)-HLA-A26-9mers-(SET1)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each


start position is specified, the length of peptide


is 9 amino acids, and the end position


for each peptide is the start position plus eight.









1
FIVPSVPKF
22


9
FPKEKIDPL
15


2
IVPSVPKFP
11


5
SVPKFPKEK
11







V2-(SET2)-HLA-A26-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each


start position is specified, the length of


peptide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









8
EAKENYGKT
15


5
KGREAKENY
12


9
AKENYGKTL
10


2
DLPKGREAK
9







V2-(SET3)-HLA-A26-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each


start position is specified, the length of peptide


is 9 amino acids, and the end position


for each peptide is the start position plus eight.









2
SSTLGEGKY
14


1
ESSTLGEGK
12


7
EGKYAGLYD
12


6
GEGKYAGLY
11


3
STLGEGKYA
10


5
LGEGKYAGL
9







V3-HLA-A26-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for


each peptide is the start position plus eight.









4
GVDVINTTY
23


6
DVINTTYVS
22


10
TTYVSNTTY
20


60
YVEKSSTFF
18


59
NYVEKSSTF
17


49
EDFIQKSTS
16


31
FICSKEQEL
14


33
CSKEQELSY
14


50
DFIQKSTSC
14


18
YVSNATGSP
12


62
EKSSTFFKI
12


23
TGSPQPSIF
11


52
IQKSTSCNY
11







V4-HLA-A26-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for


each peptide is the start position plus eight.









7
EDLPEQPTF
20


8
DLPEQPTFL
18


1
VTLYSGEDL
17







V5-HLA-A26-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









4
LTVNSSNSI
13


5
TVNSSNSIK
13







V6-HLA-A26-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









2
EIEFIVPKL
29


1
EEIEFIVPK
20


4
EFIVPKLEH
15


5
FIVPKLEHI
14







V7-HLA-A26-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









8
EDNISHELF
18


6
IVEDNISHE
17


10
NISHELFTL
17


14
ELFTLHPEP
15


2
DFHVIVEDN
14


4
HVIVEDNIS
14


9
DNISHELFT
14


5
VIVEDNISH
12


7
VEDNISHEL
10


1
HDFHVIVED
8


16
FTLHPEPPR
8


















TABLE XXVII





Pos
123456789
score















V1-HLA-B0702-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each


start position is specified, the length of


peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









988
TPSKPSWHL
26


810
GPDPQSVTL
24


151
LPCNPPKGL
23


154
NPPKGLPPL
23


539
NPRIPKLHM
23


542
IPKLHMLEL
23


991
KPSWHLSNL
23


2
EPLLLGRGL
22


133
LPKEKIDPL
21


828
APVIHGVDV
21


954
LPKKLNGNL
21


390
HPFAGDVVF
20


795
APYDVKVQA
20


1172
QPTESADSL
20


84
IPSNNSGTF
19


130
VPKLPKEKI
19


247
KQRKPKLLL
19


250
KPKLLLPPT
19


726
APDRNPQNI
19


730
NPQNIRVQA
19


62
NPEPTFSWT
18


127
VPSVPKLPK
18


352
KPQSAVYST
18


385
SPVDNHPFA
18


671
EPGRWEELT
18


758
GPGLEYRVT
18


772
APVEWEEET
18


1136
HPDPEIQSV
18


690
LPLAPFVRY
17


890
RNSGMVPSL
17


1019
KPITEESST
17


6
LGRGLIVYL
16


47
FPFDEYFQI
16


159
LPPLHIYWM
16


285
TPQVDWNKI
16


917
GPESEPYIF
16


1105
AIALLTLLL
16


218
MPMKLTVNS
15


268
ILKGEILLL
15


536
SPKNPRIPK
15


617
ADITQVTVL
15


627
VPDPPENLH
15


712
QPSQPSDHH
15


723
PPAAPDRNP
15


768
KPQGAPVEW
15


942
KVDKDTATL
15


11
VYLMFLLL
14


27
IPSSVQQVP
14


208
FPRLRTIVQ
14


280
AEGLPTPQV
14


419
ASNVHGTIL
14


451
TVVGYSAFL
14


682
QGKKTTVIL
14


684
KKTTVILPL
14


931
VPEQPTFLK
14


1035
IGKISGVNL
14


1054
EPGAEHIVR
14


1158
KPLKGSLRS
14


1214
GSSTATFPL
14


39
KQSKVQVAF
13


59
AKGNPEPTF
13


71
KDGNPFYFT
13


125
FIVPSVPKL
13


143
VEEGDPIVL
13


155
PPKGLPPLH
13


203
CCFAAFPRL
13


205
FAAFPRLRT
13


297
LPKGRETKE
13


346
PPRWTKKPQ
13


370
AEGEPQPTI
13


432
IDVVDVRPL
13


517
GKTAVTANL
13


552
CESKCDSHL
13


558
SHLKHSLKL
13


584
RIIIDGANL
13


626
DVPDPPENL
13


628
PDPPENLHL
13


638
ERQNRSVRL
13


670
EEPGRWEEL
13


693
APFVRYQFR
13


787
RVMTPAVYA
13


812
DPQSVTLYS
13


824
YPDTAPVIH
13


921
EPYIFQTPE
13


1001
ATTKYKFYL
13


1055
PGAEHIVRL
13


1094
STQGWFIGL
13


1103
MCAIALLTL
13


1106
IALLTLLLL
13







V2-(SET1)-HLA-B0702-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for


each peptide is the start position plus eight.









9
FPKEKIDPL
21


6
VPKFPKEKI
19


3
VPSVPKFPK
16







V2-(SET2)-HLA-B0702-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for


each peptide is the start position plus eight.









3
LPKGREAKE
13


9
AKENYGKTL
13


1
GDLPKGREA
8







V2-(SET3)-HLA-B0702-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for


each peptide is the start position plus eight.









5
LGEGKYAGL
13


9
KYAGLYDDI
10


3
STLGEGKYA
9







V3-HLA-B0702-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for


each peptide is the start position plus eight.









22
ATGSPQPSI
13


27
QPSIFICSK
12


25
SPQPSIFIC
11


39
LSYRNRNML
11


24
GSPQPSIFI
10


31
FICSKEQEL
10


62
EKSSTFFKI
10


2
IHGVDVINT
9


23
TGSPQPSIF
9


38
ELSYRNRNM
9


44
RNMLAEDFI
9


48
AEDFIQKST
9


60
YVEKSSTFF
9


5
VDVINTTYV
8


8
INTTYVSNT
8


11
TYVSNTTYV
8


40
SYRNRNMLA
8


53
QKSTSCNYV
8


14
SNTTYVSNA
7


15
NTTYVSNAT
7


3
HGVDVINTT
6


9
NTTYVSNTT
6


43
NRNMLAEDF
6


58
CNYVEKSST
6


59
NYVEKSSTF
6







V4-HLA-B0702-9mers


Each peptide is a portion of SEQ ID NO: 9; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for


each peptide is the start position plus eight.









9
LPEQPTFLK
14


8
DLPEQPTFL
12


1
VTLYSGEDL
11


6
GEDLPEQPT
11


7
EDLPEQPTF
9


3
LYSGEDLPE
7







V5-HLA-B0702-9mers


Each peptide is a portion of SEQ ID NO: 11; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for


each peptide is the start position plus eight.









4
LTVNSSNSI
6


6
VNSSNSIKQ
2


7
NSSNSIKQR
2


9
SNSIKQRKP
2







V6-HLA-B0702-9mers


Each peptide is a portion of SEQ ID NO: 13; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for


each peptide is the start position plus eight.









2
EIEFIVPKL
13


7
VPKLEHIEQ
10


5
FIVPKLEHI
7


4
EFIVPKLEH
6







V7-HLA-B0702-9mers


Each peptide is a portion of SEQ ID NO: 15; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for


each peptide is the start position plus eight.









19
HPEPPRWTK
15


10
NISHELFTL
12


7
VEDNISHEL
11


9
DNISHELFT
9


18
LHPEPPRWT
9


8
EDNISHELF
7


















TABLE XXVIII





Pos
123456789
score















V1-HLA-B08-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









133
LPKEKIDPL
39


245
SIKQRKPKL
34


542
IPKLHMLEL
29


268
ILKGEILLL
28


954
LPKKLNGNL
27


849
VPKDRVHGR
26


1159
PLKGSLRSL
26


130
VPKLPKEKI
24


297
LPKGRETKE
24


855
HGRLKGYQI
24


1128
SVKEKEDLH
24


377
TIKWRVNGS
23


1203
GSKEKGSVE
23


208
FPRLRTIVQ
22


219
PMKLTVNSL
22


238
EIGSKANSI
22


246
IKQRKPKLL
22


266
ITILKGEIL
22


743
EMIIKWEPL
22


863
INWWKTKSL
22


1035
IGKISGVNL
22


1042
NLTQKTHPI
22


638
ERQNRSVRL
21


670
EEPGRWEEL
21


682
QGKKTTVIL
21


1002
TTKYKFYLR
21


104
QGKYRCFAS
20


248
QRKPKLLLP
20


481
PLEGRRYHI
20


530
ATKLRVSPK
20


540
PRIPKLHML
20


865
WWKTKSLLD
20


877
HPKEVNILR
20


1156
DEKPLKGSL
20


2
EPLLLGRGL
19


226
SLKHANDSS
19


537
PKNPRIPKL
19


563
SLKLSWSKD
19


740
QPKEMIIKW
19


796
PYDVKVQAI
19


937
FLKVIKVDK
19







V2-(SET1)-HLA-B08-9mers-(SET1)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









9
FPKEKIDPL
40


6
VPKFPKEKI
23







V2-(SET2)-HLA-B08-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









3
LPKGREAKE
24


8
EAKENYGKT
18


1
GDLPKGREA
12


6
GREAKENYG
11


9
AKENYGKTL
11







V2-(SET3)-HLA-B08-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









5
LGEGKYAGL
20


7
EGKYAGLYD
13


4
TLGEGKYAG
9







V3-HLA-B08-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









31
FICSKEQEL
26


38
ELSYRNRNM
18


59
NYVEKSSTF
18


40
SYRNRNMLA
17


33
CSKEQELSY
12







V4-HLA-B08-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









8
DLPEQPTFL
18


1
VTLYSGEDL
12


7
EDLPEQPTF
8







V5-HLA-B08-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









1
PMKLTVNSS
12


9
SNSIKQRKP
12


3
KLTVNSSNS
7


4
LTVNSSNSI
6







V6-HLA-B08-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









5
FIVPKLEHI
21


7
VPKLEHIEQ
19


2
EIEFIVPKL
17







V7-HLA-B08-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









10
NISHELFTL
14


7
VEDNISHEL
12


14
ELFTLHPEP
9


5
VIVEDNISH
8


8
EDNISHELF
8


20
PEPPRWTKK
8


3
FHVIVEDNI
7


19
HPEPPRWTK
7


17
TLHPEPPRW
6


















TABLE XXIX





Pos
123456789
score















V1-HLA-B1510-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









876
THPKEVNIL
23


487
YHIYENGTL
22


558
SHLKHSLKL
21


1055
PGAEHIVRL
17


432
IDVVDVRPL
16


810
GPDPQSVTL
16


101
SHFQGKYRC
15


143
VEEGDPIVL
15


638
ERQNRSVRL
15


6
LGRGLIVYL
14


125
FIVPSVPKL
14


172
EHIEQDERV
14


214
IVQKMPMKL
14


268
ILKGEILLL
14


336
THDFHVIVE
14


537
PKNPRIPKL
14


542
IPKLHMLEL
14


718
DHHETPPAA
14


719
HHETPPAAP
14


753
SMEQNGPGL
14


831
IHGVDVINS
14


890
RNSGMVPSL
14


988
TPSKPSWHL
14


1035
IGKISGVNL
14


2
EPLLLGRGL
13


154
NPPKGLPPL
13


203
CCFAAFPRL
13


245
SIKQRKPKL
13


246
IKQRKPKLL
13


247
KQRKPKLLL
13


261
GSESSITIL
13


267
TILKGEILL
13


303
TKENYGKTL
13


591
NLTISNVTL
13


617
ADITQVTVL
13


626
DVPDPPENL
13


653
DHNSNISEY
13


670
EEPGRWEEL
13


682
QGKKTTVIL
13


778
EETVTNHTL
13


833
GVDVINSTL
13


901
FSEFHLTVL
13


930
GVPEQPTFL
13


1047
THPIEVFEP
13


1058
EHIVRLMTK
13


1100
IGLMCAIAL
13


1127
YSVKEKEDL
13


1156
DEKPLKGSL
13


1159
PLKGSLRSL
13


1182
EYGEGDHGL
13


9
GLIVYLMFL
12


11
IVYLMFLLL
12


133
LPKEKIDPL
12


151
LPCNPPKGL
12


174
IEQDERVYM
12


266
ITILKGEIL
12


358
YSTGSNGIL
12


389
NHPFAGDVV
12


451
TVVGYSAFL
12


474
QKVEEVKPL
12


540
PRIPKLHML
12


550
LHCESKCDS
12


552
CESKCDSHL
12


556
CDSHLKHSL
12


605
YCCSAHTAL
12


628
PDPPENLHL
12


657
NISEYIVEF
12


783
NHTLRVMTP
12


799
VKVQAINQL
12


850
PKDRVHGRL
12


863
INWWKTKSL
12


864
NWWKTKSLL
12


878
PKEVNILRF
12


942
KVDKDTATL
12


950
LSWGLPKKL
12


973
NDTYEIGEL
12


994
WHLSNLNAT
12


1001
ATTKYKFYL
12


1020
PITEESSTL
12


1101
GLMCAIALL
12


1103
MCAIALLTL
12


1106
IALLTLLLL
12


1135
LHPDPEIQS
12


1214
GSSTATFPL
12


10
LIVYLMFLL
11


39
KQSKVQVAF
11


80
DHRIIPSNN
11


84
IPSNNSGTF
11


98
GHISHFQGK
11


107
YRCFASNKL
11


162
LHIYWMNIE
11


163
HIYWMNIEL
11


180
VYMSQKGDL
11


219
PMKLTVNSL
11


228
KHANDSSSS
11


275
LLECFAEGL
11


289
DWNKIGGDL
11


324
YRCTASNFL
11


359
STGSNGILL
11


390
HPFAGDVVF
11


399
PREISFTNL
11


419
ASNVHGTIL
11


459
LHCEFFASP
11


517
GKTAVTANL
11


525
LDIRNATKL
11


561
KHSLKLSWS
11


609
AHTALDSAA
11


634
LHLSERQNR
11


684
KKTTVILPL
11


743
EMIIKWEPL
11


854
VHGRLKGYQ
11


898
LDAFSEFHL
11


929
EGVPEQPTF
11


946
DTATLSWGL
11


954
LPKKLNGNL
11


991
KPSWHLSNL
11


1056
GAEHIVRLM
11


1081
TRGREYAGL
11


1094
STQGWFIGL
11


1105
AIALLTLLL
11


1141
IQSVKDETF
11


1152
YSDSDEKPL
11







V2-(SET1)-HLA-B1510-9mers-(SET1)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









9
FPKEKIDPL
12


1
FIVPSVPKF
10







V2-(SET2)-HLA-B1510-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









9
AKENYGKTL
12


1
GDLPKGREA
6







V2-(SET3)-HLA-B1510-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









5
LGEGKYAGL
12


4
TLGEGKYAG
5







V3-HLA-B1510-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









2
IHGVDVINT
14


39
LSYRNRNML
12


23
TGSPQPSIF
11


31
FICSKEQEL
11


38
ELSYRNRNM
10


60
YVEKSSTFF
9


59
NYVEKSSTF
8


43
NRNMLAEDF
6







V4-HLA-B1510-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









8
DLPEQPTFL
12


7
EDLPEQPTF
11


1
VTLYSGEDL
10







V5-HLA-B1510-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









8
SSNSIKQRK
3


9
SNSIKQRKP
3


3
KLTVNSSNS
2


6
VNSSNSIKQ
2


7
NSSNSIKQR
2


1
PMKLTVNSS
1


5
TVNSSNSIK
1







V6-HLA-B1510-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









2
EIEFIVPKL
14







V7-HLA-B1510-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









18
LHPEPPRWT
15


10
NISHELFTL
12


7
VEDNISHEL
11


12
SHELFTLHP
11


3
FHVIVEDNI
10


8
EDNISHELF
7


17
TLHPEPPRW
7


















TABLE XXX





Pos
123456789
score















V1-HLA-B2705-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for


each peptide is the start position plus eight.









209
PRLRTIVQK
27


7
GRGLIVYLM
25


399
PREISFTNL
25


540
PRIPKLHML
25


734
IRVQASQPK
25


1122
NRGGKYSVK
25


107
YRCFASNKL
24


533
LRVSPKNPR
24


638
ERQNRSVRL
24


324
YRCTASNFL
22


1081
TRGREYAGL
22


786
LRVMTPAVY
21


673
GRWEELTRV
20


92
FRIPNEGHI
19


261
GSESSITIL
19


301
RETKENYGK
19


485
RRYHIYENG
19


584
RIIIDGANL
19


856
GRLKGYQIN
19


859
KGYQINWWK
19


884
LRFSGQRNS
19


890
RNSGMVPSL
19


1189
GLFSEDGSF
19


1199
GAYAGSKEK
19


8
RGLIVYLMF
18


39
KQSKVQVAF
18


268
ILKGEILLL
18


271
GEILLLECF
18


291
NKIGGDLPK
18


390
HPFAGDVVF
18


437
VRPLIQTKD
18


484
GRRYHIYEN
18


558
SHLKHSLKL
18


562
HSLKLSWSK
18


799
VKVQAINQL
18


6
LGRGLIVYL
17


9
GLIVYLMFL
17


81
HRIIPSNNS
17


125
FIVPSVPKL
17


247
KQRKPKLLL
17


267
TILKGEILL
17


284
PTPQVDWNK
17


304
KENYGKTLK
17


517
GKTAVTANL
17


525
LDIRNATKL
17


537
PKNPRIPKL
17


583
GRIIIDGAN
17


617
ADITQVTVL
17


679
TRVQGKKTT
17


684
KKTTVILPL
17


709
GRSQPSQPS
17


810
GPDPQSVTL
17


833
GVDVINSTL
17


893
GMVPSLDAF
17


929
EGVPEQPTF
17


1055
PGAEHIVRL
17


1101
GLMCAIALL
17


1150
GEYSDSDEK
17


1161
KGSLRSLNR
17


15
MFLLLKFSK
16


95
PNEGHISHF
16


106
KYRCFASNK
16


149
IVLPCNPPK
16


154
NPPKGLPPL
16


182
MSQKGDLYF
16


191
ANVEEKDSR
16


203
CCFAAFPRL
16


210
RLRTIVQKM
16


212
RTIVQKMPM
16


214
IVQKMPMKL
16


242
KANSIKQRK
16


245
SIKQRKPKL
16


329
SNFLGTATH
16


380
WRVNGSPVD
16


396
VVFPREISF
16


424
GTILANANI
16


430
ANIDVVDVR
16


436
DVRPLIQTK
16


491
ENGTLQINR
16


534
RVSPKNPRI
16


657
NISEYIVEF
16


687
TVILPLAPF
16


760
GLEYRVTWK
16


852
DRVHGRLKG
16


874
GRTHPKEVN
16


878
PKEVNILRF
16


930
GVPEQPTFL
16


942
KVDKDTATL
16


949
TLSWGLPKK
16


954
LPKKLNGNL
16


991
KPSWHLSNL
16


1074
IFQDVIETR
16


1104
CAIALLTLL
16


1106
IALLTLLLL
16


1124
GGKYSVKEK
16


1137
PDPEIQSVK
16


11
IVYLMFLLL
15


13
YLMFLLLKF
15


41
SKVQVAFPF
15


45
VAFPFDEYF
15


98
GHISHFQGK
15


124
EFIVPSVPK
15


133
LPKEKIDPL
15


157
KGLPPLHIY
15


163
HIYWMNIEL
15


188
LYFANVEEK
15


213
TIVQKMPMK
15


220
MKLTVNSLK
15


234
SSSTEIGSK
15


239
IGSKANSIK
15


241
SKANSIKQR
15


266
ITILKGEIL
15


296
DLPKGRETK
15


300
GRETKENYG
15


316
VSYQDKGNY
15


344
EEPPRWTKK
15


450
ATVVGYSAF
15


451
TVVGYSAFL
15


477
EEVKPLEGR
15


480
KPLEGRRYH
15


487
YHIYENGTL
15


527
IRNATKLRV
15


634
LHLSERQNR
15


677
ELTRVQGKK
15


696
VRYQFRVIA
15


728
DRNPQNIRV
15


744
MIIKWEPLK
15


792
AVYAPYDVK
15


835
DVINSTLVK
15


853
RVHGRLKGY
15


875
RTHPKEVNI
15


876
THPKEVNIL
15


917
GPESEPYIF
15


950
LSWGLPKKL
15


957
KLNGNLTGY
15


973
NDTYEIGEL
15


988
TPSKPSWHL
15


996
LSNLNATTK
15


1020
PITEESSTL
15


1029
GEGSKGIGK
15


1030
EGSKGIGKI
15


1035
IGKISGVNL
15


1038
ISGVNLTQK
15


1040
GVNLTQKTH
15


1045
QKTHPIEVF
15


1051
EVFEPGAEH
15


1058
EHIVRLMTK
15


1100
IGLMCAIAL
15


1110
TLLLLTVCF
15


1127
YSVKEKEDL
15


1159
PLKGSLRSL
15


2
EPLLLGRGL
14


12
VYLMFLLLK
14


59
AKGNPEPTF
14


94
IPNEGHISH
14


117
IAMSEEIEF
14


129
SVPKLPKEK
14


143
VEEGDPIVL
14


171
LEHIEQDER
14


178
ERVYMSQKG
14


219
PMKLTVNSL
14


221
KLTVNSLKH
14


244
NSIKQRKPK
14


248
QRKPKLLLP
14


299
KGRETKENY
14


305
ENYGKTLKI
14


323
NYRCTASNF
14


340
HVIVEEPPR
14


343
VEEPPRWTK
14


347
PRWTKKPQS
14


358
YSTGSNGIL
14


373
EPQPTIKWR
14


384
GSPVDNHPF
14


403
SFTNLQPNH
14


456
SAFLHCEFF
14


467
PEAVVSWQK
14


472
SWQKVEEVK
14


474
QKVEEVKPL
14


478
EVKPLEGRR
14


510
CWVENAIGK
14


530
ATKLRVSPK
14


542
IPKLHMLEL
14


552
CESKCDSHL
14


591
NLTISNVTL
14


628
PDPPENLHL
14


631
PENLHLSER
14


637
SERQNRSVR
14


661
YIVEFEGNK
14


666
EGNKEEPGR
14


690
LPLAPFVRY
14


693
APFVRYQFR
14


700
FRVIAVNEV
14


727
PDRNPQNIR
14


739
SQPKEMIIK
14


756
QNGPGLEYR
14


763
YRVTWKPQG
14


776
WEEETVTNH
14


811
PDPQSVTLY
14


843
KVTWSTVPK
14


864
NWWKTKSLL
14


867
KTKSLLDGR
14


869
KSLLDGRTH
14


877
HPKEVNILR
14


882
NILRFSGQR
14


896
PSLDAFSEF
14


901
FSEFHLTVL
14


906
LTVLAYNSK
14


915
GAGPESEPY
14


948
ATLSWGLPK
14


963
TGYLLQYQI
14


997
SNLNATTKY
14


999
LNATTKYKF
14


1001
ATTKYKFYL
14


1025
SSTLGEGSK
14


1061
VRLMTKNWG
14


1082
RGREYAGLY
14


1083
GREYAGLYD
14


1091
DDISTQGWF
14


1105
AIALLTLLL
14


1112
LLLTVCFVK
14


1115
TVCFVKRNR
14


1120
KRNRGGKYS
14


1141
IQSVKDETF
14


1156
DEKPLKGSL
14


1172
QPTESADSL
14


1182
EYGEGDHGL
14


5
LLGRGLIVY
13


19
LKFSKAIEI
13


34
VPTIIKQSK
13


63
PEPTFSWTK
13


68
SWTKDGNPF
13


73
GNPFYFTDH
13


74
NPFYFTDHR
13


84
IPSNNSGTF
13


85
PSNNSGTFR
13


102
HFQGKYRCF
13


111
ASNKLGIAM
13


127
VPSVPKLPK
13


159
LPPLHIYWM
13


200
NDYCCFAAF
13


206
AAFPRLRTI
13


211
LRTIVQKMP
13


289
DWNKIGGDL
13


370
AEGEPQPTI
13


371
EGEPQPTIK
13


419
ASNVHGTIL
13


432
IDVVDVRPL
13


441
IQTKDGENY
13


524
NLDIRNATK
13


556
CDSHLKHSL
13


557
DSHLKHSLK
13


597
VTLEDQGIY
13


626
DVPDPPENL
13


641
NRSVRLTWE
13


644
VRLTWEAGA
13


653
DHNSNISEY
13


670
EEPGRWEEL
13


672
PGRWEELTR
13


676
EELTRVQGK
13


682
QGKKTTVIL
13


689
ILPLAPFVR
13


692
LAPFVRYQF
13


721
ETPPAAPDR
13


743
EMIIKWEPL
13


746
IKWEPLKSM
13


753
SMEQNGPGL
13


755
EQNGPGLEY
13


779
ETVTNHTLR
13


847
STVPKDRVH
13


850
PKDRVHGRL
13


861
YQINWWKTK
13


863
INWWKTKSL
13


889
QRNSGMVPS
13


931
VPEQPTFLK
13


934
QPTFLKVIK
13


937
FLKVIKVDK
13


946
DTATLSWGL
13


961
NLTGYLLQY
13


976
YEIGELNDI
13


1054
EPGAEHIVR
13


1056
GAEHIVRLM
13


1094
STQGWFIGL
13


1103
MCAIALLTL
13


1113
LLTVCFVKR
13


1153
SDSDEKPLK
13


1157
EKPLKGSLR
13


1168
NRDMQPTES
13


1212
SNGSSTATF
13


1214
GSSTATFPL
13







V2-(SET1)-HLA-B2705-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









1
FIVPSVPKF
17


9
FPKEKIDPL
15


5
SVPKFPKEK
13


3
VPSVPKFPK
12


6
VPKFPKEKI
10







V2-(SET2)-HLA-B2705-9mers-(SET1)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









7
REAKENYGK
19


6
GREAKENYG
15


2
DLPKGREAK
14


5
KGREAKENY
14


9
AKENYGKTL
12







V2-(SET3)-HLA-B2705-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









5
LGEGKYAGL
14


6
GEGKYAGLY
14


2
SSTLGEGKY
13


1
ESSTLGEGK
11


9
KYAGLYDDI
11


8
GKYAGLYDD
9







V3-HLA-B2705-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









43
NRNMLAEDF
22


4
GVDVINTTY
16


59
NYVEKSSTF
16


60
YVEKSSTFF
16


10
TTYVSNTTY
15


34
SKEQELSYR
15


24
GSPQPSIFI
14


27
QPSIFICSK
14


36
EQELSYRNR
14


39
LSYRNRNML
13


46
MLAEDFIQK
13


22
ATGSPQPSI
12


23
TGSPQPSIF
12


31
FICSKEQEL
12


52
IQKSTSCNY
12


55
STSCNYVEK
12


33
CSKEQELSY
11


38
ELSYRNRNM
11


41
YRNRNMLAE
11


44
RNMLAEDFI
11


61
VEKSSTFFK
11







V4-HLA-B2705-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









7
EDLPEQPTF
18


1
VTLYSGEDL
13


8
DLPEQPTFL
13


9
LPEQPTFLK
13







V5-HLA-B2705-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









7
NSSNSIKQR
15


8
SSNSIKQRK
14


5
TVNSSNSIK
13


4
LTVNSSNSI
11







V6-HLA-B2705-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









1
EEIEFIVPK
17


2
EIEFIVPKL
15


4
EFIVPKLEH
14


5
FIVPKLEHI
10







V7-HLA-B2705-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









20
PEPPRWTKK
15


16
FTLHPEPPR
14


19
HPEPPRWTK
14


5
VIVEDNISH
13


7
VEDNISHEL
13


10
NISHELFTL
13


11
ISHELFTLH
13


8
EDNISHELF
11


3
FHVIVEDNI
10


1
HDFHVIVED
9


















TABLE XXXI





Pos
123456789
score















V1-HLA-2709-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









7
GRGLIVYLM
24


540
PRIPKLHML
22


638
ERQNRSVRL
22


673
GRWEELTRV
22


399
PREISFTNL
21


527
IRNATKLRV
21


92
FRIPNEGHI
20


107
YRCFASNKL
20


324
YRCTASNFL
20


700
FRVIAVNEV
20


728
DRNPQNIRV
20


1081
TRGREYAGL
20


485
RRYHIYENG
18


584
RIIIDGANL
17


890
RNSGMVPSL
16


8
RGLIVYLMF
15


517
GKTAVTANL
15


534
RVSPKNPRI
15


583
GRIIIDGAN
15


810
GPDPQSVTL
15


856
GRLKGYQIN
15


875
RTHPKEVNI
15


9
GLIVYLMFL
14


11
IVYLMFLLL
14


125
FIVPSVPKL
14


203
CCFAAFPRL
14


209
PRLRTIVQK
14


210
RLRTIVQKM
14


261
GSESSITIL
14


432
IDVVDVRPL
14


484
GRRYHIYEN
14


684
KKTTVILPL
14


833
GVDVINSTL
14


874
GRTHPKEVN
14


884
LRFSGQRNS
14


1083
GREYAGLYD
14


1100
IGLMCAIAL
14


1106
IALLTLLLL
14


1189
GLFSEDGSF
14


212
RTIVQKMPM
13


247
KQRKPKLLL
13


300
GRETKENYG
13


308
GKTLKIENV
13


424
GTILANANI
13


558
SHLKHSLKL
13


617
ADITQVTVL
13


688
VILPLAPFV
13


696
VRYQFRVIA
13


697
RYQFRVIAV
13


709
GRSQPSQPS
13


763
YRVTWKPQG
13


808
GSGPDPQSV
13


893
GMVPSLDAF
13


917
GPESEPYIF
13


930
GVPEQPTFL
13


942
KVDKDTATL
13


964
GYLLQYQII
13


991
KPSWHLSNL
13


1035
IGKISGVNL
13


1056
GAEHIVRLM
13


1101
GLMCAIALL
13


1121
RNRGGKYSV
13


1214
GSSTATFPL
13


2
EPLLLGRGL
12


19
LKFSKAIEI
12


23
KAIEIPSSV
12


81
HRIIPSNNS
12


135
KEKIDPLEV
12


163
HIYWMNIEL
12


206
AAFPRLRTI
12


248
QRKPKLLLP
12


266
ITILKGEIL
12


267
TILKGEILL
12


268
ILKGEILLL
12


271
GEILLLECF
12


347
PRWTKKPQS
12


380
WRVNGSPVD
12


384
GSPVDNHPF
12


394
GDVVFPREI
12


474
QKVEEVKPL
12


525
LDIRNATKL
12


533
LRVSPKNPR
12


537
PKNPRIPKL
12


542
IPKLHMLEL
12


589
GANLTISNV
12


591
NLTISNVTL
12


628
PDPPENLHL
12


644
VRLTWEAGA
12


680
RVQGKKTTV
12


734
IRVQASQPK
12


799
VKVQAINQL
12


822
EDYPDTAPV
12


852
DRVHGRLKG
12


889
QRNSGMVPS
12


963
TGYLLQYQI
12


1001
ATTKYKFYL
12


1055
PGAEHIVRL
12


1061
VRLMTKNWG
12


1105
AIALLTLLL
12


1120
KRNRGGKYS
12


1152
YSDSDEKPL
12


1172
QPTESADSL
12


4
LLLGRGLIV
11


6
LGRGLIVYL
11


10
LIVYLMELL
11


26
EIPSSVQQV
11


29
SSVQQVPTI
11


37
IIKQSKVQV
11


39
KQSKVQVAF
11


45
VAFPFDEYF
11


47
FPFDEYFQI
11


75
PFYFTDHRI
11


76
FYFTDHRII
11


122
EIEFIVPSV
11


143
VEEGDPIVL
11


154
NPPKGLPPL
11


178
ERVYMSQKG
11


180
VYMSQKGDL
11


185
KGDLYFANV
11


211
LRTIVQKMP
11


214
IVQKMPMKL
11


219
PMKLTVNSL
11


245
SIKQRKPKL
11


246
IKQRKPKLL
11


275
LLECFAEGL
11


280
AEGLPTPQV
11


289
DWNKIGGDL
11


305
ENYGKTLKI
11


333
GTATHDFHV
11


358
YSTGSNGIL
11


359
STGSNGILL
11


390
HPFAGDVVF
11


396
VVFPREISF
11


419
ASNVHGTIL
11


429
NANIDVVDV
11


437
VRPLIQTKD
11


451
TVVGYSAFL
11


487
YHIYENGTL
11


489
IYENGTLQI
11


498
NRTTEEDAG
11


579
GTEDGRIII
11


605
YCCSAHTAL
11


611
TALDSAADI
11


619
ITQVTVLDV
11


626
DVPDPPENL
11


679
TRVQGKKTT
11


682
QGKKTTVIL
11


743
EMIIKWEPL
11


753
SMEQNGPGL
11


778
EETVTNHTL
11


780
TVTNHTLRV
11


786
LRVMTPAVY
11


828
APVIHGVDV
11


850
PKDRVHGRL
11


863
INWWKTKSL
11


876
THPKEVNIL
11


929
EGVPEQPTF
11


935
PTFLKVIKV
11


954
LPKKLNGNL
11


959
NGNLTGYLL
11


970
QIINDTYEI
11


973
NDTYEIGEL
11


1020
PITEESSTL
11


1033
KGIGKISGV
11


1066
KNWGDNDSI
11


1103
MCAIALLTL
11


1104
CAIALLTLL
11


1110
TLLLLTVCF
11


1111
LLLLTVCFV
11


1127
YSVKEKEDL
11


1133
EDLHPDPEI
11


1156
DEKPLKGSL
11







V2-(SET1)-HLA-B2709-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









1
FIVPSVPKF
12


9
FPKEKIDPL
10


6
VPKFPKEKI
8







V2-(SET2)-HLA-B2709-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









6
GREAKENYG
13


9
AKENYGKTL
11


1
GDLPKGREA
6







V2-(SET3)-HLA-B2709-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









5
LGEGKYAGL
10


9
KYAGLYDDI
10


8
GKYAGLYDD
6


6
GEGKYAGLY
4







V3-HLA-B2709-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









43
NRNMLAEDF
19


39
LSYRNRNML
12


44
RNMLAEDFI
12


22
ATGSPQPSI
11


24
GSPQPSIFI
11


31
FICSKEQEL
11


41
YRNRNMLAE
11


11
TYVSNTTYV
10


5
VDVINTTYV
9


23
TGSPQPSIF
9


38
ELSYRNRNM
9


59
NYVEKSSTF
9


62
EKSSTFFKI
9


53
QKSTSCNYV
8


60
YVEKSSTFF
8







V4-HLA-B2709-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









1
VTLYSGEDL
12


7
EDLPEQPTF
12


8
DLPEQPTFL
10







V5-HLA-B2709-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









4
LTVNSSNSI
9


3
KLTVNSSNS
4







V6-HLA-B2709-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









2
EIEFIVPKL
13


5
FIVPKLEHI
10







V7-HLA-B2709-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each


start position is specified, the length of peptide


is 9 amino acids, and the end position for each


peptide is the start position plus eight.









7
VEDNISHEL
11


3
FHVIVEDNI
10


10
NISHELFTL
10


8
EDNISHELF
8


















TABLE XXXII





Pos
123456789
score















V1-HLA-B4402-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









194
EEKDSRNDY
25


271
GEILLLECF
25


670
EEPGRWEEL
25


143
VEEGDPIVL
24


372
GEPQPTIKW
24


778
EETVTNHTL
24


976
YEIGELNDI
24


482
LEGRRYHIY
23


1156
DEKPLKGSL
23


370
AEGEPQPTI
22


552
CESKCDSHL
21


206
AAFPRLRTI
20


258
TESGSESSI
20


121
EEIEFIVPS
19


617
ADITQVTVL
19


25
IEIPSSVQQ
18


141
LEVEEGDPI
18


144
EEGDPIVLP
18


344
EEPPRWTKK
18


537
PKNPRIPKL
18


540
PRIPKLHML
18


157
KGLPPLHIY
17


396
WFPREISF
17


525
LDIRNATKL
17


580
TEDGRIIID
17


903
EFHLTVLAY
17


1105
AIALLTLLL
17


1193
EDGSFIGAY
17


262
SESSITILK
16


268
ILKGEILLL
16


280
AEGLPTPQV
16


400
REISFTNLQ
16


465
ASPEAVVSW
16


657
NISEYIVEF
16


676
EELTRVQGK
16


684
KKTTVILPL
16


810
GPDPQSVTL
16


893
GMVPSLDAF
16


902
SEFHLTVLA
16


929
EGVPEQPTF
16


932
PEQPTFLKV
16


933
EQPTFLKVI
16


979
GELNDINIT
16


1139
PEIQSVKDE
16


2
EPLLLGRGL
15


6
LGRGLIVYL
15


39
KQSKVQVAF
15


45
VAFPFDEYF
15


59
AKGNPEPTF
15


92
FRIPNEGHI
15


118
AMSEEIEFI
15


125
FIVPSVPKL
15


158
GLPPLHIYW
15


169
IELEHIEQD
15


246
IKQRKPKLL
15


343
VEEPPRWTK
15


450
ATWGYSAF
15


575
FEINGTEDG
15


628
PDPPENLHL
15


638
ERQNRSVRL
15


669
KEEPGRWEE
15


687
TVILPLAPF
15


726
APDRNPQNI
15


740
QPKEMIIKW
15


743
EMIIKWEPL
15


777
EEETVTNHT
15


799
VKVQAINQL
15


853
RVHGRLKGY
15


878
PKEVNILRF
15


879
KEVNILRFS
15


920
SEPYIFQTP
15


950
LSWGLPKKL
15


961
NLTGYLLQY
15


997
SNLNATTKY
15


1030
EGSKGIGKI
15


1057
AEHIVRLMT
15


1100
IGLMCAIAL
15


1101
GLMCAIALL
15


1104
CAIALLTLL
15


1106
IALLTLLLL
15


1175
ESADSLVEY
15


1192
SEDGSFIGA
15


5
LLGRGLIVY
14


9
GLIVYLMFL
14


11
IVYLMFLLL
14


13
YLMFLLLKF
14


70
TKDGNPFYF
14


84
IPSNNSGTF
14


95
PNEGHISHF
14


120
SEEIEFIVP
14


123
IEFIVPSVP
14


151
LPCNPPKGL
14


200
NDYCCFAAF
14


237
TEIGSKANS
14


266
ITILKGEIL
14


303
TKENYGKTL
14


305
ENYGKTLKI
14


350
TKKPQSAVY
14


359
STGSNGILL
14


390
HPFAGDVVF
14


407
LQPNHTAVY
14


447
ENYATVVGY
14


456
SAFLHCEFF
14


487
YHIYENGTL
14


512
VENAIGKTA
14


558
SHLKHSLKL
14


567
SWSKDGEAF
14


572
GEAFEINGT
14


599
LEDQGIYCC
14


637
SERQNRSVR
14


653
DHNSNISEY
14


663
VEFEGNKEE
14


675
WEELTRVQG
14


720
HETPPAAPD
14


738
ASQPKEMII
14


755
EQNGPGLEY
14


759
PGLEYRVTW
14


768
KPQGAPVEW
14


838
NSTLVKVTW
14


858
LKGYQINWW
14


942
KVDKDTATL
14


944
DKDTATLSW
14


957
KLNGNLTGY
14


973
NDTYEIGEL
14


1000
NATTKYKFY
14


1023
EESSTLGEG
14


1045
QKTHPIEVF
14


1055
PGAEHIVRL
14


1060
IVRLMTKNW
14


1094
STQGWFIGL
14


1098
WFIGLMCAI
14


1110
TLLLLTVCF
14


1132
KEDLHPDPE
14


1
MEPLLLGRG
13


17
LLLKFSKAI
13


50
DEYFQIECE
13


63
PEPTFSWTK
13


96
NEGHISHFQ
13


133
LPKEKIDPL
13


135
KEKIDPLEV
13


154
NPPKGLPPL
13


174
IEQDERVYM
13


203
CCFAAFPRL
13


219
PMKLTVNSL
13


245
SIKQRKPKL
13


247
KQRKPKLLL
13


261
GSESSITIL
13


267
TILKGEILL
13


282
GLPTPQVDW
13


331
FLGTATHDF
13


417
CEASNVHGT
13


418
EASNVHGTI
13


419
ASNVHGTIL
13


446
GENYATVVG
13


474
QKVEEVKPL
13


477
EEVKPLEGR
13


479
VKPLEGRRY
13


502
EEDAGSYSC
13


503
EDAGSYSCW
13


584
RIIIDGANL
13


591
NLTISNVTL
13


626
DVPDPPENL
13


640
QNRSVRLTW
13


690
LPLAPFVRY
13


692
LAPFVRYQF
13


706
NEVGRSQPS
13


742
KEMIIKWEP
13


754
MEQNGPGLE
13


761
LEYRVTWKP
13


811
PDPQSVTLY
13


821
GEDYPDTAP
13


829
PVIHGVDVI
13


833
GVDVINSTL
13


863
INWWKTKSL
13


876
THPKEVNIL
13


890
RNSGMVPSL
13


896
PSLDAFSEF
13


915
GAGPESEPY
13


959
NGNLTGYLL
13


970
QIINDTYEI
13


986
ITTPSKPSW
13


991
KPSWHLSNL
13


1001
ATTKYKFYL
13


1050
IEVFEPGAE
13


1053
FEPGAEHIV
13


1067
NWGDNDSIF
13


1078
VIETRGREY
13


1091
DDISTQGWF
13


1103
MCAIALLTL
13


1119
VKRNRGGKY
13


1130
KEKEDLHPD
13


1152
YSDSDEKPL
13


1159
PLKGSLRSL
13


1174
TESADSLVE
13


1182
EYGEGDHGL
13


1205
KEKGSVESN
13


1210
VESNGSSTA
13


1212
SNGSSTATF
13


1214
GSSTATFPL
13


3
PLLLGRGLI
12


8
RGLIVYLMF
12


19
LKFSKAIEI
12


41
SKVQVAFPF
12


47
FPFDEYFQI
12


61
GNPEPTFSW
12


68
SWTKDGNPF
12


76
FYFTDHRII
12


99
HISHFQGKY
12


102
HFQGKYRCF
12


107
YRCFASNKL
12


117
IAMSEEIEF
12


173
HIEQDERVY
12


181
YMSQKGDLY
12


214
IVQKMPMKL
12


238
EIGSKANSI
12


260
SGSESSITI
12


304
KENYGKTLK
12


323
NYRCTASNF
12


324
YRCTASNFL
12


384
GSPVDNHPF
12


424
GTILANANI
12


432
IDVVDVRPL
12


461
CEFFASPEA
12


476
VEEVKPLEG
12


489
IYENGTLQI
12


490
YENGTLQIN
12


534
RVSPKNPRI
12


542
IPKLHMLEL
12


548
LELHCESKC
12


556
CDSHLKHSL
12


569
SKDGEAFEI
12


586
IIDGANLTI
12


605
YCCSAHTAL
12


631
PENLHLSER
12


648
WEAGADHNS
12


650
AGADHNSNI
12


682
QGKKTTVIL
12


748
WEPLKSMEQ
12


753
SMEQNGPGL
12


786
LRVMTPAVY
12


796
PYDVKVQAI
12


823
DYPDTAPVI
12


850
PKDRVHGRL
12


857
RLKGYQINW
12


864
NWWKTKSLL
12


875
RTHPKEVNI
12


901
FSEFHLTVL
12


916
AGPESEPYI
12


918
PESEPYIFQ
12


930
GVPEQPTFL
12


958
LNGNLTGYL
12


968
QYQIINDTY
12


999
LNATTKYKF
12


1022
TEESSTLGE
12


1052
VFEPGAEHI
12


1079
IETRGREYA
12


1082
RGREYAGLY
12


1090
YDDISTQGW
12


1141
IQSVKDETF
12


1144
VKDETFGEY
12


1183
YGEGDHGLF
12


1184
GEGDHGLFS
12


1189
GLFSEDGSF
12










V2-(SET1)-HLA-B4402-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









1
FIVPSVPKF
15


9
FPKEKIDPL
13


6
VPKFPKEKI
9


7
PKFPKEKID
7










V2-(SET2)-HLA-B4402-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









9
AKENYGKTL
17


5
KGREAKENY
11


7
REAKENYGK
10










V2-(SET3)-HLA-B4402-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









6
GEGKYAGLY
22


2
SSTLGEGKY
13


5
LGEGKYAGL
11


9
KYAGLYDDI
10










V3-HLA-B4402-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









48
AEDFIQKST
17


23
TGSPQPSIF
15


39
LSYRNRNML
14


4
GVDVINTTY
13


37
QELSYRNRN
13


62
EKSSTFFKI
13


43
NRNMLAEDF
12


59
NYVEKSSTF
12


10
TTYVSNTTY
11


22
ATGSPQPSI
11


33
CSKEQELSY
11


35
KEQELSYRN
11


61
VEKSSTFFK
11


24
GSPQPSIFI
10


31
FICSKEQEL
10


44
RNMLAEDFI
10


52
IQKSTSCNY
10


60
YVEKSSTFF
10










V4-HLA-B4402-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









7
EDLPEQPTF
17


6
GEDLPEQPT
12


8
DLPEQPTFL
12


1
VTLYSGEDL
11










V5-HLA-B4402-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









4
LTVNSSNSI
10


7
NSSNSIKQR
9


6
VNSSNSIKQ
4


9
SNSIKQRKP
4










V6-HLA-B4402-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









1
EEIEFIVPK
19


3
IEFIVPKLE
16


2
EIEFIVPKL
15


5
FIVPKLEHI
12










V7-HLA-B4402-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









7
VEDNISHEL
24


20
PEPPRWTKK
16


10
NISHELFTL
14


17
TLHPEPPRW
14


8
EDNISHELF
13


13
HELFTLHPE
13


















TABLE XXXIIII





Pos
123456789
score















V1-HLA-B5101-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









611
TALDSAADI
26


206
AAFPRLRTI
25


334
TATHDFHVI
25


427
LANANIDVV
25


285
TPQVDWNKI
24


418
EASNVHGTI
24


445
DGENYATVV
24


519
TAVTANLDI
24


47
FPFDEYFQI
23


130
VPKLPKEKI
23


504
DAGSYSCWV
23


1106
IALLTLLLL
23


133
LPKEKIDPL
22


260
SGSESSITI
22


429
NANIDVVDV
22


616
AADITQVTV
22


23
KAIEIPSSV
21


468
EAVVSWQKV
21


726
APDRNPQNI
21


823
DYPDTAPVI
21


873
DGRTHPKEV
21


963
TGYLLQYQI
21


115
LGIAMSEEI
20


139
DPLEVEEGD
20


151
LPCNPPKGL
20


154
NPPKGLPPL
20


578
NGTEDGRII
20


589
GANLTISNV
20


737
QASQPKEMI
20


916
AGPESEPYI
20


954
LPKKLNGNL
20


1030
EGSKGIGKI
20


1172
QPTESADSL
20


2
EPLLLGRGL
19


305
ENYGKTLKI
19


542
IPKLHMLEL
19


791
PAVYAPYDV
19


794
YAPYDVKVQ
19


810
GPDPQSVTL
19


828
APVIHGVDV
19


978
IGELNDINI
19


988
TPSKPSWHL
19


1104
CAIALLTLL
19


1136
HPDPEIQSV
19


629
DPPENLHLS
18


650
AGADHNSNI
18


690
LPLAPFVRY
18


757
NGPGLEYRV
18


795
APYDVKVQA
18


1100
IGLMCAIAL
18


6
LGRGLIVYL
17


27
IPSSVQQVP
17


433
DVVDVRPLI
17


586
IIDGANLTI
17


681
VQGKKTTVI
17


740
QPKEMIIKW
17


812
DPQSVTLYS
17


855
HGRLKGYQI
17


909
LAYNSKGAG
17


933
EQPTFLKVI
17


991
KPSWHLSNL
17


1033
KGIGKISGV
17


1055
PGAEHIVRL
17


1138
DPEIQSVKD
17


17
LLLKFSKAI
16


19
LKFSKAIEI
16


75
PFYFTDHRI
16


185
KGDLYFANV
16


297
LPKGRETKE
16


390
HPFAGDVVF
16


392
FAGDVVFPR
16


464
FASPEAVVS
16


514
NAIGKTAVT
16


573
EAFEINGTE
16


695
FVRYQFRVI
16


877
HPKEVNILR
16


899
DAFSEFHLT
16


1035
IGKISGVNL
16


1199
GAYAGSKEK
16


11
IVYLMFLLL
15


94
IPNEGHISH
15


147
DPIVLPCNP
15


208
FPRLRTIVQ
15


375
QPTIKWRVN
15


398
FPREISFTN
15


466
SPEAVVSWQ
15


480
KPLEGRRYH
15


508
YSCWVENAI
15


614
DSAADITQV
15


682
QGKKTTVIL
15


692
LAPFVRYQF
15


703
IAVNEVGRS
15


722
TPPAAPDRN
15


728
DRNPQNIRV
15


824
YPDTAPVIH
15


826
DTAPVIHGV
15


827
TAPVIHGVD
15


829
PVIHGVDVI
15


921
EPYIFQTPE
15


927
TPEGVPEQP
15


935
PTFLKVIKV
15


976
YEIGELNDI
15


1048
HPIEVFEPG
15


1054
EPGAEHIVR
15


1092
DISTQGWFI
15


1111
LLLLTVCFV
15


1158
KPLKGSLRS
15


1202
AGSKEKGSV
15


4
LLLGRGLIV
14


29
SSVQQVPTI
14


30
SVQQVPTII
14


64
EPTFSWTKD
14


76
FYFTDHRII
14


84
IPSNNSGTF
14


141
LEVEEGDPI
14


159
LPPLHIYWM
14


255
LPPTESGSE
14


279
FAEGLPTPQ
14


283
LPTPQVDWN
14


327
TASNFLGTA
14


345
EPPRWTKKP
14


370
AEGEPQPTI
14


449
YATVVGYSA
14


453
VGYSAFLHC
14


527
IRNATKLRV
14


577
INGTEDGRI
14


615
SAADITQVT
14


619
ITQVTVLDV
14


673
GRWEELTRV
14


749
EPLKSMEQN
14


758
GPGLEYRVT
14


759
PGLEYRVTW
14


836
VINSTLVKV
14


849
VPKDRVHGR
14


887
SGQRNSGMV
14


950
LSWGLPKKL
14


959
NGNLTGYLL
14


1000
NATTKYKFY
14


1027
TLGEGSKGI
14


1071
NDSIFQDVI
14


1108
LLTLLLLTV
14


1190
LFSEDGSFI
14


1201
YAGSKEKGS
14


62
NPEPTFSWT
13


74
NPFYFTDHR
13


110
FASNKLGIA
13


117
IAMSEEIEF
13


118
AMSEEIEFI
13


143
VEEGDPIVL
13


156
PKGLPPLHI
13


163
HIYWMNIEL
13


205
FAAFPRLRT
13


214
IVQKMPMKL
13


218
MPMKLTVNS
13


231
NDSSSSTEI
13


238
EIGSKANSI
13


256
PPTESGSES
13


258
TESGSESSI
13


281
EGLPTPQVD
13


357
VYSTGSNGI
13


373
EPQPTIKWR
13


388
DNHPFAGDV
13


389
NHPFAGDVV
13


408
QPNHTAVYQ
13


438
RPLIQTKDG
13


471
VSWQKVEEV
13


489
IYENGTLQI
13


522
TANLDIRNA
13


529
NATKLRVSP
13


534
RVSPKNPRI
13


539
NPRIPKLHM
13


617
ADITQVTVL
13


626
DVPDPPENL
13


680
RVQGKKTTV
13


715
QPSDHHETP
13


725
AAPDRNPQN
13


768
KPQGAPVEW
13


790
TPAVYAPYD
13


793
VYAPYDVKV
13


796
PYDVKVQAI
13


841
LVKVTWSTV
13


900
AFSEFHLTV
13


923
YIFQTPEGV
13


932
PEQPTFLKV
13


1013
TSQGCGKPI
13


1052
VFEPGAEHI
13


1056
GAEHIVRLM
13


1066
KNWGDNDSI
13


1070
DNDSIFQDV
13


1086
YAGLYDDIS
13


3
PLLLGRGLI
12


26
EIPSSVQQV
12


37
IIKQSKVQV
12


45
VAFPFDEYF
12


58
EAKGNPEPT
12


72
DGNPFYFTD
12


92
FRIPNEGHI
12


125
FIVPSVPKL
12


127
VPSVPKLPK
12


155
PPKGLPPLH
12


160
PPLHIYWMN
12


166
WMNIELEHI
12


190
FANVEEKDS
12


216
QKMPMKLTV
12


229
HANDSSSST
12


346
PPRWTKKPQ
12


352
KPQSAVYST
12


369
EAEGEPQPT
12


394
GDVVFPREI
12


426
ILANANIDV
12


444
KDGENYATV
12


462
EFFASPEAV
12


463
FFASPEAVV
12


481
PLEGRRYHI
12


516
IGKTAVTAN
12


525
LDIRNATKL
12


558
SHLKHSLKL
12


569
SKDGEAFEI
12


571
DGEAFEING
12


579
GTEDGRIII
12


591
NLTISNVTL
12


608
SAHTALDSA
12


627
VPDPPENLH
12


636
LSERQNRSV
12


649
EAGADHNSN
12


688
VILPLAPFV
12


700
FRVIAVNEV
12


723
PPAAPDRNP
12


730
NPQNIRVQA
12


772
APVEWEEET
12


822
EDYPDTAPV
12


875
RTHPKEVNI
12


876
THPKEVNIL
12


895
VPSLDAFSE
12


901
FSEFHLTVL
12


947
TATLSWGLP
12


964
GYLLQYQII
12


1010
RACTSQGCG
12


1015
QGCGKPITE
12


1042
NLTQKTHPI
12


1044
TQKTHPIEV
12


1133
EDLHPDPEI
12


1176
SADSLVEYG
12










V2-HLA-B5101-9mers-(SET1)-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









6
VPKFPKEKI
23


9
FPKEKIDPL
21


3
VPSVPKFPK
11










V2-(SET2)-HLA-B5101-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









3
LPKGREAKE
15


8
EAKENYGKT
14


9
AKENYGKTL
10


5
KGREAKENY
8










V2-(SET3)-HLA-B5101-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









5
LGEGKYAGL
16


9
KYAGLYDDI
11


7
EGKYAGLYD
7










V3-HLA-B5101-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









47
LAEDFIQKS
16


21
NATGSPQPS
14


62
EKSSTFFKI
14


3
HGVDVINTT
13


39
LSYRNRNML
13


24
GSPQPSIFI
12


25
SPQPSIFIC
12


27
QPSIFICSK
11


53
QKSTSCNYV
11


5
VDVINTTYV
10


10
TTYVSNTTY
10


11
TYVSNTTYV
10


22
ATGSPQPSI
10


44
RNMLAEDFI
10


16
TTYVSNATG
9


6
DVINTTYVS
8


23
TGSPQPSIF
8


31
FICSKEQEL
8










V4-HLA-B5101-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









8
DLPEQPTFL
15


9
LPEQPTFLK
12


1
VTLYSGEDL
10


5
SGEDLPEQP
8










V5-HLA-B5101-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









4
LTVNSSNSI
14


2
MKLTVNSSN
7










V6-HLA-B5101-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









5
FIVPKLEHI
14


7
VPKLEHIEQ
12


2
EIEFIVPKL
10


3
IEFIVPKLE
7










V7-HLA-B5101-9mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each start position is


specified, the length of peptide is 9 amino acids, and the end position


for each peptide is the start position plus eight.









3
FHVIVEDNI
13


19
HPEPPRWTK
12


10
NISHELFTL
10


7
VEDNISHEL
7


18
LHPEPPRWT
7


2
DFHVIVEDN
6


11
ISHELFTLH
6


















TABLE XXXIV





Pos
1234567890
score















V1-HLA-A1-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









810
GPDPQSVTLY
32


1192
SEDGSFIGAY
28


193
VEEKDSRNDY
27


481
PLEGRRYHIY
27


902
SEFHLTVLAY
24


1173
PTESADSLVE
23


4
LLLGRGLIVY
22


119
MSEEIEFIVP
22


309
KTLKIENVSY
22


349
WTKKPQSAVY
22


627
VPDPPENLHL
22


754
MEQNGPGLEY
22


931
VPEQPTFLKV
22


1021
ITEESSTLGE
22


1191
FSEDGSFIGA
22


406
NLQPNHTAVY
21


499
RTTEEDAGSY
21


919
ESEPYIFQTP
21


960
GNLTGYLLQY
21


996
LSNLNATTKY
21


261
GSESSITILK
20


371
EGEPQPTIKW
20


478
EVKPLEGRRY
20


579
GTEDGRIIID
20


788
VMTPAVYAPY
20


1143
SVKDETFGEY
20


1183
YGEGDHGLFS
20


43
VQVAFPFDEY
19


180
VYMSQKGDLY
19


689
ILPLAPFVRY
19


1118
FVKRNRGGKY
19


68
SWTKDGNPFY
18


236
STEIGSKANS
18


815
SVTLYSGEDY
18


1056
GAEHIVRLMT
18


1152
YSDSDEKPLK
18


78
FTDHRIIPSN
17


98
GHISHFQGKY
17


343
VEEPPRWTKK
17


500
TTEEDAGSYS
17


785
TLRVMTPAVY
17


824
YPDTAPVIHG
17


901
FSEFHLTVLA
17


1081
TRGREYAGLY
17


134
PKEKIDPLEV
16


172
EHIEQOERVY
16


257
PTESGSESSI
16


359
STGSNGILLC
16


440
LIQTKDGENY
16


446
GENYATVVGY
16


475
KVEEVKPLEG
16


569
SKDGEAFEIN
16


612
ALDSAADITQ
16


636
LSERQNRSVR
16


652
ADHNSNISEY
16


914
KGAGPESEPY
16


967
LQYQIINDTY
16


1028
LGEGSKGIGK
16


1077
DVIETRGREY
16


137
KIDPLEVEEG
15


142
EVEEGDPIVL
15


156
PKGLPPLHIY
15


298
PKGRETKENY
15


315
NVSYQDKGNY
15


434
VVDVRPLIQT
15


580
TEDGRIIIDG
15


596
NVTLEDQGIY
15


651
GADHNSNISE
15


852
DRVHGRLKGY
15


956
KKLNGNLTGY
15


999
LNATTKYKFY
15


1052
VFEPGAEHIV
15


1136
HPDPEIQSVK
15


1174
TESADSLVEY
15










V2-(SET1)-HLA-A1-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









5
PSVPKFPKEK
8


8
PKFPKEKIDP
8


4
VPSVPKFPKE
6


2
FIVPSVPKFP
5


6
SVPKFPKEKI
4


1
EFIVPSVPKF
3


10
FPKEKIDPLE
3










V2-(SET2)-HLA-A1-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









5
PKGREAKENY
15


10
AKENYGKTLK
13


1
GGDLPKGREA
11


7
GREAKENYGK
11










V2-(SET3)-HLA-A1-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









6
LGEGKYAGLY
28


2
ESSTLGEGKY
21










V3-HLA-A1-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









10
NTTYVSNTTY
22


33
ICSKEQELSY
21


4
HGVDVINTTY
16


52
FIQKSTSCNY
16


26
SPQPSIFICS
13


35
SKEQELSYRN
12


61
YVEKSSTFFK
12


37
EQELSYRNRN
11


49
AEDFIQKSTS
11


56
STSCNYVEKS
11


5
GVDVINTTYV
10


48
LAEDFIQKST
10










V4-HLA-A1-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









10
LPEQPTFLKV
21


6
SGEDLPEQPT
12


7
GEDLPEQPTF
10










V5-HLA-A1-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









9
SSNSIKQRKP
8


5
LTVNSSNSIK
7


6
VNSSNSIKQ
6


8
NSSNSIKQRK
4


10
SNSIKQRKPK
3










V6-HLA-A1-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









1
SEEIEFIVPK
12


3
EIEFIVPKLE
12


10
KLEHIEQDER
11


4
IEFIVPKLEH
6


6
FIVPKLEHIE
5










V7-HLA-A1-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









20
HPEPPRWTKK
16


8
VEDNISHELF
13


13
SHELFTLHPE
12


1
THDFHVIVED
10


7
IVEDNISHEL
10


12
ISHELFTLHP
10


17
FTLHPEPPRW
7


















TABLE XXXV





Pos
1234567890
score















V1-HLA-A0201-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









1107
ALLTLLLLTV
31


5
LLGRGLIVYL
28


426
ILANANIDVV
28


274
LLLECFAEGL
27


1102
LMCAIALLTL
27


1105
AIALLTLLLL
27


1110
TLLLLTVCFV
26


132
KLPKEKIDPL
25


267
TILKGEILLL
25


585
IIIDGANLTI
24


635
HLSERQNRSV
24


957
KLNGNLTGYL
24


9
GLIVYLMFLL
23


615
SAADITQVTV
23


835
DVINSTLVKV
23


36
TIIKQSKVQV
22


118
AMSEEIEFIV
22


158
GLPPLHIYWM
22


431
NIDVVDVRPL
22


524
NLDIRNATKL
22


840
TLVKVTWSTV
22


897
SLDAFSEFHL
22


16
FLLLKFSKAI
21


114
KLGIAMSEEI
21


150
VLPCNPPKGL
21


470
VVSWQKVEEV
21


541
RIPKLHMLEL
21


618
DITQVTVLDV
21


792
AVYAPYDVKV
21


875
RTHPKEVNIL
21


949
TLSWGLPKKL
21


953
GLPKKLNGNL
21


1034
GIGKISGVNL
21


3
PLLLGRGLIV
20


4
LLLGRGLIVY
20


10
LIVYLMFLLL
20


18
LLKFSKAIEI
20


25
IEIPSSVQQV
20


205
FAAFPRLRTI
20


213
TIVQKMPMKL
20


425
TILANANIDV
20


428
ANANIDVVDV
20


488
HIYENGTLQI
20


616
AADITQVTVL
20


862
QINWWKTKSL
20


966
LLQYQIINDT
20


1099
FIGLMCAIAL
20


1100
IGLMCAIALL
20


1189
GLFSEDGSFI
20


117
IAMSEEIEFI
19


206
AAFPRLRTIV
19


265
SITILKGEIL
19


1135
LHPDPEIQSV
19


1158
KPLKGSLRSL
19


121
EEIEFIVPSV
18


218
MPMKLTVNSL
18


245
SIKQRKPKLL
18


260
SGSESSITIL
18


266
ITILKGEILL
18


268
ILKGEILLLE
18


273
ILLLECFAEG
18


450
ATVVGYSAFL
18


526
DIRNATKLRV
18


536
SPKNPRIPKL
18


590
ANLTISNVTL
18


745
IIKWEPLKSM
18


765
VTWKPQGAPV
18


784
HTLRVMTPAV
18


889
QRNSGMVPSL
18


900
AFSEFHLTVL
18


965
YLLQYQIIND
18


972
INDTYEIGEL
18


1026
STLGEGSKGI
18


1043
LTQKTHPIEV
18


1112
LLLTVCFVKR
18


1201
YAGSKEKGSV
18


37
IIKQSKVQVA
17


82
RIIPSNNSGT
17


137
KIDPLEVEEG
17


153
CNPPKGLPPL
17


221
KLTVNSLKHA
17


253
LLLPPTESGS
17


279
FAEGLPTPQV
17


356
AVYSTGSNGI
17


413
AVYQCEASNV
17


565
KLSWSKDGEA
17


603
GIYCCSAHTA
17


687
TVILPLAPFV
17


696
VRYQFRVIAV
17


699
QFRVIAVNEV
17


795
APYDVKVQAI
17


798
DVKVQAINQL
17


809
SGPDPQSVTL
17


836
VINSTLVKVT
17


871
LLDGRTHPKE
17


899
DAFSEFHLTV
17


941
IKVDKDTATL
17


1032
SKGIGKISGV
17


1073
SIFQDVIETR
17


1101
GLMCAIALLT
17


1104
CAIALLTLLL
17


1106
IALLTLLLLT
17


8
RGLIVYLMFL
16


17
LLKFSKAIE
16


22
SKAIEIPSSV
16


124
EFIVPSVPKL
16


129
SVPKLPKEKI
16


254
LLPPTESGSE
16


326
CTASNFLGTA
16


333
GTATHDFHVI
16


365
ILLCEAEGEP
16


396
VVFPREISFT
16


421
NVHGTILANA
16


443
TKDGENYATV
16


514
NAIGKTAVTA
16


518
KTAVTANLDI
16


544
KLHMLELHCE
16


576
EINGTEDGRI
16


586
IIDGANLTIS
16


610
HTALDSAADI
16


613
LDSAADITQV
16


752
KSMEQNGPGL
16


772
APVEWEEETV
16


807
LGSGPDPQSV
16


827
TAPVIHGVDV
16


857
RLKGYQINWW
16


870
SLLDGRTHPK
16


915
GAGPESEPYI
16


937
FLKVIKVDKD
16


990
SKPSWHLSNL
16


1080
ETRGREYAGL
16


1094
STQGWFIGLM
16


1103
MCAIALLTLL
16


1166
SLNRDMQPTE
16


1181
VEYGEGDHGL
16


13
YLMFLLLKFS
15


141
LEVEEGDPIV
15


162
LHIYWMNIEL
15


165
YWMNIELEHI
15


179
RVYMSQKGDL
15


187
DLYFANVEEK
15


237
TEIGSKANSI
15


244
NSIKQRKPKL
15


252
KLLLPPTESG
15


282
GLPTPQVDWN
15


307
YGKTLKIENV
15


334
TATHDFHVIV
15


341
VIVEEPPRWT
15


458
FLHCEFFASP
15


464
FASPEAVVSW
15


515
AIGKTAVTAN
15


521
VTANLDIRNA
15


539
NPRIPKLHML
15


583
GRIIIDGANL
15


584
RIIIDGANLT
15


588
DGANLTISNV
15


598
TLEDQGIYCC
15


661
YIVEFEGNKE
15


683
GKKTTVILPL
15


702
VIAVNEVGRS
15


725
AAPDRNPQNI
15


830
VIHGVDVINS
15


833
GVDVINSTLV
15


948
ATLSWGLPKK
15


961
NLTGYLLQYQ
15


969
YQIINDTYEI
15


977
EIGELNDINI
15


980
ELNDINITTP
15


1019
KPITEESSTL
15


1029
GEGSKGIGKI
15


1037
KISGVNLTQK
15


1108
LLTLLLLTVC
15


1120
KRNRGGKYSV
15










V2-(SET1)-HLA-A0201-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









6
SVPKFPKEKI
16


9
KFPKEKIDPL
15


2
FIVPSVPKFP
10










V2-(SET2)-HLA-A0201-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









3
DLPKGREAKE
13


9
EAKENYGKTL
13


8
REAKENYGKT
8


1
GGDLPKGREA
7


2
GDLPKGREAK
6










V2-(SET3)-HLA-A0201-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









3
SSTLGEGKYA
10


4
STLGEGKYAG
9


5
TLGEGKYAGL
8










V3-HLA-A0201-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









47
MLAEDFIQKS
20


2
VIHGVDVINT
19


8
VINTTYVSNT
18


39
ELSYRNRNML
17


22
NATGSPQPSI
16


31
IFICSKEQEL
16


5
GVDVINTTYV
15


11
TTYVSNTTYV
15


3
IHGVDVINTT
13


53
IQKSTSCNYV
12


56
STSCNYVEKS
12


30
SIFICSKEQE
11


42
YRNRNMLAED
11


48
LAEDFIQKST
11


13
YVSNTTYVSN
10


14
VSNTTYVSNA
10


24
TGSPQPSIFI
10


46
NMLAEDFIQK
10


62
VEKSSTFFKI
10


7
DVINTTYVSN
9


32
FICSKEQELS
9


44
NRNMLAEDFI
9


52
FIQKSTSCNY
9










V4-HLA-A0201-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









1
SVTLYSGEDL
15


10
LPEQPTFLKV
14


3
TLYSGEDLPE
12


4
LYSGEDLPEQ
11


8
EDLPEQPTFL
11


9
DLPEQPTFLK
11










V5-HLA-A0201-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









4
KLTVNSSNSI
21










V6-HLA-A0201-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









2
EEIEFIVPKL
18


6
FIVPKLEHIE
12


10
KLEHIEQDER
12


5
EFIVPKLEHI
11


7
IVPKLEHIEQ
8










V7-HLA-A0201-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









7
IVEDNISHEL
19


10
DNISHELFTL
7


18
TLHPEPPRWT
16


6
VIVEDNISHE
15


















TABLE XXXVI





Pos
1234567890
score















V1-HLA-A0203-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









198
SRNDYCCFAA
19


608
SAHTALDSAA
19


717
SDHHETPPAA
19


421
NVHGTILANA
18


643
SVRLTWEAGA
18


1098
WFIGLMCAIA
18


1193
EDGSFIGAYA
18


199
RNDYCCFAAF
17


609
AHTALDSAAD
17


718
DHHETPPAAP
17


15
MFLLLKFSKA
10


37
IIKQSKVQVA
10


50
DEYFQIECEA
10


102
HFQGKYRCFA
10


109
CFASNKLGIA
10


182
MSQKGDLYFA
10


197
DSRNDYCCFA
10


221
KLTVNSLKHA
10


234
SSSTEIGSKA
10


271
GEILLLECFA
10


319
QDKGNYRCTA
10


326
CTASNFLGTA
10


347
PRWTKKPQSA
10


361
GSNGILLCEA
10


384
GSPVDNHPFA
10


404
FTNLQPNHTA
10


410
NHTAVYQCEA
10


419
ASNVHGTILA
10


441
IQTKOGENYA
10


448
NYATVVGYSA
10


456
SAFLHCEFFA
10


460
HCEFFASPEA
10


496
QINRTTEEDA
10


506
GSYSCWVENA
10


511
WVENAIGKTA
10


514
NAIGKTAVTA
10


521
VTANLDIRNA
10


565
KLSWSKDGEA
10


581
EDGRIIIDGA
10


600
EDQGIYCCSA
10


603
GIYCCSAHTA
10


607
CSAHTALDSA
10


641
NRSVRLTWEA
10


684
KKTTVILPLA
10


695
FVRYQFRVIA
10


716
PSDHHETPPA
10


729
RNPQNIRVQA
10


763
YRVTWKPQGA
10


783
NHTLRVMTPA
10


786
LRVMTPAVYA
10


794
YAPYDVKVQA
10


819
YSGEDYPDTA
10


891
NSGMVPSLDA
10


901
FSEFHLTVLA
10


907
TVLAYNSKGA
10


939
KVIKVDKDTA
10


992
PSWHLSNLNA
10


1002
TTKYKFYLRA
10


1048
HPIEVFEPGA
10


1078
VIETRGREYA
10


1096
QGWFIGLMCA
10


1168
NRDMQPTESA
10


1191
FSEDGSFIGA
10


1209
SVESNGSSTA
10


1215
SSTATFPLRA
10


16
FLLLKFSKAI
9


38
IKQSKVQVAF
9


51
EYFQIECEAK
9


103
FQGKYRCFAS
9


110
FASNKLGIAM
9


183
SQKGDLYFAN
9


222
LTVNSLKHAN
9


235
SSTEIGSKAN
9


272
EILLLECFAE
9


320
DKGNYRCTAS
9


327
TASNFLGTAT
9


348
RWTKKPQSAV
9


362
SNGILLCEAE
9


385
SPVDNHPFAG
9


405
TNLQPNHTAV
9


411
HTAVYQCEAS
9


420
SNVHGTILAN
9


422
VHGTILANAN
9


442
QTKDGENYAT
9


449
YATVVGYSAF
9


457
AFLHCEFFAS
9


461
CEFFASPEAV
9


497
INRTTEEDAG
9


507
SYSCWVENAI
9


512
VENAIGKTAV
9


515
AIGKTAVTAN
9


522
TANLDIRNAT
9


566
LSWSKDGEAF
9


582
OGRIIIDGAN
9


601
DQGIYCCSAH
9


604
IYCCSAHTAL
9


642
RSVRLTWEAG
9


644
VRLTWEAGAD
9


685
KTTVILPLAP
9


696
VRYQFRVIAV
9


730
NPQNIRVQAS
9


764
RVTWKPQGAP
9


784
HTLRVMTPAV
9


787
RVMTPAVYAP
9


795
APYDVKVQAI
9


820
SGEDYPDTAP
9


892
SGMVPSLDAF
9


902
SEFHLTVLAY
9


908
VLAYNSKGAG
9


940
VIKVDKDTAT
9


993
SWHLSNLNAT
9


1003
TKYKFYLRAC
9


1049
PIEVFPEGAE
9


1079
IETRGREYAG
9


1097
GWFIGLMCAI
9


1099
FIGLMCAIAL
9


1169
RDMQPTESAD
9


1192
SEDGSFIGAY
9


1194
DGSFIGAYAG
9


1210
VESNGSSTAT
9










V2-(SET1)-HLA-A0203-10mers-282P1G3


NoResultsFound.





V2-(SET2)HLA-A0203-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









1
GGDLPKGREA
10


2
GDLPKGREAK
9


3
DLPKGREAKE
8










V2-(SET3)HLA-A0203-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









3
SSTLGEGKYA
10


4
STLGEGKYAG
9


5
TLGEGKYAGL
8










V3-HLA-A0203-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









14
VSNTTYVSNA
10


40
LSYRNRNMLA
10


15
SNTTYVSNAT
9


41
SYRNRNMLAE
9


16
NTTYVSNATG
8


42
YRNRNMLAED
8










V4-HLA-A0203-10mers-


NoResultsFound.





V5-HLA-A0203-10mers-


NoResultsFound.





V6-HLA-A0203-10mers-


NoResultsFound.





V7-HLA-A0203-10mers-


NoResultsFound.


















TABLE XXXVII





Pos
1234567890
score















V1-A3-10mers-282P12G3


Each peptide is a portion of SEQ ID NO: 3; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









11
IVYLMFLLLK
30


995
HLSNLNATTK
29


342
IVEEPPRWTK
28


701
RVIAVNEVGR
27


785
TLRVMTPAVY
27


1037
KISGVNLTQK
27


1111
LLLLTVCFVK
27


406
NLQPNHTAVY
26


1121
RNRGGKYSVK
26


126
IVPSVPKLPK
25


4
LLLGRGLIVY
24


33
QVPTIIKQSK
24


187
DLYFANVEEK
24


645
RLTWEAGADH
24


870
SLLDGRTHPK
24


1118
FVKRNRGGKY
24


105
GKYRCFASNK
23


413
AVYQCEASNV
23


478
EVKPLEGRRY
23


523
ANLDIRNATK
23


689
ILPLAPFVRY
23


792
AVYAPYDVKV
23


1077
DVIETRGREY
23


312
KIENVSYQDK
22


688
VILPLAPFVR
22


691
PLAPFVRYQF
22


905
HLTVLAYNSK
22


1107
ALLTLLLLTV
22


1196
SFIGAYAGSK
22


30
SVQQVPTIIK
21


82
RIIPSNNSGT
21


176
QDERVYMSQK
21


208
FPRLRTIVQK
21


238
EIGSKANSIK
21


585
IIIDGANLTI
21


680
RVQGKKTTVI
21


704
AVNEVGRSQP
21


733
NIRVQASQPK
21


815
SVTLYSGEDY
21


930
GVPEQPTFLK
21


295
GDLPKGRETK
20


381
RVNGSPVDNH
20


532
KLRVSPKNPR
20


707
EVGRSQPSQP
20


939
KVIKVDKDTA
20


1112
LLLTVCFVKR
20


1136
HPDPEIQSVK
20


1143
SVKDETFGEY
20


3
PLLLGRGLIV
19


24
AIEIPSSVQQ
19


83
IPSNNSGTF
19


93
RIPNEGHISH
19


148
PIVLPCNPPK
19


179
RVYMSQKGDL
19


451
TVVGYSAFLH
19


488
HIYENGTLQI
19


584
RIIIDGANLT
19


603
GIYCCSAHTA
19


1057
AEHIVRLMTK
19


1088
GLYDDISTQG
19


36
TIIKQSKVQV
18


149
IVLPCNPPKG
18


210
RLRTIVQKMP
18


212
RTIVQKMPMK
18


252
KLLLPPTESG
18


253
LLLPPTESGS
18


268
ILKGEILLLE
18


273
ILLLECFAEG
18


292
KIGGDLPKGR
18


426
ILANANIDVV
18


475
KVEEVKPLEG
18


481
PLEGRRYHIY
18


511
WVENAIGKTA
18


534
RVSPKNPRIP
18


596
NVTLEDQGIY
18


643
SVRLTWEAGA
18


695
FVRYQFRVIA
18


800
KVQAINQLGS
18


834
VDVINSTLVK
18


835
DVINSTLVKV
18


857
RLKGYQINWW
18


1166
SLNRDMQPTE
18


1209
SVESNGSSTA
18


5
LLGRGLIVYL
17


44
QVAFPFDEYF
17


62
NPEPTFSWTK
17


123
IEFIVPSVPK
17


226
SLKHANDSSS
17


309
KTLKIENVSY
17


315
NVSYQDKGNY
17


322
GNYRCTASNF
17


356
AVYSTGSNGI
17


435
VDVRPLIQTK
17


436
DVRPLIQTKD
17


440
LIQTKDGENY
17


469
AVVSWQKVEE
17


499
RTTEEDAGSY
17


612
ALDSAADITQ
17


687
TVILPLAPFV
17


732
QNIRVQASQP
17


735
RVQASQPKEM
17


829
PVIHGVDVIN
17


840
TLVKVTWSTV
17


853
RVHGRLKGYQ
17


942
KVDKDTATLS
17


947
TATLSWGLPK
17


1010
RACTSQGCGK
17


1060
IVRLMTKNWG
17


1073
SIFQDVIETR
17


1159
PLKGSLRSLN
17


1179
SLVEYGEGDH
17


122
EIEFIVPSVP
16


142
EVEEGDPIVL
16


254
LLPPTESGSE
16


274
LLLECFAEGL
16


370
AEGEPQPTIK
16


395
DVVFPREISF
16


396
VVFPREISFT
16


466
SPEAVVSWQK
16


514
NAIGKTAVTA
16


559
HLKHSLKLSW
16


561
KHSLKLSWSK
16


633
NLHLSERQNR
16


677
ELTRVQGKKT
16


738
ASQPKEMIIK
16


764
RVTWKPQGAP
16


841
LVKVTWSTVP
16


850
PKDRVHGRLK
16


882
NILRFSGQRN
16


936
TFLKVIKVDK
16


957
KLNGNLTGYL
16


980
ELNDINITTP
16


982
NDINITTPSK
16


998
NLNATTKYKF
16


1101
GLMCAIALLT
16


1105
AIALLTLLLL
16


1108
LLTLLLLTVC
16


1128
SVKEKEDLHP
16


1134
DLHPDPEIQS
16


1140
EIQSVKDETF
16


1163
SLRSLNRDMQ
16


1189
GLFSEDGSFI
16


1197
FIGAYAGSKE
16


17
LLLKFSKAIE
15


37
IIKQSKVQVA
15


99
HISHFQGKYR
15


219
PMKLTVNSLK
15


243
ANSIKQRKPK
15


290
WNKIGGDLPK
15


310
TLKIENVSYQ
15


365
ILLCEAEGEP
15


421
NVHGTILANA
15


433
DVVDVRPLIQ
15


458
FLHCEFFASP
15


520
AVTANLDIRN
15


524
NLDIRNATKL
15


526
DIRNATKLRV
15


541
RIPKLHMLEL
15


546
HMLELHCESK
15


626
DVPDPPENLH
15


636
LSERQNRSVR
15


639
RQNRSVRLTW
15


710
RSQPSQPSDH
15


744
MIIKWEPLKS
15


759
PGLEYRVTWK
15


773
PVEWEEETVT
15


780
TVTNHTLRVM
15


787
RVMTPAVYAP
15


806
QLGSGPDPQS
15


860
GYQINWWKTK
15


883
ILRFSGQRNS
15


894
MVPSLDAFSE
15


948
ATLSWGLPKK
15


1008
YLRACTSQGC
15


1028
LGEGSKGIGK
15


1034
GIGKISGVNL
15


1062
RLMTKNWGDN
15


16
FLLLKFSKAI
14


18
LLKFSKAIEI
14


23
KAIEIPSSVQ
14


42
KVQVAFBFDE
14


114
KLGIAMSEEI
14


128
PSVPKLPKEK
14


137
KIDPLEVEEG
14


158
GLPPLHIYWM
14


170
ELEHIEQDER
14


172
EHIEQDERVY
14


233
SSSSTEIGSK
14


328
ASNFLGTATH
14


331
FLGTATHDFH
14


340
HVIVEEPPRW
14


343
VEEPPRWTKK
14


349
WTKKPQSAVY
14


364
GILLCEAEGE
14


366
LLCEAEGEPQ
14


386
PVDNHPFAGD
14


439
PLIQTKDGEN
14


471
VSWQKVEEVK
14


509
SCWVENAIGK
14


547
MLELHCESKC
14


565
KLSWSKDGEA
14


660
EYIVEFEGNK
14


671
EPGRWEELTR
14


743
EMIIKWEPLK
14


791
PAVYAPYDVK
14


798
DVKVQAINQL
14


817
TLYSGEDYPD
14


822
EDYPDTAPVI
14


842
VKVTWSTVPK
14


848
TVPKDRVHGR
14


880
EVNILRFSGQ
14


907
TVLAYNSKGA
14


933
EQPTFLKVIK
14


960
GNLTGYLLQY
14


967
LQYQIINDTY
14


970
QIINDTYEIG
14


986
ITTPSKPSWH
14


997
SNLNATTKYK
14


1051
EVFEPGAEHI
14


1082
RGREYAGLYD
14


1123
RGGKYSVKEK
14


1156
DEKPLKGSLR
14










V2-(SET1)-HLA-A3-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









3
IVPSVPKFPK
22


5
PSVPKFPKEK
14


2
FIVPSVPKFP
12


6
SVPKFPKEKI
11










V2-(SET2)-HLA-A3-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









2
GDLPKGREAK
17


10
AKENYGKTLK
15


3
DLPKGREAKE
13


7
GREAKENYGK
12










V2-(SET3)-HLA-A3-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









5
TLGEGKYAGL
14


1
EESSTLGEGK
13


6
LGEGKYAGLY
12


2
ESSTLGEGKY
7


4
STLGEGKYAG
6


7
GEGKYAGLYD
6


9
GKYAGLYDDI
6


10
KYAGLYDDIS
6










V3-HLA-A3-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









7
DVINTTYVSN
20


61
YVEKSSTFFK
20


1
PVIHGVDVIN
17


13
YVSNTTYVSN
17


19
YVSNATGSPQ
17


46
NMLAEDFIQK
17


55
KSTSCNYVEK
17


43
RNRNMLAEDF
15


59
CNYVEKSSTF
15


52
FIQKSTSCNY
14


30
SIFICSKEQE
13


33
ICSKEQELSY
13


4
HGVDVINTTY
12


27
PQPSIFICSK
12


39
ELSYRNRNML
12


47
MLAEDFIQKS
12


5
GVDVINTTYV
11


8
VINTTYVSNT
11


10
NTTYVSNTTY
11


23
ATGSPQPSIF
11


2
VIHGVDVINT
10


49
AEDFIQKSTS
10


34
CSKEQELSYR
9


41
SYRNRNMLAE
9










V4-HLA-A3-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









9
DLPEQPTFLK
21


3
TLYSGEDLPE
17


1
SVTLYSGEDL
15










V5-HLA-A3-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









4
KLTVNSSNSI
15


5
LTVNSSNSIK
14


10
SNSIKQRKPK
13


8
NSSNSIKQRK
11


6
TVNSSNSIKQ
10


2
PMKLTVNSSN
7


7
VNSSNSIKQR
7










V6-HLA-A3-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









10
KLEHIEQDER
17


1
SEEIEFIVPK
16


7
IVPKLEHIEQ
12


6
FIVPKLEHIE
11


4
IEFIVPKLEH
10


3
EIEFIVPKLE
9










V7-HLA-A3-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









5
HVIVEDNISH
19


19
LHPEPPRWTK
16


18
TLHPEPPRWT
15


20
HPEPPRWTKK
14


7
IVEDNISHEL
13


11
NISHELFTLH
13


6
VIVEDNISHE
12


15
ELFTLHPEPP
11


















TABLE XXXVIII





Pos
1234567890
score















V1-HLA-A26-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









1077
DVIETRGREY
35


395
DVVFPREISF
32


478
EVKPLEGRRY
32


142
EVEEGDPIVL
31


798
DVKVQAINQL
31


302
ETKENYGKTL
30


124
EFIVPSVPKL
27


835
DVINSTLVKV
27


172
EHIEQDERVY
26


852
DRVHGRLKGY
26


1051
EVFEPGAEHI
26


1080
ETRGREYAGL
26


686
TTVILPLAPF
25


433
DVVDVRPLIQ
24


1140
EIQSVKDETF
24


499
RTTEEDAGSY
23


779
ETVTNHTLRV
23


815
SVTLYSGEDY
23


929
EGVPEQPTFL
23


1118
FVKRNRGGKY
23


1143
SVKDETFGEY
23


1147
ETFGEYSDSD
23


596
NVTLEDQGIY
22


707
EVGRSQPSQP
22


1054
EPGAEHIVRL
22


121
EEIEFIVPSV
21


266
ITILKGEILL
21


315
NVSYQDKGNY
21


875
RTHPKEVNIL
21


880
EVNILRFSGQ
21


902
SEFHLTVLAY
21


1182
EYGEGDHGLF
21


396
VVFPREISFT
20


436
DVRPLIQTKD
20


450
ATVVGYSAFL
20


58
EAKGNPEPTF
19


179
RVYMSQKGDL
19


277
ECFAEGLPTP
19


309
KTLKIENVSY
19


418
EASNVHGTIL
19


626
DVPDPPENLH
19


721
ETPPAAPDRN
19


777
EEETVTNHTL
19


974
DTYEIGELND
19


1151
EYSDSDEKPL
19


1211
ESNGSSTATF
19


44
QVAFPFDEYF
18


213
TIVQKMPMKL
18


267
TILKGEILLL
18


349
WTKKPQSAVY
18


401
EISFTNLQPN
18


557
DSHLKHSLKL
18


618
DITQVTVLDV
18


656
SNISEYIVEF
18


826
DTAPVIHGVD
18


987
TTPSKPSWHL
18


10
LIVYLMFLLL
17


69
WTKDGNPFYF
17


98
GHISHFQGKY
17


573
EAFEINGTED
17


946
DTATLSWGLP
17


977
EIGELNDINI
17


1072
DSIFQDVIET
17


1105
AIALLTLLLL
17


1192
SEDGSFIGAY
17










V2-(SET1)-HLA-A26-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









1
EFIVPSVPKF
27










V2-(SET2)-HLA-A26-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









9
EAKENYGKTL
22


5
PKGREAKENY
10










V2-(SET3)-HLA-A26-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









2
ESSTLGEGKY
22


8
EGKYAGLYDD
14


1
EESSTLGEGK
12


5
TLGEGKYAGL
11


6
LGEGKYAGLY
11


4
STLGEGKYAG
10










V3-HLA-A26-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









2
EEIEFIVPKL
29


5
EFIVPKLEHI
18


3
EIEFIVPKLE
14










V4-HLA-A26-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









1
SVTLYSGEDL
21


8
EDLPEQPTFL
19


9
DLPEQPTFLK
10










V5-HLA-A26-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









6
TVNSSNSIKQ
14


5
LTVNSSNSIK
13


7
VNSSNSIKQR
6










V6-HLA-A26-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









2
EEIEFIVPKL
29


5
EFIVPKLEHI
18


3
EIEFIVPKLE
14










V7-HLA-A26-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each start position is


specified, the length of peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









10
DNISHELFTL
26


7
IVEDNISHEL
19


5
HVIVEDNISH
16


6
VIVEDNISHE
15


15
ELFTLHPEPP
14


















TABLE XXXIX





Pos
1234567890
score















V1-HLA-B0702-10mers-28P1G3


Each peptide is a portion of SEQ ID NO: 3; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









1054
EPGAEHIVRL
25


398
FPREISFTNL
24


627
VPDPPENLHL
24


218
MPMKLTVNSL
23


27
IPSSVQQVPT
22


539
NPRIPKLHML
22


1158
KPLKGSLRSL
22


536
SPKNPRIPKL
21


1019
KPITEESSTL
21


155
PPKGLPPLHI
20


795
APYDVKVQAI
20


828
APVIHGVDVI
20


849
VPKDRVHGRL
20


895
VPSLDAFSEF
19


927
TPEGVPEQPT
19


931
VPEQPTFLKV
19


2
EPLLLGRGLI
18


373
EPQPTIKWRV
18


480
KPLEGRRYHI
18


693
APFVRYQFRV
18


772
APVEWEEETV
18


877
HPKEVNILRF
18


94
IPNEGHISHF
17


790
TPAVYAPYDV
17


934
QPTFLKVIKV
17


954
LPKKLNGNLT
17


1048
HPIEVFEPGA
17


34
VPTIIKQSKV
16


74
NPFYFTDHRI
16


160
PPLHIYWMNI
16


616
AADITQVTVL
16


900
AFSEFHLTVL
16


1080
ETRGREYAGL
16


1172
QPTESADSLV
16


84
IPSNNSGTFR
15


390
HPFAGDVVFP
15


681
VQGKKTTVIL
15


1105
AIALLTLLLL
15


208
FPRLRTIVQK
14


418
EASNVHGTIL
14


450
ATVVGYSAFL
14


541
RIPKLHMLEL
14


590
ANLTISNVTL
14


637
SERQNRSVRL
14


671
EPGRWEELTR
14


715
QPSDHHETPP
14


723
PPAAPDRNPQ
14


758
GPGLEYRVTW
14


768
KPQGAPVEWE
14


810
GPDPQSVTLY
14


929
EGVPEQPTFL
14


957
KLNGNLTGYL
14


1034
GIGKISGVNL
14


1104
CAIALLTLLL
14


1151
EYSDSDEKPL
14


1213
NGSSTATFPL
14


5
LLGRGLIVYL
13


10
LIVYLMFLLL
13


106
KYRCFASNKL
13


124
EFIVPSVPKL
13


127
VPSVPKLPKE
13


132
KLPKEKIDPL
13


153
CNPPKGLPPL
13


246
IKQRKPKLLL
13


260
SGSESSITIL
13


267
TILKGEILLL
13


297
LPKGRETKEN
13


323
NYRCTASNFL
13


346
PPRWTKKPQS
13


375
QPTIKWRVNG
13


431
NIDVVDVRPL
13


516
IGKTAVTANL
13


604
IYCCSAHTAL
13


683
GKKTTVILPL
13


726
APDRNPQNIR
13


742
KEMIIKWEPL
13


752
KSMEQNGPGL
13


875
RTHPKEVNIL
13


921
EPYIFQTPEG
13


941
IKVDKDTATL
13


988
TPSKPSWHLS
13


991
KPSWHLSNLN
13


1102
LMCAIALLTL
13


1126
KYSVKEKEDL
13


1136
HPDPEIQSVK
13


8
RGLIVYLMFL
12


64
EPTFSWTKDG
12


142
EVEEGDPIVL
12


154
NPPKGLPPLH
12


202
YCCFAAFPRL
12


244
NSIKQRKPKL
12


250
KPKLLLPPTE
12


345
EPPRWTKKPQ
12


357
VYSTGSNGIL
12


358
YSTGSNGILL
12


428
ANANIDVVDV
12


473
WQKVEEVKPL
12


524
NLDIRNATKL
12


555
KCDSHLKHSL
12


557
DSHLKHSLKL
12


629
DPPENLHLSE
12


669
KEEPGRWEEL
12


680
RVQGKKTTVI
12


690
LPLAPFVRYQ
12


712
QPSQPSDHHE
12


730
NPQNIRVQAS
12


749
EPLKSMEQNG
12


809
SGPDPQSVTL
12


824
YPDTAPVIHG
12


889
QRNSGMVPSL
12


897
SLDAFSEFHL
12


945
KDTATLSWGL
12


949
TLSWGLPKKL
12


972
INDTYEIGEL
12


1093
ISTQGWFIGL
12


1099
FIGLMCAIAL
12


1100
IGLMCAIALL
12


1103
MCAIALLTLL
12


1181
VEYGEGDHGL
12










V2-(SET1)-HLA-B0702-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









9
KFPKEKIDPL
13


10
FPKEKIDPLE
11


7
VPKFPKEKID
10


1
EFIVPSVPKF
9


6
SVPKFPKEKI
7










V2-(SET2)-HLA-B0702-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









4
LPKGREAKEN
12


9
EAKENYGKTL
11


8
REAKENYGKT
9


1
GGDLPKGREA
7










V2-(SET3)-HLA-B0702-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









5
TLGEGKYAGL
12


9
GKYAGLYDDI
7


3
SSTLGEGKYA
6










V3-HLA-B0702-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









7
DVINTTYVSN
23


10
NTTYVSNTTY
20


39
ELSYRNRNML
19


23
ATGSPQPSIF
18


4
HGVDVINTTY
16


50
EDFIQKSTSC
15


1
PVIHGVDVIN
14


52
FIQKSTSCNY
14


56
STSCNYVEKS
14


60
NYVEKSSTFF
14


51
DFIQKSTSCN
13


19
YVSNATGSPQ
12


31
IFICSKEQEL
12


33
ICSKEQELSY
11


13
YVSNTTYVSN
11










V4-HLA-B0702-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









10
LPEQPTFLKV
19


8
EDLPEQPTFL
14


1
SVTLYSGEDL
10










V5-HLA-B0702-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









1
MPMKLTVNSS
13


4
KLTVNSSNSI
7










V6-HLA-B0702-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









2
EEIEFIVPKL
13


8
VPKLEHIEQD
10


5
EFIVPKLEHI
7










V7-HLA-B0702-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









20
HPEPPRWTKK
12


7
IVEDNISHEL
11


9
EDNISHELFT
10


10
DNISHELFTL
10


8
VEDNISHELF
7


18
TLHPEPPRWT
7


3
DFHVIVEDNI
6


















TABLE XL





Pos
1234567890
score















V1-HLA-B08-10mers-282P1G3


NoResultsFound.





V2-(SET1)-HLA-B08-10mers-282P1G3


NoResultsFound.





V2-(SET2)-HLA-B08-10mers-282P1G3


NoResultsFound.





V2-(SET3)-HLA-B08-10mers-282P1G3


NoResultsFound.





V3-HLA-B08-10mers-282P1G3


NoResultsFound.





V4-HLA-B08-10mers-282P1G3


NoResultsFound.





V5-HLA-B08-10mers-282P1G3


NoResultsFound.





V6-HLA-B08-10mers-282P1G3


NoResultsFound.





V7-HLA-B08-10mers-282P1G3


NoResultsFound.


















TABLE XLI





Pos
1234567890
score















V1-HLA-B1510-10mers-282P1G3


NoResultsFound.





V2-(SET1)-HLA-B1510-10mers-282P1G3


NoResultsFound.





V2-(SET2)-HLA-B1510-10mers-282P1G3


NoResultsFound.





V2-(SET3)-HLA-B1510-10mers-282P1G3


NoResultsFound.





V2-(SET3)-HLA-B1510-10mers-282P1G3


NoResultsFound.





V3-HLA-B1510-10mers-(SET3)-282P1G3


NoResultsFound.





V4-HLA-B1510-10mers-282P1G3


NoResultsFound.





V5-HLA-B1510-10mers-282P1G3


NoResultsFound.





V6-HLA-B1510-10mers-282P1G3


NoResultsFound.





V7-HLA-B1510-10mers-282P1G3


NoResultsFound.


















TABLE XLII





Pos
1234567890
score















V1-HLA-B2705-10mers-282P1G3


NoResultsFound.





V2-(SET1)-HLA-B2705-10mers-282P1G3


NoResultsFound.





V2-(SET2)-HLA-B2705-10mers-282P1G3


NoResultsFound.





V2-(SET3)-HLA-B2705-10mers-282P1G3


NoResultsFound.





V3-HLA-B2705-10mers-282P1G3


NoResultsFound.





V4-HLA-B2705-10mers-282P1G3


NoResultsFound.





V5-HLA-B2705-10mers-282P1G3


NoResultsFound.





V6-HLA-B2705-10mers-282P1G3


NoResultsFound.





V7-HLA-B2705-10mers-282P1G3


NoResultsFound.


















TABLE XLIII





Pos
1234567890
score















V1-HLA-B2709-10mers-282P1G3


NoResultsFound.





V2-(SET1)-HLA-B2709-10mers-282P1G3


NoResultsFound.





V2-(SET2)-HLA-B2709-10mers-282P1G3


NoResultsFound.





V2-(SET3)-HLA-B2709-10mers-282P1G3


NoResultsFound.





V3-HLA-B2709-10mers-282P1G3


NoResultsFound.





V4-HLA-B2709-10mers-282P1G3


NoResultsFound.





V5-HLA-B2709-10mers-282P1G3


NoResultsFound.





V6-HLA-B2709-10mers-282P1G3


NoResultsFound.





V7-HLA-B2709-10mers-282P1G3


NoResultsFound.


















TABLE XLIV





Pos
1234567890
score















V1-HLA-B4402-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









1192
SEDGSFIGAY
28


902
SEFHLTVLAY
27


777
EEETVTNHTL
25


932
PEQPTFLKVI
25


237
TEIGSKANSI
24


669
KEEPGRWEEL
24


1
MEPLLLGRGL
23


502
EEDAGSYSCW
23


1174
TESADSLVEY
23


193
VEEKDSRNDY
22


304
KENYGKTLKI
22


446
GENYATVVGY
22


637
SERQNRSVRL
22


742
KEMIIKWEPL
22


754
MEQNGPGLEY
22


1029
GEGSKGIGKI
22


1181
VEYGEGDHGL
22


928
PEGVPEQPTF
21


1132
KEDLHPDPEI
21


344
EEPPRWTKKP
20


417
CEASNVHGTI
19


25
IEIPSSVQQV
18


371
EGEPQPTIKW
18


656
SNISEYIVEF
18


1084
REYAGLYDDI
18


124
EFIVPSVPKL
17


172
EHIEQDERVY
17


262
SESSITILKG
17


281
EGLPTPQVDW
17


590
ANLTISNVTL
17


652
ADHNSNISEY
17


739
SQPKEMIIKW
17


1105
AIALLTLLLL
17


121
EEIEFIVPSV
16


143
VEEGDPIVLP
16


157
KGLPPLHIYW
16


266
ITILKGEILL
16


267
TILKGEILLL
16


280
AEGLPTPQVD
16


372
GEPQPTIKWR
16


383
NGSPVDNHPF
16


406
NLQPNHTAVY
16


464
FASPEAVVSW
16


478
EVKPLEGRRY
16


536
SPKNPRIPKL
16


580
TEDGRIIIDG
16


616
AADITQVTVL
16


810
GPDPQSVTLY
16


900
AFSEFHLTVL
16


929
EGVPEQPTFL
16


1019
KPITEESSTL
16


1054
EPGAEHIVRL
16


1104
CAIALLTLLL
16


1151
EYSDSDEKPL
16


4
LLLGRGLIVY
15


5
LLGRGLIVYL
15


60
KGNPEPTFSW
15


120
SEEIEFIVPS
15


132
KLPKEKIDPL
15


142
EVEEGDPIVL
15


144
EEGDPIVLPC
15


153
CNPPKGLPPL
15


244
NSIKQRKPKL
15


245
SIKQRKPKLL
15


260
SGSESSITIL
15


302
ETKENYGKTL
15


330
NFLGTATHDF
15


400
REISFTNLQP
15


461
CEFFASPEAV
15


627
VPDPPENLHL
15


670
EEPGRWEELT
15


676
EELTRVQGKK
15


683
GKKTTVILPL
15


691
PLAPFVRYQF
15


795
APYDVKVQAI
15


798
DVKVQAINQL
15


809
SGPDPQSVTL
15


822
EDYPDTAPVI
15


857
RLKGYQINWW
15


875
RTHPKEVNIL
15


892
SGMVPSLDAF
15


916
AGPESEPYIF
15


918
PESEPYIFQT
15


949
TLSWGLPKKL
15


960
GNLTGYLLQY
15


972
INDTYEIGEL
15


1044
TQKTHPIEVF
15


1057
AEHIVRLMTK
15


1099
FIGLMCAIAL
15


1100
IGLMCAIALL
15


1211
ESNGSSTATF
15


2
EPLLLGRGLI
14


9
GLIVYLMFLL
14


12
VYLMFLLLKF
14


16
FLLLKFSKAI
14


58
EAKGNPEPTF
14


98
GHISHFQGKY
14


101
SHFQGKYRCF
14


150
VLPCNPPKGL
14


162
LHIYWMNIEL
14


199
RNDYCCFAAF
14


271
GEILLLECFA
14


340
HVIVEEPPRW
14


343
VEEPPRWTKK
14


370
AEGEPQPTIK
14


389
NHPFAGDVVF
14


393
AGDVVFPREI
14


418
EASNVHGTIL
14


431
NIDVVDVRPL
14


524
NLDIRNATKL
14


575
FEINGTEDGR
14


585
IIIDGANLTI
14


625
LDVPDPPENL
14


639
RQNRSVRLTW
14


689
ILPLAPFVRY
14


720
HETPPAAPDR
14


725
AAPDRNPQNI
14


758
GPGLEYRVTW
14


837
INSTLVKVTW
14


877
HPKEVNILRF
14


956
KKLNGNLTGY
14


976
YEIGELNDIN
14


979
GELNDINITT
14


1053
FEPGAEHIVR
14


1059
HIVRLMTKNW
14


1077
DVIETRGREY
14


1109
LTLLLLTVCF
14


1156
DEKPLKGSLR
14


1158
KPLKGSLRSL
14


1210
VESNGSSTAT
14


10
LIVYLMFLLL
13


38
IKQSKVQVAF
13


46
AFPFDEYFQI
13


63
PEPTFSWTKD
13


94
IPNEGHISHF
13


106
KYRCFASNKL
13


123
IEFIVPSVPK
13


135
KEKIDPLEVE
13


156
PKGLPPLHIY
13


194
EEKDSRNDYC
13


196
KDSRNDYCCF
13


218
MPMKLTVNSL
13


246
IKQRKPKLLL
13


264
SSITILKGEI
13


309
KTLKIENVSY
13


358
YSTGSNGILL
13


450
ATVVGYSAFL
13


473
WQKVEEVKPL
13


507
SYSCWVENAI
13


539
NPRIPKLHML
13


548
LELHCESKCD
13


552
CESKCDSHLK
13


555
KCDSHLKHSL
13


557
DSHLKHSLKL
13


583
GRIIIDGANL
13


666
EGNKEEPGRW
13


748
WEPLKSMEQN
13


761
LEYRVTWKPQ
13


767
WKPQGAPVEW
13


774
VEWEEETVTN
13


778
EETVTNHTLR
13


788
VMTPAVYAPY
13


828
APVIHGVDVI
13


852
DRVHGRLKGY
13


862
QINWWKTKSL
13


879
KEVNILRFSG
13


895
VPSLDAFSEF
13


941
IKVDKDTATL
13


943
VDKDTATLSW
13


957
KLNGNLTGYL
13


990
SKPSWHLSNL
13


996
LSNLNATTKY
13


999
LNATTKYKFY
13


1023
EESSTLGEGS
13


1026
STLGEGSKGI
13


1051
EVFEPGAEHI
13


1079
IETRGREYAG
13


1080
ETRGREYAGL
13


1081
TRGREYAGLY
13


1089
LYDDISTQGW
13


1102
LMCAIALLTL
13


1139
PEIQSVKDET
13


1140
EIQSVKDETF
13


1143
SVKDETFGEY
13


1171
MQPTESADSL
13


1182
EYGEGDHGLF
13


1213
NGSSTATFPL
13










V2-(SET1)-HLA-B4402-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









1
EFIVPSVPKF
17


9
KFPKEKIDPL
15


6
SVPKFPKEKI
11










V2-(SET2)-HLA-B4402-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









9
EAKENYGKTL
15


5
PKGREAKENY
11


8
REAKENYGKT
11


2
GDLPKGREAK
7










V2-(SET3)-HLA-B4402-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









2
ESSTLGEGKY
15


1
EESSTLGEGK
13


6
LGEGKYAGLY
13


7
GEGKYAGLYD
11


5
TLGEGKYAGL
10


9
GKYAGLYDDI
8










V3-HLA-B4402-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









62
VEKSSTFFKI
20


39
ELSYRNRNML
15


49
AEDFIQKSTS
15


23
ATGSPQPSIF
14


24
TGSPQPSIFI
13


31
IFICSKEQEL
13


4
HGVDVINTTY
12


10
NTTYVSNTTY
12


33
ICSKEQELSY
12


36
KEQELSYRNR
12


38
QELSYRNRNM
12


43
RNRNMLAEDF
12


59
CNYVEKSSTF
11


60
NYVEKSSTFF
11


22
NATGSPQPSI
10


52
FIQKSTSCNY
10


44
NRNMLAEDFI
9










V4-HLA-B4402-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









7
GEDLPEQPTF
23


8
EDLPEQPTFL
17


1
SVTLYSGEDL
12










V5-HLA-B4402-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









4
KLTVNSSNSI
10


7
VNSSNSIKQR
7


10
SNSIKQRKPK
5










V6-HLA-B4402-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









2
EEIEFIVPKL
27


1
SEEIEFIVPK
15


5
EFIVPKLEHI
14


4
IEFIVPKLEH
13










V7-HLA-B4402-10mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each start position is


specified, the length of peptide is 10 amino acids, and the end


position for each peptide is the start position plus nine.









8
VEDNISHELF
23


21
PEPPRWTKKP
18


10
DNISHELFTL
15


7
IVEDNISHEL
12


17
FTLHPEPPRW
12


14
HELFTLHPEP
11


















TABLE XLV





Pos
1234567890
score















V1-HLA-B5101-10mers-282P1G3


NoResultsFound.





V2-(SET1)-HLA-B5101-10mers-282P1G3


NoResultsFound.





V2-(SET2)-HLA-B5101-10mers-282P1G3


NoResultsFound.





V2-(SET3)-HLA-B5101-10mers-282P1G3


NoResultsFound.





V3-HLA-B5101-10mers-(SET3)-282P1G3


NoResultsFound.





V4-HLA-B5101-10mers-282P1G3


NoResultsFound.





V5-HLA-B5101-10mers-282P1G3


NoResultsFound.





V6-HLA-B5101-10mers-282P1G3


NoResultsFound.





V7-HLA-B5101-10mers-282P1G3


NoResultsFound.
















TABLE XLVI







V1-DRB1-0101-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each start position is


specified, the length of peptide is 15 amino acids, and the end position


for each peptide is the start position plus fourteen.









Pos
123456789012345
score





263
ESSITILKGEILLLE
36


287
QVDWNKIGGDLPKGR
36


920
SEPYIFQTPEGVPEQ
33


446
GENYATVVGYSAFLH
32


1032
SKGIGKISGVNLTQK
32


1097
GWFIGLMCAIALLTL
31


120
SEEIEFIVPSVPKLP
29


522
TANLDIRNATKLRVS
29


831
IHGVDVINSTLVKVT
29


13
YLMFLLLKFSKAIEI
28


470
VVSWQKVEEVKPLEG
28


104
QGKYRCFASNKLGIA
27


127
VPSVPKLPKEKIDPL
27


461
CEFFASPEAWSWQK
27


476
VEEVKPLEGRRYHIY
27


940
VIKVDKDTATLSWGL
27


981
LNDINITTPSKPSWH
27


11
IVYLMFLLLKFSKAI
26


16
FLLLKFSKAIEIPSS
26


187
DLYFANVEEKDSRND
26


272
EILLLECFAEGLPTP
26


321
KGNYRCTASNFLGTA
26


539
NPRIPKLHMLELHCE
26


697
RYQFRVIAVNEVGRS
26


742
KEMIIKWEPLKSMEQ
26


748
WEPLKSMEQNGPGLE
26


764
RVTWKPQGAPVEWEE
26


883
ILRFSGQRNSGMVPS
26


1083
GREYAGLYDDISTQG
26


1096
QGWFIGLMCAIALLT
26


1
MEPLLLGRGLIVYLM
25


8
RGLIVYLMFLLLKFS
25


78
FTDHRIIPSNNSGTF
25


112
SNKLGIAMSEEIEFI
25


138
IDPLEVEEGDPIVLP
25


148
PIVLPCNPPKGLPPL
25


163
HIYWMNIELEHIEQD
25


208
FPRLRTIVQKMPMKL
25


354
QSAVYSTGSNGILLC
25


401
EISFTNLQPNHTAVY
25


411
HTAVYQCEASNVHGT
25


509
SCWVENAIGKTAVTA
25


581
EDGRIIIDGANLTIS
25


619
ITQVTVLDVPDPPEN
25


675
WEELTRVQGKKTTVI
25


685
KTTVILPLAPFVRYQ
25


838
NSTLVKVTWSTVPKD
25


905
HLTVLAYNSKGAGPE
25


993
SWHLSNLNATTKYKF
25


1049
PIEVFEPGAEHIVRL
25


1103
MCAIALLTLLLLTVC
25


1108
LLTLLLLTVCFVKRN
25


14
LMFLLLKFSKAIEIP
24


34
VPTIIKQSKVQVAFP
24


123
IEFIVPSVPKLPKEK
24


156
PKGLPPLHIYWMNIE
24


178
ERVYMSQKGDLYFAN
24


205
FAAFPRLRTIVQKMP
24


211
LRTIVQKMPMKLTVN
24


243
ANSIKQRKPKLLLPP
24


249
RKPKLLLPPTESGSE
24


328
ASNFLGTATHDFHVI
24


336
THDFHVIVEEPPRWT
24


601
DQGIYCCSAHTALDS
24


693
APFVRYQFRVIAVNE
24


702
VIAVNEVGRSQPSQP
24


745
IIKWEPLKSMEQNGP
24


750
PLKSMEQNGPGLEYR
24


798
DVKVQAINQLGSGPD
24


800
KVQAINQLGSGPDPQ
24


801
VQAINQLGSGPDPQS
24


839
STLVKVTWSTVPKDR
24


843
KVTWSTVPKDRVHGR
24


852
DRVHGRLKGYQINWW
24


860
GYQINWWKTKSLLDG
24


892
SGMVPSLDAFSEFHL
24


937
FLKVIKVDKDTATLS
24


947
TATLSWGLPKKLNGN
24


956
KKLNGNLTGYLLQYQ
24


975
TYEIGELNDINITTP
24


978
IGELNDINITTPSKP
24


1003
TKYKFYLRACTSQGC
24


1057
AEHIVRLMTKNWGDN
24


1087
AGLYDDISTQGWFIG
24


1105
AIALLTLLLLTVCFV
24


1187
DHGLFSEDGSFIGAY
24


38
IKQSKVQVAFPFDEY
23


89
SGTFRIPNEGHISHF
23


144
EEGDPIVLPCNPPKG
23


153
CNPPKGLPPLHIYWM
23


215
VQKMPMKLTVNSLKH
23


348
RWTKKPQSAVYSTGS
23


351
KKPQSAVYSTGSNGI
23


416
QCEASNVHGTILANA
23


429
NANIDVVDVRPLIQT
23


486
RYHIYENGTLQINRT
23


532
KLRVSPKNPRIPKLH
23


616
AADITQVTVLDVPDP
23


678
LTRVQGKKTTVILPL
23


733
NIRVQASQPKEMIIK
23


758
GPGLEYRVTWKPQGA
23


796
PYDVKVQAINQLGSG
23


955
PKKLNGNLTGYLLQY
23


1107
ALLTLLLLTVCFVKR
23


1165
RSLNRDMQPTESADS
23


1168
NRDMQPTESADSLVE
23


1204
SKEKGSVESNGSSTA
23


1207
KGSVESNGSSTATFP
23


27
IPSSVQQVPTIIKQS
22


81
HRIIPSNNSGTFRIP
22


132
KLPKEKIDPLEVEEG
22


140
PLEVEEGDPIVLPCN
22


277
ECFAEGLPTPQVDWN
22


376
PTIKWRVNGSPVDNH
22


468
EAWSWQKVEEVKPL
22


484
GRRYHIYENGTLQIN
22


686
TTVILPLAPFVRYQF
22


705
VNEVGRSQPSQPSDH
22


706
NEVGRSQPSQPSDHH
22


730
NPQNIRVQASQPKEM
22


731
PQNIRVQASQPKEMI
22


783
NHTLRVMTPAVYAPY
22


825
PDTAPVIHGVDVINS
22


828
APVIHGVDVINSTLV
22


878
PKEVNILRFSGQRNS
22


902
SEFHLTVLAYNSKGA
22


973
NDTYEIGELNDINIT
22


1100
IGLMCAIALLTLLLL
22


1102
LMCAIALLTLLLLTV
22


1138
DPEIQSVKDETFGEY
22


346
PPRWTKKPQSAVYST
21


391
PFAGDVVFPREISFT
21


431
NIDVVDVRPLIQTKD
21


529
NATKLRVSPKNPRIP
21


641
NRSVRLTWEAGADHN
21


683
GKKTTVILPLAPFVR
21


881
VNILRFSGQRNSGMV
21


889
QRNSGMVPSLDAFSE
21


1095
TQGWFIGLMCAIALL
21


1166
SLNRDMQPTESADSL
21


1198
IGAYAGSKEKGSVES
21


10
LIVYLMFLLLKFSKA
20


18
LLKFSKAIEIPSSVQ
20


28
PSSVQQVPTIIKQSK
20


44
QVAFPFDEYFQIECE
20


73
GNPFYFTDHRIIPSN
20


109
CFASNKLGIAMSEEI
20


362
SNGILLCEAEGEPQP
20


421
NVHGTILANANIDVV
20


434
VVDVRPLIQTKDGEN
20


473
WQKVEEVKPLEGRRY
20


572
GEAFEINGTEDGRII
20


582
DGRIIIDGANLTISN
20


593
TISNVTLEDQGIYCC
20


684
KKTTVILPLAPFVRY
20


692
LAPFVRYQFRVIAVN
20


782
TNHTLRVMTPAVYAP
20


908
VLAYNSKGAGPESEP
20


928
PEGVPEQPTFLKVIK
20


966
LLQYQIINDTYEIGE
20


1039
SGVNLTQKTHPIEVF
20


1117
CFVKRNRGGKYSVKE
20


32
QQVPTIIKQSKVQVA
19


75
PFYFTDHRIIPSNNS
19


122
EIEFIVPSVPKLPKE
19


265
SITILKGEILLLECF
19


268
ILKGEILLLECFAEG
19


377
TIKWRVNGSPVDNHP
19


455
YSAFLHCEFFASPEA
19


602
QGIYCCSAHTALDSA
19


659
SEYIVEFEGNKEEPG
19


898
LDAFSEFHLTVLAYN
19


922
PYIFQTPEGVPEQPT
19


932
PEQPTFLKVIKVDKD
19


952
WGLPKKLNGNLTGYL
19


1028
LGEGSKGIGKISGVN
19


1038
ISGVNLTQKTHPIEV
19


1061
VRLMTKNWGDNDSIF
19


1106
IALLTLLLLTVCFVK
19


1124
GGKYSVKEKEDLHPD
19


1149
FGEYSDSDEKPLKGS
19


4
LLLGRGLIVYLMFLL
18


64
EPTFSWTKDGNPFYF
18


74
NPFYFTDHRIIPSNN
18


100
ISHFQGKYRCFASNK
18


107
YRCFASNKLGIAMSE
18


114
KLGIAMSEEIEFIVP
18


130
VPKLPKEKIDPLEVE
18


199
RNDYCCFAAFPRLRT
18


209
PRLRTIVQKMPMKLT
18


216
QKMPMKLTVNSLKHA
18


219
PMKLTVNSLKHANDS
18


251
PKLLLPPTESGSESS
18


270
KGEILLLECFAEGLP
18


300
GRETKENYGKTLKIE
18


355
SAVYSTGSNGILLCE
18


393
AGDVVFPREISFTNL
18


452
VVGYSAFLHCEFFAS
18


460
HCEFFASPEAVVSWQ
18


492
NGTLQINRTTEEDAG
18


505
AGSYSCWVENAIGKT
18


524
NLDIRNATKLRVSPK
18


542
IPKLHMLELHCESKC
18


566
LSWSKDGEAFEINGT
18


574
AFEINGTEDGRIIID
18


645
RLTWEAGADHNSNIS
18


672
PGRWEELTRVQGKKT
18


743
EMIIKWEPLKSMEQN
18


763
YRVTWKPQGAPVEWE
18


778
EETVTNHTLRVMTPA
18


784
HTLRVMTPAVYAPYD
18


813
PQSVTLYSGEDYPDT
18


862
QINWWKTKSLLDGRT
18


866
WKTKSLLDGRTHPKE
18


907
TVLAYNSKGAGPESE
18


909
LAYNSKGAGPESEPY
18


934
QPTFLKVIKVDKDTA
18


951
SWGLPKKLNGNLTGY
18


979
GELNDINITTPSKPS
18


996
LSNLNATTKYKFYLR
18


1006
KFYLRACTSQGCGKP
18


1021
ITEESSTLGEGSKGI
18


1024
ESSTLGEGSKGIGKI
18


1050
IEVFEPGAEHIVRLM
18


1116
VCFVKRNRGGKYSVK
18


1126
KYSVKEKEOLHPOPE
18


1161
KGSLRSLNRDMQPTE
18


1180
LVEYGEGDHGLFSED
18


1194
DGSFIGAYAGSKEKG
18


6
LGRGLIVYLMFLLLK
17


12
VYLMFLLLKFSKAIE
17


20
KFSKAIEIPSSVQQV
17


22
SKAIEIPSSVQQVPT
17


35
PTIIKQSKVQVAFPF
17


42
KVQVAFPFDEYFQIE
17


49
FDEYFQIECEAKGNP
17


90
GTFRIPNEGHISHFQ
17


97
EGHISHFQGKYRCFA
17


135
KEKIDPLEVEEGDPI
17


150
VLPCNPPKGLPPLHI
17


171
LEHIEQDERVYMSQK
17


177
DERVYMSQKGDLYFA
17


179
RVYMSQKGDLYFANV
17


212
RTIVQKMPMKLTVNS
17


221
KLTVNSLKHANDSSS
17


222
LTVNSLKHANDSSSS
17


224
VNSLKHANDSSSSTE
17


232
DSSSSTEIGSKANSI
17


240
GSKANSIKQRKPKLL
17


242
KANSIKQRKPKLLLP
17


269
LKGEILLLECFAEGL
17


276
LECFAEGLPTPQVDW
17


290
WNKIGGDLPKGRETK
17


305
ENYGKTLKIENVSYQ
17


326
CTASNFLGTATHDFH
17


356
AVYSTGSNGILLCEA
17


364
GILLCEAEGEPQPTI
17


375
QPTIKWRVNGSPVDN
17


385
SPVDNHPFAGDVVFP
17


396
VVFPREISFTNLQPN
17


442
QTKDGENYATVVGYS
17


449
YATVVGYSAFLHCEF
17


453
VGYSAFLHCEFFASP
17


494
TLQINRTTEEDAGSY
17


510
CWVENAIGKTAVTAN
17


519
TAVTANLDIRNATKL
17


561
KHSLKLSWSKDGEAF
17


575
FEINGTEDGRIIIDG
17


584
RIIIDGANLTISNVT
17


608
SAHTALDSAADITQV
17


633
NLHLSERQNRSVRLT
17


643
SVRLTWEAGADHNSN
17


700
FRVIAVNEVGRSQPS
17


740
QPKEMIIKWEPLKSM
17


761
LEYRVTWKPQGAPVE
17


762
EYRVTWKPQGAPVEW
17


790
TPAVYAPYDVKVQAI
17


795
APYDVKVQAINQLGS
17


802
QAINQLGSGPDPQSV
17


833
GVDVINSTLVKVTWS
17


836
VINSTLVKVTWSTVP
17


891
NSGMVPSLDAFSEFH
17


903
EFHLTVLAYNSKGAG
17


943
VDKDTATLSWGLPKK
17


961
NLTGYLLQYQIINDT
17


962
LTGYLLQYQIINDTY
17


967
LQYQIINDTYEIGEL
17


1009
LRACTSQGCGKPITE
17


1010
RACTSQGCGKPITEE
17


1058
EHIVRLMTKNWGDND
17


1072
DSIFQDVIETRGREY
17


1075
FQDVIETRGREYAGL
17


1076
QDVIETRGREYAGLY
17


1154
DSDEKPLKGSLRSLN
17


1178
DSLVEYGEGDHGLFS
17


1188
HGLFSEDGSFIGAYA
17


1195
GSFIGAYAGSKEKGS
17


1210
VESNGSSTATFPLRA
17










V2-(SET1)-HLA-DRB1-0101-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









Pos
123456789012345
score





2
SEEIEFIVPSVPKFP
29


5
IEFIVPSVPKFPKEK
24


14
KFPKEKIDPLEVEEG
22


12
VPKFPKEKIDPLEVE
20


4
EIEFIVPSVPKFPKE
19


9
VPSVPKFPKEKIDPL
19


3
EEIEFIVPSVPKFPK
15


6
EFIVPSVPKFPKEKI
14










V2-(SET2)-HLA-DRB1-0101-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









Pos
123456789012345
score





4
KIGGDLPKGREAKEN
18


12
GREAKENYGKTLKIE
18


2
WNKIGGDLPKGREAK
17


14
EAKENYGKTLKIENV
16


6
GGDLPKGREAKENYG
11


7
GDLPKGREAKENYGK
11


3
NKIGGDLPKGREAKE
8










V2-(SET3)-HLA-DRB1-0101-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









Pos
123456789012345
score





13
EGKYAGLYDDISTQG
26


6
EESSTLGEGKYAGLY
20


8
SSTLGEGKYAGLYDD
18


1
GKPITEESSTLGEGK
16










V3-HLA-DRB1-0101-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









Pos
123456789012345
score





8
IHGVDVINTTYVSNT
29


15
NTTYVSNTTYVSNAT
25


21
NTTYVSNATGSPQPS
25


46
SYRNRNMLAEDFIQK
24


2
PDTAPVIHGVDVINT
22


27
NATGSPQPSIFICSK
20


20
SNTTYVSNATGSPQP
19


9
HGVDVINTTYVSNTT
18


34
PSIFICSKEQELSYR
18


47
YRNRNMLAEDFIQKS
18


35
SIFICSKEQELSYRN
17


50
RNMLAEDFIQKSTSC
17


22
TTYVSNATGSPQPSI
16


23
TYVSNATGSPQPSIF
16


42
EQELSYRNRNMLAED
16


53
LAEDFIQKSTSCNYV
16


54
AEDFIQKSTSCNYVE
16


55
EDFIQKSTSCNYVEK
16


14
INTTYVSNTTYVSNA
15


52
MLAEDFIQKSTSCNY
15


5
APVIHGVDVINTTYV
14


24
YVSNATGSPQPSIFI
14


26
SNATGSPQPSIFICS
14


32
PQPSIFICSKEQELS
14


62
TSCNYVEKSSTFFKI
14










V4-HLA-DRB1-0101-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









Pos
123456789012345 
score





4
PQSVTLYSGEDLPEQ
26


12
GEDLPEQPTFLKVIK
20


3
DPQSVTLYSGEDLPE
15


11
SGEDLPEQPTFLKVI
15


8
TLYSGEDLPEQPTFL
14










V5-HLA-DRB1-0101-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









Pos
123456789012345
score





15
SNSIKQRKPKLLLPP
24


3
VQKMPMKLTVNSSNS
23


7
PMKLTVNSSNSIKQR
23


6
MPMKLTVNSSNSIKQ
20


4
QKMPMKLTVNSSNSI
18


12
VNSSNSIKQRKPKLL
17


14
SSNSIKQRKPKLLLP
17


9
KLTVNSSNSIKQRKP
16










V6-HLA-DRB1-0101-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









Pos
123456789012345
score





6
SEEIEFIVPKLEHIE
22


10
EFIVPKLEHIEQDER
19


8
EIEFIVPKLEHIEQD
18


2
GIAMSEEIEFIVPKL
16


3
IAMSEEIEFIVPKLE
15


13
VPKLEHIEQDERVYM
15


14
PKLEHIEQDERVYMS
11










V7-HLA-DRB1-0101-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









Pos
123456789012345
score





18
SHELFTLHPEPPRWT
30


6
THDFHVIVEDNISHE
24


10
HVIVEDNISHELFTL
22


15
DNISHELFTLHPEPP
22


7
HDFHVIVEDNISHEL
19










V7-HLA-DRB1-0101-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









Pos
123456789012345
score





24
LHPEPPRWTKKPQSA
16


21
LFTLHPEPPRWTKKP
15


11
VIVEDNISHELFTLH
14


















TABLE XLVII





Pos
123456789012345
score















V1-HLA-DRB1-0301-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









171
LEHIEQDERVYMSQK
28


1132
KEDLHPDPEIQSVKD
28


66
TFSWTKDGNPFYFTD
26


114
KLGIAMSEEIEFIVP
26


191
ANVEEKDSRNDYCCF
26


594
ISNVTLEDQGIYCCS
26


951
SWGLPKKLNGNLTGY
26


1164
LRSLNRDMQPTESAD
26


177
DERVYMSQKGDLYFA
25


313
IENVSYQDKGNYRCT
25


393
AGDVVFPREISFTNL
25


689
ILPLAPFVRYQFRVI
25


895
VPSLDAFSEFHLTVL
25


996
LSNLNATTKYKFYLR
25


582
DGRIIIDGANLTISN
24


265
SITILKGEILLLECF
23


938
LKVIKVDKDTATLSW
23


623
TVLDVPDPPENLHLS
22


2
EPLLLGRGLIVYLMF
21


7
GRGLIVYLMFLLLKF
21


8
RGLIVYLMFLLLKFS
21


243
ANSIKQRKPKLLLPP
21


272
EILLLECFAEGLPTP
21


290
WNKIGGDLPKGRETK
21


476
VEEVKPLEGRRYHIY
21


687
TVILPLAPFVRYQFR
21


786
LRVMTPAVYAPYDVK
21


813
PQSVTLYSGEDYPDT
21


940
VIKVDKDTATLSWGL
21


968
QYQIINDTYEIGELN
21


1032
SKGIGKISGVNLTQK
21


1103
MCAIALLTLLLLTVC
21


3
PLLLGRGLIVYLMFL
20


15
MFLLLKFSKAIEIPS
20


179
RVVMSQKGDLYFANV
20


211
LRTIVQKMPMKLTVN
20


263
ESSITILKGEILLLE
20


404
FTNLQPNHTAVYQCE
20


631
PENLHLSERQNRSVR
20


748
WEPLKSMEQNGPGLE
20


783
NHTLRVMTPAVYAPY
20


796
PYDVKVQAINQLGSG
20


846
WSTVPKDRVHGRLKG
20


867
KTKSLLDGRTHPKEV
20


947
TATLSWGLPKKLNGN
20


1071
NDSIFQDVIETRGRE
20


1075
FQDVIETRGREYAGL
20


1086
YAGLYDDISTQGWFI
20


1097
GWFIGLMCAIALLTL
20


1116
VCFVKRNRGGKYSVK
20


1138
DPEIQSVKDETFGEY
20


1172
QPTESADSLVEYGEG
20


1188
HGLFSEDGSFIGAYA
20


34
VPTIIKQSKVQVAFP
19


44
QVAFPFDEYFQIECE
19


81
HRIIPSNNSGTFRIP
19


122
EIEFIVPSVPKLPKE
19


124
EFIVPSVPKLPKEKI
19


130
VPKLPKEKIDPLEVE
19


140
PLEVEEGDPIVLPCN
19


148
PIVLPCNPPKGLPPL
19


217
KMPMKLTVNSLKHAN
19


251
PKLLLPPTESGSESS
19


434
VVDVRPLIQTKDGEN
19


438
RPLIQTKDGENYATV
19


439
PLIQTKDGENYATVV
19


467
PEAVVSWQKVEEVKP
19


520
AVTANLDIRNATKLR
19


522
TANLDIRNATKLRVS
19


530
ATKLRVSPKNPRIPK
19


539
NPRIPKLHMLELHCE
19


557
DSHLKHSLKLSWSKD
19


576
EINGTEDGRIIIDGA
19


624
VLDVPDPPENLHLSE
19


641
NRSVRLTWEAGADHN
19


762
EYRVTWKPQGAPVEW
19


860
GYQINWWKTKSLLDG
19


881
VNILRFSGQRNSGMV
19


893
GMVPSLDAFSEFHLT
19


955
PKKLNGNLTGYLLQY
19


1058
EHIVRLMTKNWGDND
19


1100
IGLMCAIALLTLLLL
19


1141
IQSVKDETFGEYSDS
19


1150
GEYSDSDEKPLKGSL
19


1157
EKPLKGSLRSLNRDM
19


1177
ADSLVEYGEGDHGLF
19


12
VYLMFLLLKFSKAIE
18


24
AIEIPSSVQQVPTII
18


42
KVQVAFPFDEYFQIE
18


80
DHRIIPSNNSGTFRI
18


127
VPSVPKLPKEKIDPL
18


146
GDPIVLPCNPPKGLP
18


264
SSITILKGEILLLEC
18


307
YGKTLKIENVSYQDK
18


328
ASNFLGTATHDFHVI
18


340
HVIVEEPPRWTKKPQ
18


363
NGILLCEAEGEPQPT
18


389
NHPFAGDVVFPREIS
18


423
HGTILANANIDVVDV
18


429
NANIDVVDVRPLIQT
18


430
ANIDVVDVRPLIQTK
18


479
VKPLEGRRYHIYENG
18


524
NLDIRNATKLRVSPK
18


545
LHMLELHCESKCDSH
18


565
KLSWSKDGEAFEING
18


583
GRIIIDGANLTISNV
18


588
DGANLTISNVTLEDQ
18


693
APFVRYQFRVIAVNE
18


699
QFRVIAVNEVGRSQP
18


702
VIAVNEVGRSQPSQP
18


741
PKEMIIKWEPLKSME
18


806
QLGSGPDPQSVTLYS
18


906
LTVLAYNSKGAGPES
18


977
EIGELNDINITTPSK
18


983
DINITTPSKPSWHLS
18


1025
SSTLGEGSKGIGKIS
18


1064
MTKNWGDNDSIFQDV
18


1113
LLTVCFVKRNRGGKY
18


1161
KGSLRSLNRDMQPTE
18


31
VQQVPTIIKQSKVQV
17


36
TIIKQSKVQVAFPFD
17


52
YFQIECEAKGNPEPT
17


96
NEGHISHFQGKYRCF
17


116
GIAMSEEIEFIVPSV
17


164
IYWMNIELEHIEQDE
17


168
NIELEHIEQDERVYM
17


208
FPRLRTIVQKMPMKL
17


236
STEIGSKANSIKQRK
17


244
NSIKQRKPKLLLPPT
17


273
ILLLECFAEGLPTPQ
17


294
GGDLPKGRETKENYG
17


375
QPTIKWRVNGSPVDN
17


547
MLELHCESKCDSHLK
17


625
LDVPDPPENLHLSER
17


633
NLHLSERQNRSVRLT
17


647
TWEAGADHNSNISEY
17


660
EYIVEFEGNKEEPGR
17


722
TPPAAPDRNPQNIRV
17


771
GAPVEWEEETVTNHT
17


792
AVYAPYDVKVQAINQ
17


798
DVKVQAINQLGSGPD
17


851
KDRVHGRLKGYQINW
17


898
LDAFSEFHLTVLAYN
17


921
EPYIFQTPEGVPEQP
17


934
QPTFLKVIKVDKDTA
17


937
FLKVIKVDKDTATLS
17


1038
ISGVNLTQKTHPIEV
17


1076
QDVIETRGREYAGLY
17


50
DEYFQIECEAKGNPE
16


56
ECEAKGNPEPTFSWT
16


74
NPFYFTDHRIIPSNN
16


170
ELEHIEQDERVYMSQ
16


202
YCCFAAFPRLRTIVQ
16


242
KANSIKQRKPKLLLP
16


283
LPTPQVDWNKIGGDL
16


460
HCEFFASPEAVVSWQ
16


494
TLQINRTTEEDAGSY
16


553
ESKCDSHLKHSLKLS
16


969
YQIINDTYEIGELND
16


46
AFPFDEYFQIECEAK
15


89
SGTFRIPNEGHISHF
15


160
PPLHIYWMNIELEHI
15


187
DLYFANVEEKDSRND
15


270
KGEILLLECFAEGLP
15


296
DLPKGRETKENYGKT
15


355
SAVYSTGSNGILLCE
15


381
RVNGSPVDNHPFAGD
15


484
GRRYHIYENGTLQIN
15


659
SEYIVEFEGNKEEPG
15


662
IVEFEGNKEEPGRWE
15


830
VIHGVDVINSTLVKV
15


861
YQINWWKTKSLLDGR
15


926
QTPEGVPEQPTFLKV
15


998
NLNATTKYKFYLRAC
15


1050
IEVFEPGAEHIVRLM
15


1115
TVCFVKRNRGGKYSV
15


1124
GGKYSVKEKEDLHPD
15


1153
SDSDEKPLKGSLRSL
15


1
MEPLLLGRGLIVYLM
14


10
LIVYLMFLLLKFSKA
14


14
LMFLLLKFSKAIEIP
14


18
LLKFSKAIEIPSSVQ
14


100
ISHFQGKYRCFASNK
14


147
DPIVLPCNPPKGLPP
14


215
VQKMPMKLTVNSLKH
14


221
KLTVNSLKHANDSSS
14


250
KPKLLLPPTESGSES
14


271
GEILLLECFAEGLPT
14


336
THDFHVIVEEPPRWT
14


362
SNGILLCEAEGEPQP
14


551
HCESKGDSHLKHSLK
14


742
KEMIIKWEPLKSMEQ
14


775
EWEEETVTNHTLRVM
14


880
EVNILRFSGQRNSGM
14


883
ILRFSGQRNSGMVPS
14


891
NSGMVPSLDAFSEFH
14


965
YLLQYQIINDTYEIG
14


1018
GKPITEESSTLGEGS
14


1072
DSIFQDVIETRGREY
14


1088
GLYDDISTQGWFIGL
14


1105
AIALLTLLLLTVCFV
14


1110
TLLLLTVCFVKRNRG
14


1126
KYSVKEKEDLHPDPE
14


1149
FGEYSDSDEKPLKGS
14


9
GLIVYLMFLLLKFSK
13


13
YLMFLLLKFSKAIEI
13


35
PTIIKQSKVQVAFPF
13


123
IEFIVPSVPKLPKEK
13


141
LEVEEGDPIVLPCNP
13


181
YMSQKGDLYFANVEE
13


266
TILKGEILLLECFA 
13


339
FHVIVEEPPRWTKKP
13


354
QSAVYSTGSNGILLC
13


449
YATVVGYSAFLHCEF
13


473
WQKVEEVKPLEGRRY
13


486
RYHIYENGTLQINRT
13


544
KLHMLELHCESKCDS
13


595
SNVTLEDQGIYCCSA
13


608
SAHTALDSAADITQV
13


621
QVTVLDVPDPPENLH
13


685
KTTVILPLAPFVRYQ
13


686
TTVILPLAPFVRYQF
13


833
GVDVINSTLVKVTWS
13


928
PEGVPEQPTFLKVIK
13


935
PTFLKVIKVDKDTAT
13


959
NGNLTGYLLQYQIIN
13


963
TGYLLQYQIINDTYE
13


1098
WFIGLMCAIALLTLL
13


1106
IALLTLLLLTVCFVK
13


1107
ALLTLLLLTVCFVKR
13


1108
LLTLLLLTVCFVKRN
13


1109
LTLLLLTVCFVKRNR
13


1128
SVKEKEDLHPDPEIQ
13


1148
TFGEYSDSDEKPLKG
13


1168
NRDMQPTESAOSLVE
13










V2-(SET1)-HLA-DRB1-0301-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









4
EIEFIVPSVPKFPKE
19


6
EFIVPSVPKFPKEKI
18


9
VPSVPKFPKEKIDPL
18


12
VPKFPKEKIDPLEVE
17


5
IEFIVPSVPKFPKEK
12


15
FPKEKIDPLEVEEGD
12


2
SEEIEFIVPSVPKFP
10










V2-(SET2)-HLA-DRB1-0301-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









2
WNKIGGDLPKGREAK
21


6
GGDLPKGREAKENYG
18


8
DLPKGREAKENYGKT
15


5
IGGDLPKGREAKENY
11


9
LPKGREAKENYGKTL
10


12
GREAKENYGKTLKIE
10










V2-(SET3)-HLA-DRB1-0301-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









8
SSTLGEGKYAGLYDD
21


5
TEESSTLGEGKYAGL
16


1
GKPITEESSTLGEGK
14


15
KYAGLYDDISTQGWF
12


7
ESSTLGEGKYAGLYD
10










V3-HLA-DRB1-0301-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each pepfide is the start posifion plus fourteen.









34
PSIFICSKEQELSYR
25


42
EQELSYRNRNMLAED
25


50
RNMLAEDFIQKSTSC
23


35
SIFICSKEQELSYRN
19


55
EDFIQKSTSCNYVEK
19


33
QPSIFICSKEQELSY
17


13
VINTTYVSNTTYVSN
16


7
VIHGVDVINTTYVSN
15


36
IFICSKEQELSYRNR
15


4
TAPVIHGVDVINTTY
12


10
GVDViNTTYVSNTTY
12


49
NRNMLAEDFIQKSTS
12










V4-HLA-DRB1-0301-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









4
PQSVTLYSGEDLPEQ
21


10
YSGEDLPEQPTFLKV
16


6
SVTLYSGEDLPEQPT
13


12
GEDLPEQPTFLKVIK
13


8
TLYSGEDLPEQPTFL
12


11
SGEDLPEQPTFLKVI
11


5
QSVTLYSGEDLPEQP
11










V5-HLA-DRB1-0301-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









15
SNSIKQRKPKLLLPP
21


5
KMPMKLTVNSSNSIK
18


14
SSNSIKQRKPKLLLP
16


3
VQKMPMKLTVNSSNS
14


7
PMKLTVNSSNSIKQR
13


9
KLTVNSSNSIKQRKP
12


















TABLE XLVIII





Pos
123456789012345
score















V2-(SET1)-HLA-0401-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









12
VPKFPKEKIDPLEVE
22


6
EFIVPSVPKFPKEKI
20


9
VPSVPKFPKEKIDPL
20


3
EEIEFIVPSVPKFPK
18


2
SEEIEFIVPSVPKFP
14


1
MSEEIEFIVPSVPKF
12


14
KFPKEKIDPLEVEEG
12


4
EIEFIVPSVPKFPKE
10







V2-(SET2)-HLA-0401-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









2
WNKIGGDLPKGREAK
14


11
KGREAKENYGKTLKI
12


14
EAKENYGKTLKIENV
12


6
GGDLPKGREAKENYG
8


5
IGGDLPKGREAKENY
7


10
PKGREAKENYGKTLK
7


3
NKIGGDLPKGREAKE
6


4
KIGGDLPKGREAKEN
6


8
DLPKGREAKENYGKT
6


9
LPKGREAKENYGKTL
6


12
GREAKENYGKTLKIE
6


13
REAKENYGKTLKIEN
6










V2-(SET3)-HLA1-0401-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









13
EGKYAGLYDDISTQG
22


1
GKPITEESSTLGEGK
20


8
SSTLGEGKYAGLYDD
14


15
KYAGLYDDISTQGWF
12










V3-HLA-0401-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









8
IHGVDVINTTYVSNT
26


15
NTTYVSNTTYVSNAT
22


5
APVIHGVDVINTTYV
20


22
TTYVSNATGSPQPSI
20


49
NRNMLAEDFIQKSTS
20


50
RNMLAEDFIQKSTSC
20


32
PQPSIFICSKEQELS
18


38
ICSKEQELSYRNRNM
18


51
NMLAEDFIQKSTSCN
18


54
AEDFIQKSTSCNYVE
17


21
NTTYVSNATGSPQPS
16


34
PSIFICSKEQELSYR
16


35
SIFICSKEQELSYRN
15


42
EQELSYRNRNMLAED
15


4
TAPVIHGVDVINTTY
14


10
GVDVINTTYVSNTTY
14


11
VDVINTTYVSNTTYV
14


16
TTYVSNTTYVSNATG
14


55
EDFIQKSTSCNYVEK
14


1
YPDTAPVIHGVDVIN
12


2
PDTAPVIHGVDVINT
12


6
PVIHGVDVINTTYVS
12


7
VIHGVDVINTTYVSN
12


9
HGVDVINTTYVSNTT
12


12
DVINTTYVSNTTYVS
12


13
VINTTYVSNTTYVSN
12


14
INTTYVSNTTYVSNA
12


18
YVSNTTYVSNATGSP
12


19
VSNTTYVSNATGSPQ
12


25
VSNATGSPQPSIFIC
12


27
NATGSPQPSIFICSK
12


30
GSPQPSIFICSKEQE
12


40
SKEQELSYRNRNMLA
12


41
KEQELSYRNRNMLAE
12


43
QELSYRNRNMLAEDF
12


47
YRNRNMLAEDFIQKS
12


48
RNRNMLAEDFIQKST
12


52
MLAEDFIQKSTSCNY
12


61
STSCNYVEKSSTFFK
12


62
TSCNYVEKSSTFFKI
12










V4-HLA-0401-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









7
VTLYSGEDLPEQPTF
22


12
GEDLPEQPTFLKVIK
20


4
PQSVTLYSGEDLPEQ
14


3
DPQSVTLYSGEDLPE
12


9
LYSGEDLPEQPTFLK
12


11
SGEDLPEQPTFLKVI
12


15
LPEQPTFLKVIKVDK
12










V5-HLA-0401-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









5
KMPMKLTVNSSNSIK
20


7
PMKLTVNSSNSIKQR
20


9
KLTVNSSNSIKQRKP
20


6
MPMKLTVNSSNSIKQ
18


3
VQKMPMKLTVNSSNS
15


4
QKMPMKLTVNSSNSI
12


8
MKLTVNSSNSIKQRK
12


12
VNSSNSIKQRKPKLL
12


15
SNSIKQRKPKLLLPP
9










V6-HLA-0401-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









10
EFIVPKLEHIEQDER
26


2
GIAMSEEIEFIVPKL
20


13
VPKLEHIEQDERVYM
20


8
EIEFIVPKLEHIEQD
16


6
SEEIEFIVPKLEHIE
14


1
LGIAMSEEIEFIVPK
12


4
AMSEEIEFIVPKLEH
12


5
MSEEIEFIVPKLEHI
12


14
PKLEHIEQDERVYMS
12










V7-HLA-0401-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









6
THDFHVIVEDNISHE
22


8
DFHVIVEDNISHELF
20


10
HVIVEDNISHELFTL
20


21
LFTLHPEPPRWTKKP
20


2
LGTATHDFHVIVEDN
18


9
FHVIVEDNISHELFT
14


14
EDNISHELFTLHPEP
14


18
SHELFTLHPEPPRWT
14


5
ATHDFHVIVEDNISH
12


7
HDFHVIVEDNISHEL
12


11
VIVEDNISHELFTLH
12


15
DNISHELFTLHPEPP
12


22
FTLHPEPPRWTKKPQ
12


19
HELFTLHPEPPRWTK
10


















TABLE XLIX





Pos
123456789012345
score















V1-HLA-DRB1-1101-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 3; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









672
PGRWEELTRVQGKKT
30


1113
LLTVCFVKRNRGGKY
28


74
NPFYFTDHRIIPSNN
26


702
VIAVNEVGRSQPSQP
26


843
KVTWSTVPKDRVHGR
26


937
FLKVIKVDKDTATLS
26


1058
EHIVRLMTKNWGDND
26


336
THDFHVIVEEPPRWT
25


760
GLEYRVTWKPQGAPV
25


100
ISHFQGKYRCFASNK
24


446
GENYATVVGYSAFLH
24


934
QPTFLKVIKVDKDTA
24


949
TLSWGLPKKLNGNLT
24


1072
DSIFQDVIETRGREY
24


13
YLMFLLLKFSKAIEI
23


287
QVDWNKIGGDLPKGR
23


470
VVSWQKVEEVKPLEG
23


1083
GREYAGLYDDISTQG
23


1096
QGWFIGLMCAIALLT
23


187
DLYFANVEEKDSRND
22


12
VYLMFLLLKFSKAIE
21


120
SEEIEFIVPSVPKLP
21


127
VPSVPKLPKEKIDPL
21


619
ITQVTVLDVPDPPEN
21


693
APFVRYQFRVIAVNE
21


1025
SSTLGEGSKGIGKIS
21


31
VQQVPTIIKQSKVQV
20


52
YFQIECEAKGNPEPT
20


73
GNPFYFTDHRIIPSN
20


94
PNEGHISHFQGKYRI
20


124
EFIVPSVPKLPKEKI
20


202
YCCFAAFPRLRTIVQ
20


208
FPRLRTIVQKMPMKL
20


221
KLTVNSLKHANDSSS
20


547
MLELHCESKCDSHLK
20


739
SQPKEMIIKWEPLKS
20


877
HPKEVNILRFSGQRN
20


906
LTVLAYNSKGAGPES
20


1029
GEGSKGIGKISGVNL
20


1038
ISGVNLTQKTHPIEV
20


1076
QDVIETRGREYAGLY
20


1161
KGSLRSLNRDMQPTE
20


1124
GGKYSVKEKEDLHPD
19


1180
LVEYGEGDHGLFSED
19


11
IVYLMFLLLKFSKAI
18


75
PFYFTDHRIIPSNNS
18


135
KEKIDPLEVEEGDPI
18


304
KENYGKTLKIENVSY
18


401
EISFTNLQPNHTAVY
18


452
VVGYSAFLHCEFFAS
18


473
WQKVEEVKPLEGRRY
18


602
QGIYCCSAHTALDSA
18


748
WEPLKSMEQNGPGLE
18


798
DVKVQAINQLGSGPD
18


828
APVIHGVDVINSTLV
18


978
IGELNDINITTPSKP
18


1002
TTKYKFYLRACTSQG
18


1115
TVCFVKRNRGGKYSV
18


49
FDEYFQIECEAKGNP
17


50
DEYFQIECEAKGNPE
17


107
YRCFASNKLGIAMSE
17


205
FAAFPRLRTIVQKMP
17


461
CEFFASPEAVVSWQK
17


697
RYQFRVIAVNEVGRS
17


1040
GVNLTQKTHPIEVFE
17


1188
HGLFSEDGSFIGAYA
17


18
LLKFSKAIEIPSSVQ
16


64
EPTFSWTKDGNPFYF
16


163
HIYWMNIELEHIEQD
16


209
PRLRTIVQKMPMKLT
16


291
NKIGGDLPKGRETKE
16


373
EPQPTIKWRVNGSPV
16


392
FAGDVVFPREISFTN
16


428
ANANIDVVDVRPLIQ
16


455
YSAFLHCEFFASPEA
16


524
NLDIRNATKLRVSPK
16


572
GEAFEINGTEDGRII
16


645
RLTWEAGADHNSNIS
16


662
IVEFEGNKEEPGRWE
16


689
ILPLAPFVRYQFRVI
16


745
IIKWEPLKSMEQNGP
16


792
AVYAPYDVKVQAINQ
16


835
DVINSTLVKVTWSTV
16


849
VPKDRVHGRLKGYQI
16


863
INWWKTKSLLDGRTH
16


966
LLQYQIINDTYEIGE
16


983
DINITTPSKPSWHLS
16


1005
YKFYLRACTSQGCGK
16


1054
EPGAEHIVRLMTKNW
16


1087
AGLYDDISTQGWFIG
16


1198
IGAYAGSKEKGSVES
16


15
MFLLLKFSKAIEIPS
15


24
AIEIPSSVQQVPTII
15


123
IEFIVPSVPKLPKEK
15


149
IVLPCNPPKGLPPLH
15


219
PMKLTVNSLKHANDS
15


263
ESSITILKGEILLLE
15


382
VNGSPVDNHPFAGDV
15


476
VEEVKPLEGRRYHIY
15


530
ATKLRVSPKNPRIPK
15


554
SKCDSHLKHSLKLSW
15


661
YIVEFEGNKEEPGRW
15


675
WEELTRVQGKKTTVI
15


682
QGKKTTVILPLAPFV
15


683
GKKTTVILPLAPFVR
15


700
FRVIAVNEVGRSQPS
15


758
GPGLEYRVTWKPQGA
15


824
YPDTAPVIHGVDVIN
15


859
KGYQINWWKTKSLLD
15


869
KSLLDGRTHPKEVNI
15


882
NILRFSGQRNSGMVP
15


903
EFHLTVLAYNSKGAG
15


948
ATLSWGLPKKLNGNL
15


996
LSNLNATTKYKFYLR
15


1018
GKPITEESSTLGEGS
15


1106
IALLTLLLLTVCFVK
15


1157
EKPLKGSLRSLNRDM
15


1197
FIGAYAGSKEKGSVE
15


34
VPTIIKQSKVQVAFP
14


91
TFRIPNEGHISHFQG
14


129
SVPKLPKEKIDPLEV
14


165
YWMNIELEHIEQDER
14


171
LEHIEQDERVYMSQK
14


177
DERVYMSQKGDLYFA
14


186
GDLYFANVEEKDSRN
14


234
SSSTEIGSKANSIKQ
14


240
GSKANSIKQRKPKLL
14


262
SESSITILKGEILLL
14


284
PTPQVDWNKIGGDLP
14


313
IENVSYQDKGNYRCT
14


317
SYQDKGNYRCTASNF
14


329
SNFLGTATHDFHVIV
14


340
HVIVEEPPRWTKKPQ
14


344
EEPPRWTKKPQSAVY
14


375
QPTIKWRVNGSPVDN
14


408
QPNHTAVYQCEASNV
14


429
NANIDVVDVRPLIQT
14


467
PEAVVSWQKVEEVKP
14


491
ENGTLQINRTTEEDA
14


510
CWVENAIGKTAVTAN
14


526
DIRNATKLRVSPKNP
14


532
KLRVSPKNPRIPKLH
14


536
SPKNPRIPKLHMLEL
14


543
PKLHMLELHCESKCD
14


557
DSHLKHSLKLSWSKD
14


616
AADITQVTVLDVPDP
14


631
PENLHLSERQNRSVR
14


656
SNISEYIVEFEGNKE
14


712
QPSQPSDHHETPPAA
14


727
PDRNPQNIRVQASQP
14


762
EYRVTWKPQGAPVEW
14


783
NHTLRVMTPAVYAPY
14


794
YAPYDVKVQAINQLG
14


831
IHGVDVINSTLVKVT
14


836
VINSTLVKVTWSTVP
14


839
STLVKVTWSTVPKDR
14


845
TWSTVPKDRVHGRLK
14


851
KDRVHGRLKGYQINW
14


867
KTKSLLDGRTHPKEV
14


921
EPYIFQTPEGVPEQP
14


935
PTFLKVIKVDKDTAT
14


1128
SVKEKEDLHPDPEIQ
14










V2-(SET2)-HLA-DRB1-1101-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









3
NKIGGDLPKGREAKE
16


13
REAKENYGKTLKIEN
9


5
IGGDLPKGREAKENY
8


8
DLPKGREAKENYGKT
8


1
DWNKIGGDLPKGREA
7


4
KIGGDLPKGREAKEN
7


6
GGDLPKGREAKENYG
7










V2-(SET3)-HLA-DRB1-1101-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 5; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









13
EGKYAGLYDDISTQG
23


1
GKPITEESSTLGEGK
15


7
ESSTLGEGKYAGLYD
14










V3-HLA-DRB1-1101-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 7; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









52
MLAEDFIQKSTSCNY
20


15
NTTYVSNTTYVSNAT
17


21
NTTYVSNATGSPQPS
16


1
YPDTAPVIHGVDVIN
15


33
QPSIFICSKEQELSY
15


19
VSNTTYVSNATGSPQ
14


42
EQELSYRNRNMLAED
14


61
STSCNYVEKSSTFFK
14


8
IHGVDVINTTYVSNT
13


35
SIFICSKEQELSYRN
13


50
RNMLAEDFIQKSTSC
13


5
APVIHGVDVINTTYV
12


34
PSIFICSKEQELSYR
10


40
SKEQELSYRNRNMLA
10


44
ELSYRNRNMLAEDFI
10


54
AEDFIQKSTSCNYVE
10


4
TAPVIHGVDVINTTY
9










V4-HLA-DRB1-1101-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 9; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









1
GPDPQSVTLYSGEDL
12


7
VTLYSGEDLPEQPTF
10


15
LPEQPTFLKVIKVDK
9


4
PQSVTLYSGEDLPEQ
8


5
QSVTLYSGEDLPEQP
7


12
GEDLPEQPTFLKVIK
7


3
DPQSVTLYSGEDLPE
6


6
SVTLYSGEDLPEQPT
6


8
TLYSGEDLPEQPTFL
6


9
LYSGEDLPEQPTFLK
6










V5-HLA-DRB1-1101-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 11; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









12
VNSSNSIKQRKPKLL
14


3
VQKMPMKLTVNSSNS
12


4
QKMPMKLTVNSSNSI
12


6
MPMKLTVNSSNSIKQ
12


11
TVNSSNSIKQRKPKL
10


7
PMKLTVNSSNSIKQR
9


14
SSNSIKQRKPKLLLP
9


1
TIVQKMPMKLTVNSS
8


5
KMPMKLTVNSSNSIK
8


13
NSSNSIKQRKPKLLL
8


2
IVQKMPMKLTVNSSN
7


9
KLTVNSSNSIKQRKP
7


15
SNSIKQRKPKLLLPP
7










V6-HLA-DRB1-1101-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 13; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









10
EFIVPKLEHIEQDER
20


6
SEEIEFIVPKLEHIE
15


7
EEIEFIVPKLEHIEQ
15


3
IAMSEEIEFIVPKLE
13


13
VPKLEHIEQDERVYM
12


8
EIEFIVPKLEHIEQD
11










V7-HLA-DRB1-1101-15mers-282P1G3


Each peptide is a portion of SEQ ID NO: 15; each start position is


specified, the length of peptide is 15 amino acids, and the end


position for each peptide is the start position plus fourteen.









6
THDFHVIVEDNISHE
19


18
SHELFTLHPEPPRWT
19


11
VIVEDNISHELFTLH
15


17
ISHELFTLHPEPPRW
14


26
PEPPRWTKKPQSAVY
14


7
HDFHVIVEDNISHEL
13


15
DNISHELFTLHPEPP
12


19
HELFTLHPEPPRWTK
12


2
LGTATHDFHVIVEDN
11
















TABLE L





Protein Characteristics of 282P1G3


















Bioinformatic



282P1G3 v.1
Program
Outcome





ORF
ORF finder


Protein length

1224 aa


Trans-
TM Pred
2TM, aa 6-25, 1098-1116


membrane
HMMTop
one TM, aa 1098-1117


region
Sosui
2TM, aa 3-25, 1096-1118



TMHMM
one TM, aa 1097-1119


Signal Peptide
Signal P
yes, cleave aa 24-25


pI
pI/MW tool
pI 5.54


Molecular
pI/MW tool
136.6 kD


weight


Localization
PSORT
46% plasma membrane, 10% micobody



PSORT II
44% endoplasmic, 11% vacuolar


Motifs
Pfam
Ig domain, Fibronectin type III repeat



Prints
Cadherin, Fibronectin type III repeat



Blocks
Fibronectin type III repeat






Bioinformatic


v.3
Program
Outcome





ORF
ORF finder


Protein length

893 aa


Trans-
TM Pred
one TM, aa 3-19, N-terminus in


membrane
HMMTop
one TM, aa 1-25, N-terminus out


region
Sosui
one TM, aa 3-25



TMHMM
none


Signal Peptide
Signal P
none


pI
pI/MW tool
pI 5.49


Molecular
pI/MW tool
100.2 kD


weight


Localization
PSORT
78% secreted, 19% lysosomal



PSORT II
52% nuclear, 17% mitochondreal


Motifs
Pfam
Ig domain, Fibronectin type III repeat



Prints
Cadherin, Fibronectin type III repeat



Blocks
Fibronectin type III repeat
















TABLE LI







Exon boundaries of transcript 282P1G03 v.1












Exon Number
Start
End
Length
















1
1
97
97



2
98
177
80



3
178
362
185



4
363
468
106



5
469
656
188



6
657
779
123



7
780
950
171



8
951
998
48



9
999
1119
121



10
1120
1304
185



11
1305
1436
132



12
1437
1577
141



13
1578
1689
112



14
1690
1906
217



15
1907
2022
116



16
2023
2147
125



17
2148
2249
102



18
2250
2447
198



19
2448
2518
71



20
2519
2741
223



21
2742
2857
116



22
2858
3062
205



23
3063
3185
123



24
3186
3365
180



25
3366
3524
159



26
3525
3656
132



27
3657
3729
73



28
3730
7650
3921

















TABLE LIIa





Nucleotide sequence of transcript variant 282P1G03 v.2 (SEQ ID NO: 151)
















cggaccctgc gcgcccccgt cccggctccc ggccggctcg ggggagaagg cgcccgaggg
60


 


gaggcgccgg acagatcgcg tttcggaggc ggcgcaggtg ctgtaaactg caaaccataa
120


 


tcctgtctta atactgcaaa caaatcatag tggaactaag gggaacttaa tttactgttt
180


 


ccaggttaac taaggtctca gctgtaaacc aaaagtgaga ggagacatta agattttcat
240


 


tcttaccggg ttgtcttctt cctgaagagc aatggagccg cttttacttg gaagaggact
300


 


aatcgtatat ctaatgttcc tcctgttaaa attctcaaaa gcaattgaaa taccatcttc
360


 


agttcaacag gttccaacaa tcataaaaca gtcaaaagtc caagttgcct ttcccttcga
420


 


tgagtatttt caaattgaat gtgaagctaa aggaaatcca gaaccaacat tttcgtggac
480


 


taaggatggc aacccttttt atttcactga ccatcggata attccatcga acaattcagg
540


 


aacattcagg atcccaaacg aggggcacat atctcacttt caagggaaat accgctgctt
600


 


tgcttcaaat aaactgggaa tcgctatgtc agaagaaata gaatttatag ttccaagtgt
660


 


tccaaaattc ccaaaagaaa aaattgaccc tcttgaagtg gaggagggag atccaattgt
720


 


cctcccatgc aatcctccca aaggcctccc acctttacac atttattgga tgaatattga
780


 


attagaacac atcgaacaag atgaaagagt atacatgagc caaaagggag atctatactt
840


 


cgcaaacgtg gaagaaaagg acagtcgcaa tgactactgt tgctttgctg catttccaag
900


 


attaaggact attgtacaga aaatgccaat gaaactaaca gttaacagtt taaagcatgc
960


 


taatgactca agttcatcca cagaaattgg ttccaaggca aattccatca agcaaagaaa
1020


 


acccaaactg ctgttgcctc ccactgagag tggcagtgag tcttcaatta ccatcctcaa
1080


 


aggggaaatc ttgctgcttg agtgttttgc tgaaggcttg ccaactccac aggttgattg
1140


 


gaacaaaatt ggtggtgact taccaaaggg gagagaagca aaagaaaatt atggcaagac
1200


 


tttgaagata gagaatgtct cctaccagga caaaggaaat tatcgctgca cagccagcaa
1260


 


tttcttggga acagccactc acgattttca cgttatagta gaagagcctc ctcgctggac
1320


 


aaagaagcct cagagtgctg tgtatagcac cggaagcaat ggcatcttgt tatgtgaggc
1380


 


tgaaggagaa cctcaaccca caatcaagtg gagagtcaat ggctccccag ttgacaatca
1440


 


tccatttgct ggtgatgttg tcttccccag ggaaatcagt tttaccaacc ttcaaccaaa
1500


 


tcatactgct gtgtaccagt gtgaagcctc aaatgtccat ggaactatcc ttgccaatgc
1560


 


caatattgat gttgtggatg tccgtccatt gatacaaacc aaagatggag aaaattacgc
1620


 


tacagtggtt gggtacagtg ctttcttaca ttgcgagttc tttgcttcac ctgaggcagt
1680


 


cgtgtcctgg cagaaggtgg aagaagtgaa acccctggag ggcaggcggt atcatatcta
1740


 


tgaaaatggc acattgcaga tcaacagaac caccgaagaa gatgctgggt cttactcatg
1800


 


ttgggtagaa aatgctatag gaaaaactgc agtcacagcc aatttggata ttagaaatgc
1860


 


tacaaaactt agagtttctc ctaagaatcc tcgtatcccc aaattgcata tgcttgaatt
1920


 


acattgtgaa agcaaatgtg actcacattt gaaacacagt ttgaagttgt cctggagtaa
1980


 


agatggagaa gcctttgaaa ttaatggcac agaagatggc aggataatta ttgatggagc
2040


 


taatttgacc atatctaatg taactttaga ggaccaaggt atttactgct gttcagctca
2100


 


tactgctcta gacagtgctg ccgatataac tcaagtaact gttcttgatg ttccggatcc
2160


 


accagaaaac cttcacttgt ctgaaagaca gaacaggagt gttcggctga cctgggaagc
2220


 


tggagctgac cacaacagca atattagcga gtatattgtt gaatttgaag gaaacaaaga
2280


 


agagcctgga aggtgggagg aactgaccag agtccaagga aagaaaacca cagttatctt
2340


 


acctttggct ccatttgtga gataccagtt cagggtcata gccgtgaacg aagtagggag
2400


 


aagtcagcct agccagccgt cagaccatca tgaaacacca ccagcagctc cagataggaa
2460


 


tccacaaaac ataagggttc aagcctctca acccaaggaa atgattataa agtgggagcc
2520


 


tttgaaatcc atggagcaga atggaccagg cctagagtac agagtgacct ggaagccaca
2580


 


gggagcccca gtggagtggg aagaagaaac agtcacaaac cacacattgc gggtgatgac
2640


 


gcctgctgtc tatgcccctt atgatgtcaa ggtccaggct atcaatcaac taggatctgg
2700


 


gcctgaccct cagtcagtga ctctctattc tggagaagac tatcctgata cagctccagt
2760


 


gatccatggg gtggacgtta taaacagtac attagttaaa gttacctggt caacagttcc
2820


 


aaaggacaga gtacatggac gtctgaaagg ctatcagata aattggtgga aaacaaaaag
2880


 


tctgttggat ggaagaacac atcccaaaga agtgaacatt ctaagatttt caggacaaag
2940


 


aaactctgga atggttcctt ccttagatgc ctttagtgaa tttcatttaa cagtcttagc
3000


 


ctataactct aaaggagctg gtcctgaaag tgagccttat atatttcaaa caccagaagg
3060


 


agtacctgaa cagccaactt ttctaaaggt catcaaagtt gataaagaca ctgccacttt
3120


 


atcttgggga ctacctaaga aattaaatgg aaacttaact ggctatcttt tgcaatatca
3180


 


gataataaat gacacctacg agattggaga attaaatgat attaacatta caactccatc
3240


 


aaagcccagc tggcacctct caaacctgaa tgcaactacc aagtacaaat tctacttgag
3300


 


ggcttgcact tcacagggct gtggaaaacc gatcacggag gaaagctcca ccttaggaga
3360


 


agggaaatat gctggtttat atgatgacat ctccactcaa ggctggttta ttggactgat
3420


 


gtgtgcgatt gctcttctca cactactatt attaactgtt tgctttgtga agaggaatag
3480


 


aggtggaaag tactcagtta aagaaaagga agatttgcat ccagacccag aaattcagtc
3540


 


agtaaaagat gaaacctttg gtgaatacag tgacagtgat gaaaagcctc tcaaaggaag
3600


 


ccttcggtcc cttaataggg atatgcagcc tactgaaagt gctgacagct tagtcgaata
3660


 


cggagaggga gaccatggtc tcttcagtga agatggatca tttattggtg cctacgctgg
3720


 


atctaaggag aagggatctg ttgaaagcaa tggaagttct acagcaactt ttccccttcg
3780


 


ggcataaaca caacatatgt aagcaacgct actggttcac cccaaccttc catatttatc
3840


 


tgttcaaagg agcaagaact ttcatatagg aatagaaaca tgctggccga agatttcatc
3900


 


cagaagtcaa catcctgcaa ttatgttgaa aagagtagta ctttcttcaa aatataaaat
3960


 


gccaagcact tcaggcctat gttttgctta tattgttttc aggtgctcaa aatgcaaaac
4020


 


acaaaacaaa tcctgcattt agatacacct caactaaatc caaagtcccc attcagtata
4080


 


ttccatattt gcctgatttt actattcggt gtgtttgcat agatgttgct acttggtggg
4140


 


tttttctccg tatgcacatt ggtatacagt ctctgagaac tggcttggtg actttgcttc
4200


 


actacaggtt aaaagaccat aagcaaactg gttatttaaa atgtaaaaag gaatatgaaa
4260


 


gtcttattaa aacacttcat tgaaaatata cagtctaaat ttattattta aattttacta
4320


 


gcaaaagtct taggtgaaca atcaactagt atttgttgag ctcctatttg cccagagatg
4380


 


gtcatattta aacagaagta tacgtttttc agtttcaaca tgaatttttt tatttctgtc
4440


 


agttatgaca tccacgagca tcactttttg tgtctgtttt tttttttttc ttggactaaa
4500


 


ttcaactgca tggaagcggt ggtcagaagg ttgttttata cgagaacagg cagaaagtgc
4560


 


ccattgttca ggattctaat agctacatct acttaatatc ttcatttcta aattgactgc
4620


 


ttttaccttt ttctcatgtt tatataatgg tatgcttgca tatatttcat gaatacattg
4680


 


tacatattat gttaatattt acacaattta aaatatagat gtgttttatt ttgaagtgag
4740


 


aaaatgaaca ttaacaggca tgtttgtaca gctagaatat attagtaaga tactgttttt
4800


 


cgtcattcca gagctacaac taataacacg aggttccaaa gctgaagact ttgtataaag
4860


 


tatttgggtt ttgttcttgt attgctttct ttcaacagtt tcaaaataaa atatcataca
4920


 


aatattgagg gaaatgtttt catatttttc aaaataggtt tttattgttg aatgtacatc
4980


 


taccccagcc cctcaaaaga aaaactgttt acatagaaat tcctacacat acgtttgcgt
5040


 


atatgttatt ttaaacatct ttgtggtgag aattttttcc ccgatattct ccttctgtca
5100


 


aagtcagaac aaattcaggg aatttatttt ctggcagttg tgctccagtc cttttaaaat
5160


 


tgtacatgaa catgttttag aaacaatatg gaggatgatg catacatgtc ggtcaagttc
5220


 


agcgctcgac attttatgga aagatttttt taaccttacc acgaaatact taactactgt
5280


 


ttaagtgaat tgacttattt cactttagtt tttgaactgt gattattggt atactgttat
5340


 


atcctcaact tggatttatg gtaacccctt ttagttcatg gagaccaaaa tttggggtat
5400


 


ttataatagt cagcgcagga atgcacatgg aatatctact tgtccttttg aacctcacga
5460


 


gtcatccaga atgtatagac aggaaaagca tgtcttattt aaaactgtaa tttatgggct
5520


 


caggatctga ccgcagtccc gggagtaagc atttcaaagg gggaaggcag tgtggtccct
5580


 


accctgtgtg aatgtgagga tgtagacatc catcagtgca actcgagctc catcctcctc
5640


 


cgatttctaa ggctccagtt ttctggaggg acagtcatca tgttttgatt tatctgggag
5700


 


aaaactgtgg tgcacagctt gtgaggaggg caaggttgtg acgttcgagc ttagttctgg
5760


 


tgttattctg tctcctcttc tttgtcatca gccaaaacgt ggtttttaaa gagagtcatg
5820


 


caggttagaa ataatgtcaa aaatatttag gaatttaata acctttaagt cagaaactaa
5880


 


aacaaatact gaaatattag ctcttcctac acttcgtgtt cccctttagc tgcctgaaaa
5940


 


tcaagattgc tcctactcag atcttctgag tggctaaaac ttatggatat gaaaaatgag
6000


 


attgaatgat gactatgctt tgctatcatt gttacctttc ctcaatacta tttggcaact
6060


 


actgggactc ttcagcacaa aaggaataga tctatgattg accctgattt taattgtgaa
6120


 


attatatgat tcatatattt tatgaatcag aataaccttc aaataaaata aatctaagtc
6180


 


ggttaaaatg gatttcatga ttttccctca gaaaatgagt aacggagtcc acggcgtgca
6240


 


atggtaatta taaattggtg atgcttgttt gcaaattgcc cactcgtgat aagtcaacag
6300


 


ccaatattta aaactttgtt cgttactggc tttaccctaa ctttctctag tctactgtca
6360


 


atatcatttt aatgtaattg attgtatata gtctcaagaa tggttggtgg gcatgagttc
6420


 


ctagagaact gtccaagggt tgggaaaatc caaattctct tcctggctcc agcactgatt
6480


 


ttgtacataa acattaggca ggttgcttaa cctttttatt tcaaactctc tcaactctaa
6540


 


agtgctaata ataatctcag ttaccttatc tttgtcacag ggtgttcttt tttatgaaga
6600


 


aaaatttgaa aatgataaaa gctaagatgc cttctaactt cataagcaaa cctttaacta
6660


 


attatgtatc tgaaagtcac ccccacatac caactcaact tttttcctgt gaacacataa
6720


 


atatattttt atagaaaaac aaatctacat aaaataaatc tactgtttag tgagcagtat
6780


 


gacttgtaca tgccattgaa aattattaat cagaagaaaa ttaagcaggg tctttgctat
6840


 


acaaaagtgt tttccactaa ttttgcatgc gtatttataa gaaaaatgtg aatttggtgg
6900


 


ttttattcta tcggtataaa ggcatcgata ttttagatgc acccgtgttt gtaaaaatgt
6960


 


agagcacaat ggaattatgc tggaagtctc aaataatatt tttttcctat tttatactca
7020


 


tggaagagat aagctaaaga ggggacaata atgagaaatg ttggtgtgct tttctaagca
7080


 


tttaaaacat aattgccaat tgaaacccta aatatgttta cataccatta agatatgatt
7140


 


catgtaacaa tgttaaatta attataatgg gattgggttt gttatctgtg gtagtatata
7200


 


tcctagtgtt cctatagtga aataagtagg gttcagccaa agctttcttt gttttgtacc
7260


 


ttaaattgtt cgattacgtc atcaaaagag atgaaaggta tgtagaacag gttcacgtga
7320


 


ttaccttttt cttttggctt ggattaatat tcatagtaga actttataaa acgtgtttgt
7380


 


attgtaggtg gtgtttgtat tatgcttatg actatgtatg gtttgaaaat attttcatta
7440


 


tacatgaaat tcaactttcc aaataaaagt tctacttcat gtaatccaaa a
7491
















TABLE LIIIa





Nucleotide sequence alignment of 282P1G03 v.1 (SEQ ID NO:152)


and 282P1G03 v.2 (SEQ ID NO:153)





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 











Note:


Two SNP at 668 and 1178.













TABLE LIVa





Peptide sequences of protein coded by 282P1G03 v.2 (SEQ ID NO: 154)
















MEPLLLGRGL IVYLMFLLLK FSKAIEIPSS VQQVPTIIKQ SKVQVAFPFD EYFQIECEAK
60


 


GNPEPTFSWT KDGNPFYFTD HRIIPSNNSG TFRIPNEGHI SHFQGKYRCF ASNKLGIAMS
120


 


EEIEFIVPSV PKFPKEKIDP LEVEEGDPIV LPCNPPKGLP PLHIYWMNIE LEHIEQDERV
180


 


YMSQKGDLYF ANVEEKDSRN DYCCFAAFPR LRTIVQKMPM KLTVNSLKHA NDSSSSTEIG
240


 


SKANSIKQRK PKLLLPPTES GSESSITILK GEILLLECFA EGLPTPQVDW NKIGGDLPKG
300


 


REAKENYGKT LKIENVSYQD KGNYRCTASN FLGTATHDFH VIVEEPPRWT KKPQSAVYST
360


 


GSNGILLCEA EGEPQPTIKW RVNGSPVDNH PFAGDVVFPR EISFTNLQPN HTAVYQCEAS
420


 


NVHGTILANA NIDVVDVRPL IQTKDGENYA TVVGYSAFLH CEFFASPEAV VSWQKVEEVK
480


 


PLEGRRYHIY ENGTLQINRT TEEDAGSYSC WVENAIGKTA VTANLDIRNA TKLRVSPKNP
540


 


RIPKLHMLEL HCESKCDSHL KHSLKLSWSK DGEAFEINGT EDGRIIIDGA NLTISNVTLE
600


 


DQGIYCCSAH TALDSAADIT QVTVLDVPDP PENLHLSERQ NRSVRLTWEA GADHNSNISE
660


 


YIVEFEGNKE EPGRWEELTR VQGKKTTVIL PLAPFVRYQF RVIAVNEVGR SQPSQPSDHH
720


 


ETPPAAPDRN PQNIRVQASQ PKEMIIKWEP LKSMEQNGPG LEYRVTWKPQ GAPVEWEEET
780


 


VTNHTLRVMT PAVYAPYDVK VQAINQLGSG PDPQSVTLYS GEDYPDTAPV IHGVDVINST
840


 


LVKVTWSTVP KDRVHGRLKG YQINWWKTKS LLDGRTHPKE VNILRFSGQR NSGMVPSLDA
900


 


FSEFHLTVLA YNSKGAGPES EPYIFQTPEG VPEQPTFLKV IKVDKDTATL SWGLPKKLNG
960


 


NLTGYLLQYQ IINDTYEIGE LNDINITTPS KPSWHLSNLN ATTKYKFYLR ACTSQGCGKP
1020


 


ITEESSTLGE GKYAGLYDDI STQGWFIGLM CAIALLTLLL LTVCFVKRNR GGKYSVKEKE
1080


 


DLHPDPEIQS VKDETFGEYS DSDEKPLKGS LRSLNRDMQP TESADSLVEY GEGDHGLFSE
1140


 


DGSFIGAYAG SKEKGSVESN GSSTATFPLR A
1171
















TABLE LVa





Amino acid sequence alignment of 282P1G03 v.1 (SEQ ID NO:155)


and 282P1G03 v.2 (SEQ ID NO:156)





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE LIIb





Nucleotide sequence of transcript variant 282P1G03 v.3
















cggaccctgc gcgcccccgt cccggctccc ggccggctcg ggggagaagg cgcccgaggg
60


 


gaggcgccgg acagatcgcg tttcggaggc ggcgcaggtg ctgtaaactg caaaccataa
120


 


tcctgtctta atactgcaaa caaatcatag tggaactaag gggaacttaa tttactgttt
180


 


ccaggttaac taaggtctca gctgtaaacc aaaagtgaga ggagacatta agattttcat
240


 


tcttaccggg ttgtcttctt cctgaagagc aatggagccg cttttacttg gaagaggact
300


 


aatcgtatat ctaatgttcc tcctgttaaa attctcaaaa gcaattgaaa taccatcttc
360


 


agttcaacag gttccaacaa tcataaaaca gtcaaaagtc caagttgcct ttcccttcga
420


 


tgagtatttt caaattgaat gtgaagctaa aggaaatcca gaaccaacat tttcgtggac
480


 


taaggatggc aacccttttt atttcactga ccatcggata attccatcga acaattcagg
540


 


aacattcagg atcccaaacg aggggcacat atctcacttt caagggaaat accgctgctt
600


 


tgcttcaaat aaactgggaa tcgctatgtc agaagaaata gaatttatag ttccaagtgt
660


 


tccaaaactc ccaaaagaaa aaattgaccc tcttgaagtg gaggagggag atccaattgt
720


 


cctcccatgc aatcctccca aaggcctccc acctttacac atttattgga tgaatattga
780


 


attagaacac atcgaacaag atgaaagagt atacatgagc caaaagggag atctatactt
840


 


cgcaaacgtg gaagaaaagg acagtcgcaa tgactactgt tgctttgctg catttccaag
900


 


attaaggact attgtacaga aaatgccaat gaaactaaca gttaacagtt taaagcatgc
960


 


taatgactca agttcatcca cagaaattgg ttccaaggca aattccatca agcaaagaaa
1020


 


acccaaactg ctgttgcctc ccactgagag tggcagtgag tcttcaatta ccatcctcaa
1080


 


aggggaaatc ttgctgcttg agtgttttgc tgaaggcttg ccaactccac aggttgattg
1140


 


gaacaaaatt ggtggtgact taccaaaggg gagagaaaca aaagaaaatt atggcaagac
1200


 


tttgaagata gagaatgtct cctaccagga caaaggaaat tatcgctgca cagccagcaa
1260


 


tttcttggga acagccactc acgattttca cgttatagta gaagagcctc ctcgctggac
1320


 


aaagaagcct cagagtgctg tgtatagcac cggaagcaat ggcatcttgt tatgtgaggc
1380


 


tgaaggagaa cctcaaccca caatcaagtg gagagtcaat ggctccccag ttgacaatca
1440


 


tccatttgct ggtgatgttg tcttccccag ggaaatcagt tttaccaacc ttcaaccaaa
1500


 


tcatactgct gtgtaccagt gtgaagcctc aaatgtccat ggaactatcc ttgccaatgc
1560


 


caatattgat gttgtggatg tccgtccatt gatacaaacc aaagatggag aaaattacgc
1620


 


tacagtggtt gggtacagtg ctttcttaca ttgcgagttc tttgcttcac ctgaggcagt
1680


 


cgtgtcctgg cagaaggtgg aagaagtgaa acccctggag ggcaggcggt atcatatcta
1740


 


tgaaaatggc acattgcaga tcaacagaac caccgaagaa gatgctgggt cttactcatg
1800


 


ttgggtagaa aatgctatag gaaaaactgc agtcacagcc aatttggata ttagaaatgc
1860


 


tacaaaactt agagtttctc ctaagaatcc tcgtatcccc aaattgcata tgcttgaatt
1920


 


acattgtgaa agcaaatgtg actcacattt gaaacacagt ttgaagttgt cctggagtaa
1980


 


agatggagaa gcctttgaaa ttaatggcac agaagatggc aggataatta ttgatggagc
2040


 


taatttgacc atatctaatg taactttaga ggaccaaggt atttactgct gttcagctca
2100


 


tactgctcta gacagtgctg ccgatataac tcaagtaact gttcttgatg ttccggatcc
2160


 


accagaaaac cttcacttgt ctgaaagaca gaacaggagt gttcggctga cctgggaagc
2220


 


tggagctgac cacaacagca atattagcga gtatattgtt gaatttgaag gaaacaaaga
2280


 


agagcctgga aggtgggagg aactgaccag agtccaagga aagaaaacca cagttatctt
2340


 


acctttggct ccatttgtga gataccagtt cagggtcata gccgtgaacg aagtagggag
2400


 


aagtcagcct agccagccgt cagaccatca tgaaacacca ccagcagctc cagataggaa
2460


 


tccacaaaac ataagggttc aagcctctca acccaaggaa atgattataa agtgggagcc
2520


 


tttgaaatcc atggagcaga atggaccagg cctagagtac agagtgacct ggaagccaca
2580


 


gggagcccca gtggagtggg aagaagaaac agtcacaaac cacacattgc gggtgatgac
2640


 


gcctgctgtc tatgcccctt atgatgtcaa ggtccaggct atcaatcaac taggatctgg
2700


 


gcctgaccct cagtcagtga ctctctattc tggagaagac tatcctgata cagctccagt
2760


 


gatccatggg gtggacgtta taaacacaac atatgtaagc aacgctactg gttcacccca
2820


 


accttccata tttatctgtt caaaggagca agaactttca tataggaata gaaacatgct
2880


 


ggccgaagat ttcatccaga agtcaacatc ctgcaattat gttgaaaaga gtagtacttt
2940


 


cttcaaaata taaaatgcca agcacttcag gcctatgttt tgcttatatt gttttcaggt
3000


 


gctcaaaatg caaaacacaa aacaaatcct gcatttagat acacctcaac taaatccaaa
3060


 


gtccccattc agtatattcc atatttgcct gattttacta ttcggtgtgt ttgcatagat
3120


 


gttgctactt ggtgggtttt tctccgtatg cacattggta tacagtctct gagaactggc
3180


 


ttggtgactt tgcttcacta caggttaaaa gaccataagc aaactggtta tttaaaatgt
3240


 


aaaaaggaat atgaaagtct tattaaaaca cttcattgaa aatatacagt ctaaatttat
3300


 


tatttaaatt ttactagcaa aagtcttagg tgaacaatca actagtattt gttgagctcc
3360


 


tatttgccca gagatggtca tatttaaaca gaagtatacg tttttcagtt tcaacatgaa
3420


 


tttttttatt tctgtcagtt atgacatcca cgagcatcac tttttgtgtc tgtttttttt
3480


 


tttttcttgg actaaattca actgcatgga agcggtggtc agaaggttgt tttatacgag
3540


 


aacaggcaga aagtgcccat tgttcaggat tctaatagct acatctactt aatatcttca
3600


 


tttctaaatt gactgctttt acctttttct catgtttata taatggtatg cttgcatata
3660


 


tttcatgaat acattgtaca tattatgtta atatttacac aatttaaaat atagatgtgt
3720


 


tttattttga agtgagaaaa tgaacattaa caggcatgtt tgtacagcta gaatatatta
3780


 


gtaagatact gtttttcgtc attccagagc tacaactaat aacacgaggt tccaaagctg
3840


 


aagactttgt ataaagtatt tgggttttgt tcttgtattg ctttctttca acagtttcaa
3900


 


aataaaatat catacaaata ttgagggaaa tgttttcata tttttcaaaa taggttttta
3960


 


ttgttgaatg tacatctacc ccagcccctc aaaagaaaaa ctgtttacat agaaattcct
4020


 


acacatacgt ttgcgtatat gttattttaa acatctttgt ggtgagaatt ttttccccga
4080


 


tattctcctt ctgtcaaagt cagaacaaat tcagggaatt tattttctgg cagttgtgct
4140


 


ccagtccttt taaaattgta catgaacatg ttttagaaac aatatggagg atgatgcata
4200


 


catgtcggtc aagttcagcg ctcgacattt tatggaaaga tttttttaac cttaccacga
4260


 


aatacttaac tactgtttaa gtgaattgac ttatttcact ttagtttttg aactgtgatt
4320


 


attggtatac tgttatatcc tcaacttgga tttatggtaa ccccttttag ttcatggaga
4380


 


ccaaaatttg gggtatttat aatagtcagc gcaggaatgc acatggaata tctacttgtc
4440


 


cttttgaacc tcacgagtca tccagaatgt atagacagga aaagcatgtc ttatttaaaa
4500


 


ctgtaattta tgggctcagg atctgaccgc agtcccggga gtaagcattt caaaggggga
4560


 


aggcagtgtg gtccctaccc tgtgtgaatg tgaggatgta gacatccatc agtgcaactc
4620


 


gagctccatc ctcctccgat ttctaaggct ccagttttct ggagggacag tcatcatgtt
4680


 


ttgatttatc tgggagaaaa ctgtggtgca cagcttgtga ggagggcaag gttgtgacgt
4740


 


tcgagcttag ttctggtgtt attctgtctc ctcttctttg tcatcagcca aaacgtggtt
4800


 


tttaaagaga gtcatgcagg ttagaaataa tgtcaaaaat atttaggaat ttaataacct
4860


 


ttaagtcaga aactaaaaca aatactgaaa tattagctct tcctacactt cgtgttcccc
4920


 


tttagctgcc tgaaaatcaa gattgctcct actcagatct tctgagtggc taaaacttat
4980


 


ggatatgaaa aatgagattg aatgatgact atgctttgct atcattgtta cctttcctca
5040


 


atactatttg gcaactactg ggactcttca gcacaaaagg aatagatcta tgattgaccc
5100


 


tgattttaat tgtgaaatta tatgattcat atattttatg aatcagaata accttcaaat
5160


 


aaaataaatc taagtcggtt aaaatggatt tcatgatttt ccctcagaaa atgagtaacg
5220


 


gagtccacgg cgtgcaatgg taattataaa ttggtgatgc ttgtttgcaa attgcccact
5280


 


cgtgataagt caacagccaa tatttaaaac tttgttcgtt actggcttta ccctaacttt
5340


 


ctctagtcta ctgtcaatat cattttaatg taattgattg tatatagtct caagaatggt
5400


 


tggtgggcat gagttcctag agaactgtcc aagggttggg aaaatccaaa ttctcttcct
5460


 


ggctccagca ctgattttgt acataaacat taggcaggtt gcttaacctt tttatttcaa
5520


 


actctctcaa ctctaaagtg ctaataataa tctcagttac cttatctttg tcacagggtg
5580


 


ttctttttta tgaagaaaaa tttgaaaatg ataaaagcta agatgccttc taacttcata
5640


 


agcaaacctt taactaatta tgtatctgaa agtcaccccc acataccaac tcaacttttt
5700


 


tcctgtgaac acataaatat atttttatag aaaaacaaat ctacataaaa taaatctact
5760


 


gtttagtgag cagtatgact tgtacatgcc attgaaaatt attaatcaga agaaaattaa
5820


 


gcagggtctt tgctatacaa aagtgttttc cactaatttt gcatgcgtat ttataagaaa
5880


 


aatgtgaatt tggtggtttt attctatcgg tataaaggca tcgatatttt agatgcaccc
5940


 


gtgtttgtaa aaatgtagag cacaatggaa ttatgctgga agtctcaaat aatatttttt
6000


 


tcctatttta tactcatgga agagataagc taaagagggg acaataatga gaaatgttgg
6060


 


tgtgcttttc taagcattta aaacataatt gccaattgaa accctaaata tgtttacata
6120


 


ccattaagat atgattcatg taacaatgtt aaattaatta taatgggatt gggtttgtta
6180


 


tctgtggtag tatatatcct agtgttccta tagtgaaata agtagggttc agccaaagct
6240


 


ttctttgttt tgtaccttaa attgttcgat tacgtcatca aaagagatga aaggtatgta
6300


 


gaacaggttc acgtgattac ctttttcttt tggcttggat taatattcat agtagaactt
6360


 


tataaaacgt gtttgtattg taggtggtgt ttgtattatg cttatgacta tgtatggttt
6420


 


gaaaatattt tcattataca tgaaattcaa ctttccaaat aaaagttcta cttcatgtaa
6480


 


tccaaaa
6487
















TABLE LIIIb





Nucleotide sequence alignment of 282P1G03 v.1


(SEQ ID NO: 158) and 282P1G03 v.3 (SEQ ID NO: 159)





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE LIVb





Peptide seguences of protein coded by 282P1G03 v.3 (SEQ ID NO: 160)















MEPLLLGRGL IVYLMFLLLK FSKAIEIPSS VQQVPTIIKQ SKVQVAFPFD EYFQIECEAK  60


 


GNPEPTFSWT KDGNPFYFTD HRIIPSNNSG TFRIPNEGHI SHFQGKYRCF ASNKLGIANS 120


 


EEIEFIVPSV PKLPKEKIDP LEVEEGDPIV LPCNPPKGLP PLHIYWMNIE LEHIEQDERV 180


 


YMSQKGDLYF ANVEEKDSRN DYCCFAAFPR LRTIVQKMPM KLTVNSLKHA NDSSSSTEIG 240


 


SKANSIKQRK PKLLLPPTES GSESSITILK GEILLLECFA EGLPTPQVDW NKIGGDLPKG 300


 


RETKENYGKT LKIENVSYQD KGNYRCTASN FLGTATHDFH VIVEEPPRWT KKPQSAVYST 360


 


GSNGILLCEA EGEPQPTIKW RVNGSPVDNH PFAGDVVFPR EISFTNLQPN HTAVYQCEAS 420


 


NVHGTILANA NIDVVDVRPL IQTKDGENYA TVVGYSAFLH CEFFASPEAV VSWQKVEEVK 480


 


PLEGRRYHIY ENGTLQINRT TEEDAGSYSC WVENAIGKTA VTANLDIRNA TKLRVSPKNP 540


 


RIPKLHMLEL HCESKCDSHL KHSLKLSWSK DGEAFEINGT EDGRIIIDGA NLTISNVTLE 600


 


DQGIYCCSAH TALDSAADIT QVTVLDVPDP PENLHLSERQ NRSVRLTWEA GADHNSNISE 660


 


YIVEFEGNKE EPGRWEELTR VQGKKTTVIL PLAPFVRYQF RVIAVNEVGR SQPSQPSDHH 720


 


ETPPAAPDRN PQNIRVQASQ PKEMIIKWEP LKSMEQNGPG LEYRVTWKPQ GAPVEWEEET 780


 


VTNHTLRVMT PAVYAPYDVK VQAINQLGSG PDPQSVTLYS GEDYPDTAPV IHGVDVINTT 840


 


YVSNATGSPQ PSIFICSKEQ ELSYRNRNML AEDFIQKSTS CNYVEKSSTF FKI        893













TABLE LVb





Amino acid sequence alignment of 282P1G03 v.1


(SEQ ID NO: 161) and 282P1G03 v.3 (SEQ ID NO: 162)





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE LIIc





Nucleotide sequence of transcript variant 282P1G03 v.4 (SEQ ID NO: 163)















cggaccctgc gcgcccccgt cccggctccc ggccggctcg ggggagaagg cgcccgaggg   60


 


gaggcgccgg acagatcgcg tttcggaggc ggcgcaggtg ctgtaaactg caaaccataa  120


 


tcctgtctta atactgcaaa caaatcatag tggaactaag gggaacttaa tttactgttt  180


 


ccaggttaac taaggtctca gctgtaaacc aaaagtgaga ggagacatta agattttcat  240


 


tcttaccggg ttgtcttctt cctgaagagc aatggagccg cttttacttg gaagaggact  300


 


aatcgtatat ctaatgttcc tcctgttaaa attctcaaaa gcaattgaaa taccatcttc  360


 


agttcaacag gttccaacaa tcataaaaca gtcaaaagtc caagttgcct ttcccttcga  420


 


tgagtatttt caaattgaat gtgaagctaa aggaaatcca gaaccaacat tttcgtggac  480


 


taaggatggc aacccttttt atttcactga ccatcggata attccatcga acaattcagg  540


 


aacattcagg atcccaaacg aggggcacat atctcacttt caagggaaat accgctgctt  600


 


tgcttcaaat aaactgggaa tcgctatgtc agaagaaata gaatttatag ttccaagtgt  660


 


tccaaaactc ccaaaagaaa aaattgaccc tcttgaagtg gaggagggag atccaattgt  720


 


cctcccatgc aatcctccca aaggcctccc acctttacac atttattgga tgaatattga  780


 


attagaacac atcgaacaag atgaaagagt atacatgagc caaaagggag atctatactt  840


 


cgcaaacgtg gaagaaaagg acagtcgcaa tgactactgt tgctttgctg catttccaag  900


 


attaaggact attgtacaga aaatgccaat gaaactaaca gttaacagtt taaagcatgc  960


 


taatgactca agttcatcca cagaaattgg ttccaaggca aattccatca agcaaagaaa 1020


 


acccaaactg ctgttgcctc ccactgagag tggcagtgag tcttcaatta ccatcctcaa 1080


 


aggggaaatc ttgctgcttg agtgttttgc tgaaggcttg ccaactccac aggttgattg 1140


 


gaacaaaatt ggtggtgact taccaaaggg gagagaaaca aaagaaaatt atggcaagac 1200


 


tttgaagata gagaatgtct cctaccagga caaaggaaat tatcgctgca cagccagcaa 1260


 


tttcttggga acagccactc acgattttca cgttatagta gaagagcctc ctcgctggac 1320


 


aaagaagcct cagagtgctg tgtatagcac cggaagcaat ggcatcttgt tatgtgaggc 1380


 


tgaaggagaa cctcaaccca caatcaagtg gagagtcaat ggctccccag ttgacaatca 1440


 


tccatttgct ggtgatgttg tcttccccag ggaaatcagt tttaccaacc ttcaaccaaa 1500


 


tcatactgct gtgtaccagt gtgaagcctc aaatgtccat ggaactatcc ttgccaatgc 1560


 


caatattgat gttgtggatg tccgtccatt gatacaaacc aaagatggag aaaattacgc 1620


 


tacagtggtt gggtacagtg ctttcttaca ttgcgagttc tttgcttcac ctgaggcagt 1680


 


cgtgtcctgg cagaaggtgg aagaagtgaa acccctggag ggcaggcggt atcatatcta 1740


 


tgaaaatggc acattgcaga tcaacagaac caccgaagaa gatgctgggt cttactcatg 1800


 


ttgggtagaa aatgctatag gaaaaactgc agtcacagcc aatttggata ttagaaatgc 1860


 


tacaaaactt agagtttctc ctaagaatcc tcgtatcccc aaattgcata tgcttgaatt 1920


 


acattgtgaa agcaaatgtg actcacattt gaaacacagt ttgaagttgt cctggagtaa 1980


 


agatggagaa gcctttgaaa ttaatggcac agaagatggc aggataatta ttgatggagc 2040


 


taatttgacc atatctaatg taactttaga ggaccaaggt atttactgct gttcagctca 2100


 


tactgctcta gacagtgctg ccgatataac tcaagtaact gttcttgatg ttccggatcc 2160


 


accagaaaac cttcacttgt ctgaaagaca gaacaggagt gttcggctga cctgggaagc 2220


 


tggagctgac cacaacagca atattagcga gtatattgtt gaatttgaag gaaacaaaga 2280


 


agagcctgga aggtgggagg aactgaccag agtccaagga aagaaaacca cagttatctt 2340


 


acctttggct ccatttgtga gataccagtt cagggtcata gccgtgaacg aagtagggag 2400


 


aagtcagcct agccagccgt cagaccatca tgaaacacca ccagcagctc cagataggaa 2460


 


tccacaaaac ataagggttc aagcctctca acccaaggaa atgattataa agtgggagcc 2520


 


tttgaaatcc atggagcaga atggaccagg cctagagtac agagtgacct ggaagccaca 2580


 


gggagcccca gtggagtggg aagaagaaac agtcacaaac cacacattgc gggtgatgac 2640


 


gcctgctgtc tatgcccctt atgatgtcaa ggtccaggct atcaatcaac taggatctgg 2700


 


gcctgaccct cagtcagtga ctctctattc tggagaagac ttacctgaac agccaacttt 2760


 


tctaaaggtc atcaaagttg ataaagacac tgccacttta tcttggggac tacctaagaa 2820


 


attaaatgga aacttaactg gctatctttt gcaatatcag ataataaatg acacctacga 2880


 


gattggagaa ttaaatgata ttaacattac aactccatca aagcccagct ggcacctctc 2940


 


aaacctgaat gcaactacca agtacaaatt ctacttgagg gcttgcactt cacagggctg 3000


 


tggaaaaccg atcacggagg aaagctccac cttaggagaa gggagtaaag gtatcgggaa 3060


 


gatatcagga gtaaatctta ctcaaaagac tcacccaata gaggtatttg agccgggagc 3120


 


tgaacatata gttcgcctaa tgactaagaa ttggggcgat aacgatagca tttttcaaga 3180


 


tgtaattgag acaagaggga gagaatatgc tggtttatat gatgacatct ccactcaagg 3240


 


ctggtttatt ggactgatgt gtgcgattgc tcttctcaca ctactattat taactgtttg 3300


 


ctttgtgaag aggaatagag gtggaaagta ctcagttaaa gaaaaggaag atttgcatcc 3360


 


agacccagaa attcagtcag taaaagatga aacctttggt gaatacagtg acagtgatga 3420


 


aaagcctctc aaaggaagcc ttcggtccct taatagggat atgcagccta ctgaaagtgc 3480


 


tgacagctta gtcgaatacg gagagggaga ccatggtctc ttcagtgaag atggatcatt 3540


 


tattggtgcc tacgctggat ctaaggagaa gggatctgtt gaaagcaatg gaagttctac 3600


 


agcaactttt ccccttcggg cataaacaca acatatgtaa gcaacgctac tggttcaccc 3660


 


caaccttcca tatttatctg ttcaaaggag caagaacttt catataggaa tagaaacatg 3720


 


ctggccgaag atttcatcca gaagtcaaca tcctgcaatt atgttgaaaa gagtagtact 3780


 


ttcttcaaaa tataaaatgc caagcacttc aggcctatgt tttgcttata ttgttttcag 3840


 


gtgctcaaaa tgcaaaacac aaaacaaatc ctgcatttag atacacctca actaaatcca 3900


 


aagtccccat tcagtatatt ccatatttgc ctgattttac tattcggtgt gtttgcatag 3960


 


atgttgctac ttggtgggtt tttctccgta tgcacattgg tatacagtct ctgagaactg 4020


 


gcttggtgac tttgcttcac tacaggttaa aagaccataa gcaaactggt tatttaaaat 4080


 


gtaaaaagga atatgaaagt cttattaaaa cacttcattg aaaatataca gtctaaattt 4140


 


attatttaaa ttttactagc aaaagtctta ggtgaacaat caactagtat ttgttgagct 4200


 


cctatttgcc cagagatggt catatttaaa cagaagtata cgtttttcag tttcaacatg 4260


 


aattttttta tttctgtcag ttatgacatc cacgagcatc actttttgtg tctgtttttt 4320


 


tttttttctt ggactaaatt caactgcatg gaagcggtgg tcagaaggtt gttttatacg 4380


 


agaacaggca gaaagtgccc attgttcagg attctaatag ctacatctac ttaatatctt 4440


 


catttctaaa ttgactgctt ttaccttttt ctcatgttta tataatggta tgcttgcata 4500


 


tatttcatga atacattgta catattatgt taatatttac acaatttaaa atatagatgt 4560


 


gttttatttt gaagtgagaa aatgaacatt aacaggcatg tttgtacagc tagaatatat 4620


 


tagtaagata ctgtttttcg tcattccaga gctacaacta ataacacgag gttccaaagc 4680


 


tgaagacttt gtataaagta tttgggtttt gttcttgtat tgctttcttt caacagtttc 4740


 


aaaataaaat atcatacaaa tattgaggga aatgttttca tatttttcaa aataggtttt 4800


 


tattgttgaa tgtacatcta ccccagcccc tcaaaagaaa aactgtttac atagaaattc 4860


 


ctacacatac gtttgcgtat atgttatttt aaacatcttt gtggtgagaa ttttttcccc 4920


 


gatattctcc ttctgtcaaa gtcagaacaa attcagggaa tttattttct ggcagttgtg 4980


 


ctccagtcct tttaaaattg tacatgaaca tgttttagaa acaatatgga ggatgatgca 5040


 


tacatgtcgg tcaagttcag cgctcgacat tttatggaaa gattttttta accttaccac 5100


 


gaaatactta actactgttt aagtgaattg acttatttca ctttagtttt tgaactgtga 5160


 


ttattggtat actgttatat cctcaacttg gatttatggt aacccctttt agttcatgga 5220


 


gaccaaaatt tggggtattt ataatagtca gcgcaggaat gcacatggaa tatctacttg 5280


 


tccttttgaa cctcacgagt catccagaat gtatagacag gaaaagcatg tcttatttaa 5340


 


aactgtaatt tatgggctca ggatctgacc gcagtcccgg gagtaagcat ttcaaagggg 5400


 


gaaggcagtg tggtccctac cctgtgtgaa tgtgaggatg tagacatcca tcagtgcaac 5460


 


tcgagctcca tcctcctccg atttctaagg ctccagtttt ctggagggac agtcatcatg 5520


 


ttttgattta tctgggagaa aactgtggtg cacagcttgt gaggagggca aggttgtgac 5580


 


gttcgagctt agttctggtg ttattctgtc tcctcttctt tgtcatcagc caaaacgtgg 5640


 


tttttaaaga gagtcatgca ggttagaaat aatgtcaaaa atatttagga atttaataac 5700


 


ctttaagtca gaaactaaaa caaatactga aatattagct cttcctacac ttcgtgttcc 5760


 


cctttagctg cctgaaaatc aagattgctc ctactcagat cttctgagtg gctaaaactt 5820


 


atggatatga aaaatgagat tgaatgatga ctatgctttg ctatcattgt tacctttcct 5880


 


caatactatt tggcaactac tgggactctt cagcacaaaa ggaatagatc tatgattgac 5940


 


cctgatttta attgtgaaat tatatgattc atatatttta tgaatcagaa taaccttcaa 6000


 


ataaaataaa tctaagtcgg ttaaaatgga tttcatgatt ttccctcaga aaatgagtaa 6060


 


cggagtccac ggcgtgcaat ggtaattata aattggtgat gcttgtttgc aaattgccca 6120


 


ctcgtgataa gtcaacagcc aatatttaaa actttgttcg ttactggctt taccctaact 6180


 


ttctctagtc tactgtcaat atcattttaa tgtaattgat tgtatatagt ctcaagaatg 6240


 


gttggtgggc atgagttcct agagaactgt ccaagggttg ggaaaatcca aattctcttc 6300


 


ctggctccag cactgatttt gtacataaac attaggcagg ttgcttaacc tttttatttc 6360


 


aaactctctc aactctaaag tgctaataat aatctcagtt accttatctt tgtcacaggg 6420


 


tgttcttttt tatgaagaaa aatttgaaaa tgataaaagc taagatgcct tctaacttca 6480


 


taagcaaacc tttaactaat tatgtatctg aaagtcaccc ccacatacca actcaacttt 6540


 


tttcctgtga acacataaat atatttttat agaaaaacaa atctacataa aataaatcta 6600


 


ctgtttagtg agcagtatga cttgtacatg ccattgaaaa ttattaatca gaagaaaatt 6660


 


aagcagggtc tttgctatac aaaagtgttt tccactaatt ttgcatgcgt atttataaga 6720


 


aaaatgtgaa tttggtggtt ttattctatc ggtataaagg catcgatatt ttagatgcac 6780


 


ccgtgtttgt aaaaatgtag agcacaatgg aattatgctg gaagtctcaa ataatatttt 6840


 


tttcctattt tatactcatg gaagagataa gctaaagagg ggacaataat gagaaatgtt 6900


 


ggtgtgcttt tctaagcatt taaaacataa ttgccaattg aaaccctaaa tatgtttaca 6960


 


taccattaag atatgattca tgtaacaatg ttaaattaat tataatggga ttgggtttgt 7020


 


tatctgtggt agtatatatc ctagtgttcc tatagtgaaa taagtagggt tcagccaaag 7080


 


ctttctttgt tttgtacctt aaattgttcg attacgtcat caaaagagat gaaaggtatg 7140


 


tagaacaggt tcacgtgatt acctttttct tttggcttgg attaatattc atagtagaac 7200


 


tttataaaac gtgtttgtat tgtaggtggt gtttgtatta tgcttatgac tatgtatggt 7260


 


ttgaaaatat tttcattata catgaaattc aactttccaa ataaaagttc tacttcatgt 7320


 


aatccaaaa                                                         7329













TABLE LIIIc





Nucleotide sequence alignment of 282P1G03 v.1


(SEQ ID NO: 164) and 282P1G03 v.4 (SEQ ID NO: 165)





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE LIVc





Peptide sequences of protein coded by 28P1G03 v.4 (SEQ ID NO: 166)















MEPLLLGRGL IVYLMFLLLK FSKAIEIPSS VQQVPTIIKQ SKVQVAFPFD EYFQIECEAK   60


 


GNPEPTFSWT KDGNPFYFTD HRIIPSNNSG TFRIPNEGHI SHFQGKYRCF ASNKLGIAMS  120


 


EEIEFIVPSV PKLPKEKIDP LEVEEGDPIV LPCNPPKGLP PLHIYWMNIE LEHIEQDERV  180


 


YMSQKGDLYF ANVEEKDSRN DYCCFAAFPR LRTIVQKMPM KLTVNSLKHA NDSSSSTEIG  240


 


SKANSIKQRK PKLLLPPTES GSESSITILK GEILLLECFA EGLPTPQVOW NKIGGDLPKG  300


 


RETKENYGKT LKIENVSYQD KGNYRCTASN FLGTATHDFH VIVEEPPRWT KKPQSAVYST  360


 


GSNGILLCEA EGEPQPTIKW RVNGSPVDNH PFAGDVVFPR EISFTNLQPN HTAVYQCEAS  420


 


NVHGTILANA NIDVVDVRPL IQTKDGENYA TVVGYSAFLH CEFFASPEAV VSWQKVEEVK  480


 


PLEGRRYHIY ENGTLQINRT TEEDAGSYSC WVENAIGKTA VTANLDIRNA TKLRVSPKNP  540


 


RIPKLHMLEL HCESKCDSHL KHSLKLSWSK DGEAFEINGT EDGRIIIDGA NLTISNVTLE  600


 


DQGIYCCSAH TALDSAADIT QVTVLDVPDP PENLHLSERQ NRSVRLTWEA GADHNSNISE  660


 


YIVEFEGNKE EPGRWEELTR VQGKKTTVIL PLAPFVRYQF RVIAVNEVGR SQPSQPSDHH  720


 


ETPPAAPDRN PQNIRVQASQ PKEMIIKWEP LKSMEQNGPG LEYRVTWKPQ GAPVEWEEET  780


 


VTNHTLRVMT PAVYAPYDVK VQAINQLGSG PDPQSVTLYS GEDLPEQPTF LKVIKVDKDT  840


 


ATLSWGLPKK LNGNLTGYLL QYQIINDTYE IGELNDINIT TPSKPSWHLS NLNATTKYKF  900


 


YLRACTSQGC GKPITEESST LGEGSKGIGK ISGVNLTQKT HPIEVFEPGA EHIVRLMTKN  960


 


WGDNDSIFQD VIETRGREYA GLYDDISTQG WFIGLMCAIA LLTLLLLTVC FVKRNRGGKY 1020


 


SVKEKEDLHP DPEIQSVKDE TFGEYSDSDE KPLKGSLRSL NRDMQPTESA DSLVEYGEGD 1080


 


HGLFSEDGSF IGAYAGSKEK GSVESNGSST ATFPLRA                          1117













TABLE LVc





Amino acid sequence alignment of 282P1G03 v.1 (SEQ ID NO:167)


and 282P1G03 v.4 (SEQ ID NO:168)





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE LIId





Nucleotide sequence of transcript variant 282P1G03 v.5 (SEQ ID NO: 169)















cggaccctgc gcgcccccgt cccggctccc ggccggctcg ggggagaagg cgcccgaggg   60


 


gaggcgccgg acagatcgcg tttcggaggc ggcgcaggtg ctgtaaactg caaaccataa  120


 


tcctgtctta atactgcaaa caaatcatag tggaactaag gggaacttaa tttactgttt  180


 


ccaggttaac taaggtctca gctgtaaacc aaaagtgaga ggagacatta agattttcat  240


 


tcttaccggg ttgtcttctt cctgaagagc aatggagccg cttttacttg gaagaggact  300


 


aatcgtatat ctaatgttcc tcctgttaaa attctcaaaa gcaattgaaa taccatcttc  360


 


agttcaacag gttccaacaa tcataaaaca gtcaaaagtc caagttgcct ttcccttcga  420


 


tgagtatttt caaattgaat gtgaagctaa aggaaatcca gaaccaacat tttcgtggac  480


 


taaggatggc aacccttttt atttcactga ccatcggata attccatcga acaattcagg  540


 


aacattcagg atcccaaacg aggggcacat atctcacttt caagggaaat accgctgctt  600


 


tgcttcaaat aaactgggaa tcgctatgtc agaagaaata gaatttatag ttccaagtgt  660


 


tccaaaactc ccaaaagaaa aaattgaccc tcttgaagtg gaggagggag atccaattgt  720


 


cctcccatgc aatcctccca aaggcctccc acctttacac atttattgga tgaatattga  780


 


attagaacac atcgaacaag atgaaagagt atacatgagc caaaagggag atctatactt  840


 


cgcaaacgtg gaagaaaagg acagtcgcaa tgcatcatgt tgctttgctg catttccaag  900


 


attaaggact attgtacaga aaatgccaat gaaactaaca gttaacagtt caaattccat  960


 


caagcaaaga aaacccaaac tgctgttgcc tcccactgag agtggcagtg agtcttcaat 1020


 


taccatcctc aaaggggaaa tcttgctgct tgagtgtttt gctgaaggct tgccaactcc 1080


 


acaggttgat tggaacaaaa ttggtggtga cttaccaaag gggagagaaa caaaagaaaa 1140


 


ttatggcaag actttgaaga tagagaatgt ctcctaccag gacaaaggaa attatcgctg 1200


 


cacagccagc aatttcttgg gaacagccac tcacgatttt cacgttatag tagaagagcc 1260


 


tcctcgctgg acaaagaagc ctcagagtgc tgtgtatagc accggaagca atggcatctt 1320


 


gttatgtgag gctgaaggag aacctcaacc cacaatcaag tggagagtca atggctcccc 1380


 


agttgacaat catccatttg atggtgatgt tgtcttcccc agggaaatca gttttaccaa 1440


 


ccttcaacca aatcatactg cagtgtacca gtgtgaagcc tcaaatgtcc atggaactat 1500


 


ccttgccaat gccaatattg atgttgtgga tgtccgtcca ttgatacaaa ccaaagatgg 1560


 


agaaaattac gctacagtgg ttgggtacag tgctttctta cattgcgagt tctttgcttc 1620


 


acctgaggca gtcgtgtcct ggcagaaggt ggaagaagtg aaacccctgg agggcaggcg 1680


 


gtatcatatc tatgaaaatg gcacattgca gatcaacaga accaccgaag aagatgctgg 1740


 


gtcttactcg tgttgggtag aaaatgctat aggaaaaact gcagtcacag ccaatttgga 1800


 


tattagaaat gctacaaaac ttagagtttc tcctaagaat cctcgtatcc ccaaattgca 1860


 


tatgcttgaa ttacattgtg aaagcaaatg tgactcacat ttgaaacaca gtttgaagtt 1920


 


gtcctggagt aaagatggag aagcctttga aattaatggc acagaagatg gcaggataat 1980


 


tattgatgga gctaatttga ccatatctaa tgtaacttta gaggaccaag gtatttactg 2040


 


ctgttcagct catactgctc tagacagtgc tgccgatata actcaagtaa ctgttcttga 2100


 


tgttccggat ccaccagaaa accttcactt gtctgaaaga cagaacagga gtgttcggct 2160


 


gacctgggaa gctggagctg accacaacag caatattagc gagtatattg ttgaatttga 2220


 


aggaaacaaa gaagagcctg gaaggtggga ggaactgacc agagtccaag gaaagaaaac 2280


 


cacagttatc ttacctttgg ctccatttgt gagataccag ttcagggtca tagccgtgaa 2340


 


cgaagtaggg agaagtcagc ctagccagcc gtcagaccat catgaaacac caccagcagc 2400


 


tccagatagg aatccacaaa acataagggt tcaagcctct caacccaagg aaatgattat 2460


 


aaagtgggag cctttgaaat ccatggagca gaatggacca ggcctagagt acagagtgac 2520


 


ctggaagcca cagggagccc cagtggagtg ggaagaagaa acagtcacaa accacacatt 2580


 


gcgggtgatg acgcctgctg tctatgcccc ttatgatgtc aaggtccagg ctatcaatca 2640


 


actaggatct gggcctgacc ctcagtcagt gactctctat tctggagaag actatcctga 2700


 


tacagctcca gtgatccatg gggtggacgt tataaacagt acattagtta aagttacctg 2760


 


gtcaacagtt ccaaaggaca gagtacatgg acgtctgaaa ggctatcaga taaattggtg 2820


 


gaaaacaaaa agtctgttgg atggaagaac acatcccaaa gaagtgaaca ttctaagatt 2880


 


ttcaggacaa agaaactctg gaatggttcc ttccttagat gcctttagtg aatttcattt 2940


 


aacagtctta gcctataact ctaaaggagc tggtcctgaa agtgagcctt atatatttca 3000


 


aacaccagaa ggagtacctg aacagccaac ttttctaaag gtcatcaaag ttgataaaga 3060


 


cactgccact ttatcttggg gactacctaa gaaattaaat ggaaacttaa ctggctatct 3120


 


tttgcaatat cagataataa atgacaccta cgagattgga gaattaaatg atattaacat 3180


 


tacaactcca tcaaagccca gctggcacct ctcaaacctg aatgcaacta ccaagtacaa 3240


 


attctacttg agggcttgca cttcacaggg ctgtggaaaa ccgatcacgg aggaaagctc 3300


 


caccttagga gaagggagta aaggtatcgg gaagatatca ggagtaaatc ttactcaaaa 3360


 


gactcaccca atagaggtat ttgagccggg agctgaacat atagttcgcc taatgactaa 3420


 


gaattggggc gataacgata gcatttttca agatgtaatt gagacaagag ggagagaata 3480


 


tgctggttta tatgatgaca tctccactca aggctggttt attggactga tgtgtgcgat 3540


 


tgctcttctc acactactat tattaactgt ttgctttgtg aagaggaata gaggtggaaa 3600


 


gtactcagtt aaagaaaagg aagatttgca tccagaccca gaaattcagt cagtaaaaga 3660


 


tgaaaccttt ggtgaataca gtgacagtga tgaaaagcct ctcaaaggaa gccttcggtc 3720


 


ccttaatagg gatatgcagc ctactgaaag tgctgacagc ttagtcgaat acggagaggg 3780


 


agaccatggt ctcttcagtg aagatggatc atttattggt gcctacgctg gatctaagga 3840


 


gaagggatct gttgaaagca atggaagttc tacagcaact tttccccttc gggcataaac 3900


 


acaacatatg taagcaacgc tactggttca ccccaacctt ccatatttat ctgttcaaag 3960


 


gagcaagaac tttcatatag gaatagaaac atgctggccg aagatttcat ccagaagtca 4020


 


acatcctgca attatgttga aaagagtagt actttcttca aaatataaaa tgccaagcac 4080


 


ttcaggccta tgttttgctt atattgtttt caggtgctca aaatgcaaaa cacaaaacaa 4140


 


atcctgcatt tagatacacc tcaactaaat ccaaagtccc cattcagtat attccatatt 4200


 


tgcctgattt tactattcgg tgtgtttgca tagatgttgc tacttggtgg gtttttctcc 4260


 


gtatgcacat tggtatacag tctctgagaa ctggcttggt gactttgctt cactacaggt 4320


 


taaaagacca taagcaaact ggttatttaa aatgtaaaaa ggaatatgaa agtcttatta 4380


 


aaacacttca ttgaaaatat acagtctaaa tttattattt aaattttact agcaaaagtc 4440


 


ttaggtgaac aatcaactag tatttgttga gctcctattt gcccagagat ggtcatattt 4500


 


aaacagaagt atacgttttt cagtttcaac atgaattttt ttatttctgt cagttatgac 4560


 


atccacgagc atcacttttt gtgtctgttt tttttttttt cttggactaa attcaactgc 4620


 


atggaagcgg tggtcagaag gttgttttat acgagaacag gcagaaagtg cccattgttc 4680


 


aggattctaa tagctacatc tacttaatat cttcatttct aaattgactg cttttacctt 4740


 


tttctcatgt ttatataatg gtatgcttgc atatatttca tgaatacatt gtacatatta 4800


 


tgttaatatt tacacaattt aaaatataga tgtgttttat tttgaagtga gaaaatgaac 4860


 


attaacaggc atgtttgtac agctagaata tattagtaag atactgtttt tcgtcattcc 4920


 


agagctacaa ctaataacac gaggttccaa agctgaagac tttgtataaa gtatttgggt 4980


 


tttgttcttg tattgctttc tttcaacagt ttcaaaataa aatatcatac aaatattgag 5040


 


ggaaatgttt tcatattttt caaaataggt ttttattgtt gaatgtacat ctaccccagc 5100


 


ccctcaaaag aaaaactgtt tacatagaaa ttcctacaca tacgtttgcg tatatgttat 5160


 


tttaaacatc tttgtggtga gaattttttc cccgatattc tccttctgtc aaagtcagaa 5220


 


caaattcagg gaatttattt tctggcagtt gtgctccagt ccttttaaaa ttgtacatga 5280


 


acatgtttta gaaacaatat ggaggatgat gcatacatgt cggtcaagtt cagcgctcga 5340


 


cattttatgg aaagattttt ttaaccttac cacgaaatac ttaactactg tttaagtgaa 5400


 


ttgacttatt tcactttagt ttttgaactg tgattattgg tatactgtta tatcctcaac 5460


 


ttggatttat ggtaacccct tttagttcat ggagaccaaa atttggggta tttataatag 5520


 


tcagcgcagg aatgcacatg gaatatctac ttgtcctttt gaacctcacg agtcatccag 5580


 


aatgtataga caggaaaagc atgtcttatt taaaactgta atttatgggc tcaggatctg 5640


 


accgcagtcc cgggagtaag catttcaaag ggggaaggca gtgtggtccc taccctgtgt 5700


 


gaatgtgagg atgtagacat ccatcagtgc aactcgagct ccatcctcct ccgatttcta 5760


 


aggctccagt tttctggagg gacagtcatc atgttttgat ttatctggga gaaaactgtg 5820


 


gtgcacagct tgtgaggagg gcaaggttgt gacgttcgag cttagttctg gtgttattct 5880


 


gtctcctctt ctttgtcatc agccaaaacg tggtttttaa agagagtcat gcaggttaga 5940


 


aataatgtca aaaatattta ggaatttaat aacctttaag tcagaaacta aaacaaatac 6000


 


tgaaatatta gctcttccta cacttcgtgt tcccctttag ctgcctgaaa atcaagattg 6060


 


ctcctactca gatcttctga gtggctaaaa cttatggata tgaaaaatga gattgaatga 6120


 


tgactatgct ttgctatcat tgttaccttt cctcaatact atttggcaac tactgggact 6180


 


cttcagcaca aaaggaatag atctatgatt gaccctgatt ttaattgtga aattatatga 6240


 


ttcatatatt ttatgaatca gaataacctt caaataaaat aaatctaagt cggttaaaat 6300


 


ggatttcatg attttccctc agaaaatgag taacggagtc cacggcgtgc aatggtaatt 6360


 


ataaattggt gatgcttgtt tgcaaattgc ccactcgtga taagtcaaca gccaatattt 6420


 


aaaactttgt tcgttactgg ctttacccta actttctcta gtctactgtc aatatcattt 6480


 


taatgtaatt gattgtatat agtctcaaga atggttggtg ggcatgagtt cctagagaac 6540


 


tgtccaaggg ttgggaaaat ccaaattctc ttcctggctc cagcactgat tttgtacata 6600


 


aacattaggc aggttgctta acctttttat ttcaaactct ctcaactcta aagtgctaat 6660


 


aataatctca gttaccttat ctttgtcaca gggtgttctt ttttatgaag aaaaatttga 6720


 


aaatgataaa agctaagatg ccttctaact tcataagcaa acctttaact aattatgtat 6780


 


ctgaaagtca cccccacata ccaactcaac ttttttcctg tgaacacata aatatatttt 6840


 


tatagaaaaa caaatctaca taaaataaat ctactgttta gtgagcagta tgacttgtac 6900


 


atgccattga aaattattaa tcagaagaaa attaagcagg gtctttgcta tacaaaagtg 6960


 


ttttccacta attttgcatg cgtatttata agaaaaatgt gaatttggtg gttttattct 7020


 


atcggtataa aggcatcgat attttagatg cacccgtgtt tgtaaaaatg tagagcacaa 7080


 


tggaattatg ctggaagtct caaataatat ttttttccta ttttatactc atggaagaga 7140


 


taagctaaag aggggacaat aatgagaaat gttggtgtgc ttttctaagc atttaaaaca 7200


 


taattgccaa ttgaaaccct aaatatgttt acataccatt aagatatgat tcatgtaaca 7260


 


atgttaaatt aattataatg ggattgggtt tgttatctgt ggtagtatat atcctagtgt 7320


 


tcctatagtg aaataagtag ggttcagcca aagctttctt tgttttgtac cttaaattgt 7380


 


tcgattacgt catcaaaaga gatgaaaggt atgtagaaca ggttcacgtg attacctttt 7440


 


tcttttggct tggattaata ttcatagtag aactttataa aacgtgtttg tattgtaggt 7500


 


ggtgtttgta ttatgcttat gactatgtat ggtttgaaaa tattttcatt atacatgaaa 7560


 


ttcaactttc caaataaaag ttctacttca tgtaatccaa aa                    7602













TABLE LIIId





Nucleotide sequence alignment of 282P1G03 v.1 (SEQ ID NO: 170) and


282P1G03 v.5 (SEQ ID NO: 171)





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE LIVd





Peptide sequences of protein coded by 282P1G03 v.5 (SEQ ID NO: 172)















MEPLLLGRGL IVYLMFLLLK FSKAIEIPSS VQQVPTIIKQ SKVQVAFPFD EYFQIECEAK   60


 


GNPEPTFSWT KDGNPFYFTD HRIIPSNNSG TFRIPNEGHI SHFQGKYRCF ASNKLGIAMS  120


 


EEIEFIVPSV PKLPKEKIDP LEVEEGDPIV LPCNPPKGLP PLHIYWMNIE LEHIEQDERV  180


 


YMSQKGDLYF ANVEEKDSRN DYCCFAAFPR LRTIVQKMPM KLTVNSSNSI KQRKPKLLLP  240


 


PTESGSESSI TILKGEILLL ECFAEGLPTP QVDWNKIGGD LPKGRETKEN YGKTLKIENV  300


 


SYQDKGNYRC TASNFLGTAT HDFHVIVEEP PRWTKKPQSA VYSTGSNGIL LCEAEGEPQP  360


 


TIKWRVNGSP VDNHPFAGDV VFPREISFTN LQPNHTAVYQ CEASNVHGTI LANANIDVVD  420


 


VRPLIQTKDG ENYATVVGYS AFLHCEFFAS PEAVVSWQKV EEVKPLEGRR YHIYENGTLQ  480


 


INRTTEEDAG SYSCWVENAI GKTAVTANLD IRNATKLRVS PKNPRIPKLH MLELHCESKC  540


 


DSHLKHSLKL SWSKDGEAFE INGTEDGRII IDGANLTISN VTLEDQGIYC CSAHTALDSA  600


 


ADITQVTVLD VPDPPENLHL SERQNRSVRL TWEAGADHNS NISEYIVEFE GNKEEPGRWE  660


 


ELTRVQGKKT TVILPLAPFV RYQFRVIAVN EVGRSQPSQP SDHHETPPAA PDRNPQNIRV  720


 


QASQPKEMII KWEPLKSMEQ NGPGLEYRVT WKPQGAPVEW EEETVTNHTL RVMTPAVYAP  780


 


YDVKVQAINQ LGSGPDPQSV TLYSGEDYPD TAPVIHGVDV INSTLVKVTW STVPKDRVHG  840


 


RLKGYQINWW KTKSLLDGRT HPKEVNILRF SGQRNSGMVP SLDAFSEFHL TVLAYNSKGA  900


 


GPESEPYIFQ TPEGVPEQPT FLKVIKVDKD TATLSWGLPK KLNGNLTGYL LQYQIINDTY  960


 


EIGELNDINI TTPSKPSWHL SNLNATTKYK FYLRACTSQG CGKPITEESS TLGEGSKGIG 1020


 


KISGVNLTQK THPIEVFEPG AEHIVRLMTK NWGDNDSIFQ DVIETRGREY AGLYDDISTQ 1080


 


GWFIGLMCAI ALLTLLLLTV CFVKRNRGGK YSVKEKEDLH PDPEIQSVKD ETFGEYSDSD 1140


 


EKPLKGSLRS LNRDMQPTES ADSLVEYGEG DHGLFSEDGS FIGAYAGSKE KGSVESNGSS 1200


 


TATFPLRA                                                          1208
















TABLE LVd





Amino acid sequence alignment of 282P1G03 v.1 (SEQ ID NO: 173) and


282P1G03 v.5 (SEQ ID NO: 174)





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE LIIe





Nucleotide sequence of transcript variant 282P1G03 v.6 (SEQ ID NO: 175)















cggaccctgc gcgcccccgt cccggctccc ggccggctcg ggggagaagg cgcccgaggg   60


 


gaggcgccgg acagatcgcg tttcggaggc ggcgcaggtg ctgtaaactg caaaccataa  120


 


tcctgtctta atactgcaaa caaatcatag tggaactaag gggaacttaa tttactgttt  180


 


ccaggttaac taaggtctca gctgtaaacc aaaagtgaga ggagacatta agattttcat  240


 


tcttaccggg ttgtcttctt cctgaagagc aatggagccg cttttacttg gaagaggact  300


 


aatcgtatat ctaatgttcc tcctgttaaa attctcaaaa gcaattgaaa taccatcttc  360


 


agttcaacag gttccaacaa tcataaaaca gtcaaaagtc caagttgcct ttcccttcga  420


 


tgagtatttt caaattgaat gtgaagctaa aggaaatcca gaaccaacat tttcgtggac  480


 


taaggatggc aacccttttt atttcactga ccatcggata attccatcga acaattcagg  540


 


aacattcagg atcccaaacg aggggcacat atctcacttt caagggaaat accgctgctt  600


 


tgcttcaaat aaactgggaa tcgttatgtc agaagaaata gaatttatag ttccaaaatt  660


 


agaacacatc gaacaagatg aaagagtata catgagccaa aagggagatc tatacttcgc  720


 


aaacgtggaa gaaaaggaca gtcgcaatga ctactgttgc tttgctgcat ttccaagatt  780


 


aaggactatt gtacagaaaa tgccaatgaa actaacagtt aacagtttaa agcatgctaa  840


 


tgactcaagt tcatccacag aaattggttc caaggcaaat tccatcaagc aaagaaaacc  900


 


caaactgctg ttgcctccca ctgagagtgg cagtgagtct tcaattacca tcctcaaagg  960


 


ggaaatcttg ctgcttgagt gttttgctga aggcttgcca actccacagg ttgattggaa 1020


 


caaaattggt ggtgacttac caaaggggag agaaacaaaa gaaaattatg gcaagacttt 1080


 


gaagatagag aatgtctcct accaggacaa aggaaattat cgctgcacag ccagcaattt 1140


 


cttgggaaca gccactcacg attttcacgt tatagtagaa gagcctcctc gctggacaaa 1200


 


gaagcctcag agtgctgtgt atagcaccgg aagcaatggc atcttgttat gtgaggctga 1260


 


aggagaacct caacccacaa tcaagtggag agtcaatggc tccccagttg acaatcatcc 1320


 


atttgctggt gatgttgtct tccccaggga aatcagtttt accaaccttc aaccaaatca 1380


 


tactgctgtg taccagtgtg aagcctcaaa tgtccatgga actatccttg ccaatgccaa 1440


 


tattgatgtt gtggatgtcc gtccattgat acaaaccaaa gatggagaaa attacgctac 1500


 


agtggttggg tacagtgctt tcttacattg cgagttcttt gcttcacctg aggcagtcgt 1560


 


gtcctggcag aaggtggaag aagtgaaacc cctggagggc aggcggtatc atatctatga 1620


 


aaatggcaca ttgcagatca acagaaccac cgaagaagat gctgggtctt actcatgttg 1680


 


ggtagaaaat gctataggaa aaactgcagt cacagccaat ttggatatta gaaatgctac 1740


 


aaaacttaga gtttctccta agaatcctcg tatccccaaa ttgcatatgc ttgaattaca 1800


 


ttgtgaaagc aaatgtgact cacatttgaa acacagtttg aagttgtcct ggagtaaaga 1860


 


tggagaagcc tttgaaatta atggcacaga agatggcagg ataattattg atggagctaa 1920


 


tttgaccata tctaatgtaa ctttagagga ccaaggtatt tactgctgtt cagctcatac 1980


 


tgctctagac agtgctgccg atataactca agtaactgtt cttgatgttc cggatccacc 2040


 


agaaaacctt cacttgtctg aaagacagaa caggagtgtt cggctgacct gggaagctgg 2100


 


agctgaccac aacagcaata ttagcgagta tattgttgaa tttgaaggaa acaaagaaga 2160


 


gcctggaagg tgggaggaac tgaccagagt ccaaggaaag aaaaccacag ttatcttacc 2220


 


tttggctcca tttgtgagat accagttcag ggtcatagcc gtgaacgaag tagggagaag 2280


 


tcagcctagc cagccgtcag accatcatga aacaccacca gcagctccag ataggaatcc 2340


 


acaaaacata agggttcaag cctctcaacc caaggaaatg attataaagt gggagccttt 2400


 


gaaatccatg gagcagaatg gaccaggcct agagtacaga gtgacctgga agccacaggg 2460


 


agccccagtg gagtgggaag aagaaacagt cacaaaccac acattgcggg tgatgacgcc 2520


 


tgctgtctat gccccttatg atgtcaaggt ccaggctatc aatcaactag gatctgggcc 2580


 


tgaccctcag tcagtgactc tctattctgg agaagactat cctgatacag ctccagtgat 2640


 


ccatggggtg gacgttataa acagtacatt agttaaagtt acctggtcaa cagttccaaa 2700


 


ggacagagta catggacgtc tgaaaggcta tcagataaat tggtggaaaa caaaaagtct 2760


 


gttggatgga agaacacatc ccaaagaagt gaacattcta agattttcag gacaaagaaa 2820


 


ctctggaatg gttccttcct tagatgcctt tagtgaattt catttaacag tcttagccta 2880


 


taactctaaa ggagctggtc ctgaaagtga gccttatata tttcaaacac cagaaggagt 2940


 


acctgaacag ccaacttttc taaaggtcat caaagttgat aaagacactg ccactttatc 3000


 


ttggggacta cctaagaaat taaatggaaa cttaactggc tatcttttgc aatatcagat 3060


 


aataaatgac acctacgaga ttggagaatt aaatgatatt aacattacaa ctccatcaaa 3120


 


gcccagctgg cacctctcaa acctgaatgc aactaccaag tacaaattct acttgagggc 3180


 


ttgcacttca cagggctgtg gaaaaccgat cacggaggaa agctccacct taggagaagg 3240


 


gagtaaaggt atcgggaaga tatcaggagt aaatcttact caaaagactc acccaataga 3300


 


ggtatttgag ccgggagctg aacatatagt tcgcctaatg actaagaatt ggggcgataa 3360


 


cgatagcatt tttcaagatg taattgagac aagagggaga gaatatgctg gtttatatga 3420


 


tgacatctcc actcaaggct ggtttattgg actgatgtgt gcgattgctc ttctcacact 3480


 


actattatta actgtttgct ttgtgaagag gaatagaggt ggaaagtact cagttaaaga 3540


 


aaaggaagat ttgcatccag acccagaaat tcagtcagta aaagatgaaa cctttggtga 3600


 


atacagtgac agtgatgaaa agcctctcaa aggaagcctt cggtccctta atagggatat 3660


 


gcagcctact gaaagtgctg acagcttagt cgaatacgga gagggagacc atggtctctt 3720


 


cagtgaagat ggatcattta ttggtgccta cgctggatct aaggagaagg gatctgttga 3780


 


aagcaatgga agttctacag caacttttcc ccttcgggca taaacacaac atatgtaagc 3840


 


aacgctactg gttcacccca accttccata tttatctgtt caaaggagca agaactttca 3900


 


tataggaata gaaacatgct ggccgaagat ttcatccaga agtcaacatc ctgcaattat 3960


 


gttgaaaaga gtagtacttt cttcaaaata taaaatgcca agcacttcag gcctatgttt 4020


 


tgcttatatt gttttcaggt gctcaaaatg caaaacacaa aacaaatcct gcatttagat 4080


 


acacctcaac taaatccaaa gtccccattc agtatattcc atatttgcct gattttacta 4140


 


ttcggtgtgt ttgcatagat gttgctactt ggtgggtttt tctccgtatg cacattggta 4200


 


tacagtctct gagaactggc ttggtgactt tgcttcacta caggttaaaa gaccataagc 4260


 


aaactggtta tttaaaatgt aaaaaggaat atgaaagtct tattaaaaca cttcattgaa 4320


 


aatatacagt ctaaatttat tatttaaatt ttactagcaa aagtcttagg tgaacaatca 4380


 


actagtattt gttgagctcc tatttgccca gagatggtca tatttaaaca gaagtatacg 4440


 


tttttcagtt tcaacatgaa tttttttatt tctgtcagtt atgacatcca cgagcatcac 4500


 


tttttgtgtc tgtttttttt tttttcttgg actaaattca actgcatgga agcggtggtc 4560


 


agaaggttgt tttatacgag aacaggcaga aagtgcccat tgttcaggat tctaatagct 4620


 


acatctactt aatatcttca tttctaaatt gactgctttt acctttttct catgtttata 4680


 


taatggtatg cttgcatata tttcatgaat acattgtaca tattatgtta atatttacac 4740


 


aatttaaaat atagatgtgt tttattttga agtgagaaaa tgaacattaa caggcatgtt 4800


 


tgtacagcta gaatatatta gtaagatact gtttttcgtc attccagagc tacaactaat 4860


 


aacacgaggt tccaaagctg aagactttgt ataaagtatt tgggttttgt tcttgtattg 4920


 


ctttctttca acagtttcaa aataaaatat catacaaata ttgagggaaa tgttttcata 4980


 


tttttcaaaa taggttttta ttgttgaatg tacatctacc ccagcccctc aaaagaaaaa 5040


 


ctgtttacat agaaattcct acacatacgt ttgcgtatat gttattttaa acatctttgt 5100


 


ggtgagaatt ttttccccga tattctcctt ctgtcaaagt cagaacaaat tcagggaatt 5160


 


tattttctgg cagttgtgct ccagtccttt taaaattgta catgaacatg ttttagaaac 5220


 


aatatggagg atgatgcata catgtcggtc aagttcagcg ctcgacattt tatggaaaga 5280


 


tttttttaac cttaccacga aatacttaac tactgtttaa gtgaattgac ttatttcact 5340


 


ttagtttttg aactgtgatt attggtatac tgttatatcc tcaacttgga tttatggtaa 5400


 


ccccttttag ttcatggaga ccaaaatttg gggtatttat aatagtcagc gcaggaatgc 5460


 


acatggaata tctacttgtc cttttgaacc tcacgagtca tccagaatgt atagacagga 5520


 


aaagcatgtc ttatttaaaa ctgtaattta tgggctcagg atctgaccgc agtcccggga 5580


 


gtaagcattt caaaggggga aggcagtgtg gtccctaccc tgtgtgaatg tgaggatgta 5640


 


gacatccatc agtgcaactc gagctccatc ctcctccgat ttctaaggct ccagttttct 5700


 


ggagggacag tcatcatgtt ttgatttatc tgggagaaaa ctgtggtgca cagcttgtga 5760


 


ggagggcaag gttgtgacgt tcgagcttag ttctggtgtt attctgtctc ctcttctttg 5820


 


tcatcagcca aaacgtggtt tttaaagaga gtcatgcagg ttagaaataa tgtcaaaaat 5880


 


atttaggaat ttaataacct ttaagtcaga aactaaaaca aatactgaaa tattagctct 5940


 


tcctacactt cgtgttcccc tttagctgcc tgaaaatcaa gattgctcct actcagatct 6000


 


tctgagtggc taaaacttat ggatatgaaa aatgagattg aatgatgact atgctttgct 6060


 


atcattgtta cctttcctca atactatttg gcaactactg ggactcttca gcacaaaagg 6120


 


aatagatcta tgattgaccc tgattttaat tgtgaaatta tatgattcat atattttatg 6180


 


aatcagaata accttcaaat aaaataaatc taagtcggtt aaaatggatt tcatgatttt 6240


 


ccctcagaaa atgagtaacg gagtccacgg cgtgcaatgg taattataaa ttggtgatgc 6300


 


ttgtttgcaa attgcccact cgtgataagt caacagccaa tatttaaaac tttgttcgtt 6360


 


actggcttta ccctaacttt ctctagtcta ctgtcaatat cattttaatg taattgattg 6420


 


tatatagtct caagaatggt tggtgggcat gagttcctag agaactgtcc aagggttggg 6480


 


aaaatccaaa ttctcttcct ggctccagca ctgattttgt acataaacat taggcaggtt 6540


 


gcttaacctt tttatttcaa actctctcaa ctctaaagtg ctaataataa tctcagttac 6600


 


cttatctttg tcacagggtg ttctttttta tgaagaaaaa tttgaaaatg ataaaagcta 6660


 


agatgccttc taacttcata agcaaacctt taactaatta tgtatctgaa agtcaccccc 6720


 


acataccaac tcaacttttt tcctgtgaac acataaatat atttttatag aaaaacaaat 6780


 


ctacataaaa taaatctact gtttagtgag cagtatgact tgtacatgcc attgaaaatt 6840


 


attaatcaga agaaaattaa gcagggtctt tgctatacaa aagtgttttc cactaatttt 6900


 


gcatgcgtat ttataagaaa aatgtgaatt tggtggtttt attctatcgg tataaaggca 6960


 


tcgatatttt agatgcaccc gtgtttgtaa aaatgtagag cacaatggaa ttatgctgga 7020


 


agtctcaaat aatatttttt tcctatttta tactcatgga agagataagc taaagagggg 7080


 


acaataatga gaaatgttgg tgtgcttttc taagcattta aaacataatt gccaattgaa 7140


 


accctaaata tgtttacata ccattaagat atgattcatg taacaatgtt aaattaatta 7200


 


taatgggatt gggtttgtta tctgtggtag tatatatcct agtgttccta tagtgaaata 7260


 


agtagggttc agccaaagct ttctttgttt tgtaccttaa attgttcgat tacgtcatca 7320


 


aaagagatga aaggtatgta gaacaggttc acgtgattac ctttttcttt tggcttggat 7380


 


taatattcat agtagaactt tataaaacgt gtttgtattg taggtggtgt ttgtattatg 7440


 


cttatgacta tgtatggttt gaaaatattt tcattataca tgaaattcaa ctttccaaat 7500


 


aaaagttcta cttcatgtaa tccaaaa                                     7527













TABLE LIIIe





Nucleotide sequence alignment of 282P1G03 v.1 (SEQ ID NO: 176) and


282P1G03 v.6 (SEQ ID NO: 177)





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE LIVe





Peptide sequences of protein coded by 282P1G03 v.6 (SEQ ID NO: 178)















MEPLLLGRGL IVYLMFLLLK FSKAIEIPSS VQQVPTIIKQ SKVQVAFPFD EYFQIECEAK   60


 


GNPEPTFSWT KDGNPFYFTD HRIIPSNNSG TFRIPNEGHI SHFQGKYRCF ASNKLGIAMS  120


 


EEIEFIVPKL EHIEQDERVY MSQKGDLYFA NVEEKDSRND YCCFAAFPRL RTIVQKMPMK  180


 


LTVNSLKHAN DSSSSTEIGS KANSIKQRKP KLLLPPTESG SESSITILKG EILLLECFAE  240


 


GLPTPQVDWN KIGGDLPKGR ETKENYGKTL KIENVSYQDK GNYRCTASNF LGTATHDFHV  300


 


IVEEPPRWTK KPQSAVYSTG SNGILLCEAE GEPQPTIKWR VNGSPVDNHP FAGDVVFPRE  360


 


ISFTNLQPNH TAVYQCEASN VHGTILANAN IDVVDVRPLI QTKDGENYAT VVGYSAFLHC  420


 


EFFASPEAVV SWQKVEEVKP LEGRRYHIYE NGTLQINRTT EEDAGSYSCW VENAIGKTAV  480


 


TANLDIRNAT KLRVSPKNPR IPKLHMLELH CESKCDSHLK HSLKLSWSKD GEAFEINGTE  540


 


DGRIIIDGAN LTISNVTLED QGIYCCSAHT ALDSAADITQ VTVLDVPDPP ENLHLSERQN  600


 


RSVRLTWEAG ADHNSNISEY IVEFEGNKEE PGRWEELTRV QGKKTTVILP LAPFVRYQFR  660


 


VIAVNEVGRS QPSQPSDHHE TPPAAPDRNP QNIRVQASQP KEMIIKWEPL KSMEQNGPGL  720


 


EYRVTWKPQG APVEWEEETV TNHTLRVMTP AVYAPYDVKV QAINQLGSGP DPQSVTLYSG  780


 


EDYPDTAPVI HGVDVINSTL VKVTWSTVPK DRVHGRLKGY QINWWKTKSL LDGRTHPKEV  840


 


NILRFSGQRN SGMVPSLDAF SEFHLTVLAY NSKGAGPESE PYIFQTPEGV PEQPTFLKVI  900


 


KVDKDTATLS WGLPKKLNGN LTGYLLQYQI INDTYEIGEL NDINITTPSK PSWHLSNLNA  960


 


TTKYKFYLRA CTSQGCGKPI TEESSTLGEG SKGIGKISGV NLTQKTHPIE VFEPGAEHIV 1020


 


RLMTKNWGDN DSIFQDVIET RGREYAGLYD DISTQGWFIG LMCAIALLTL LLLTVCFVKR 1080


 


NRGGKYSVKE KEDLHPDPEI QSVKDETFGE YSDSDEKPLK GSLRSLNRDM QPTESADSLV 1140


 


EYGEGDHGLF SEDGSFIGAY AGSKEKGSVE SNGSSTATFP LRA                   1183
















TABLE LVe





Amino acid sequence alignment of 282P1G03 v.1 (SEQ ID NO: 179) and


282P1G03 v.6 (SEQ ID NO: 180)





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE LIIf





Nucleotide sequence of transcript variant 282P1G03 v.7 (SEQ ID NO: 181)
















cggaccctgc gcgcccccgt cccggctccc ggccggctcg ggggagaagg cgcccgaggg
60


 


gaggcgccgg acagatcgcg tttcggaggc ggcgcaggtg ctgtaaactg caaaccataa
120


 


tcctgtctta atactgcaaa caaatcatag tggaactaag gggaacttaa tttactgttt
180


 


ccaggttaac taaggtctca gctgtaaacc aaaagtgaga ggagacatta agattttcat
240


 


tcttaccggg ttgtcttctt cctgaagagc aatggagccg cttttacttg gaagaggact
300


 


aatcgtatat ctaatgttcc tcctgttaaa attctcaaaa gcaattgaaa taccatcttc
360


 


agttcaacag gttccaacaa tcataaaaca gtcaaaagtc caagttgcct ttcccttcga
420


 


tgagtatttt caaattgaat gtgaagctaa aggaaatcca gaaccaacat tttcgtggac
480


 


taaggatggc aacccttttt atttcactga ccatcggata attccatcga acaattcagg
540


 


aacattcagg atcccaaacg aggggcacat atctcacttt caagggaaat accgctgctt
600


 


tgcttcaaat aaactgggaa tcgctatgtc agaagaaata gaatttatag ttccaagtgt
660


 


tccaaaactc ccaaaagaaa aaattgaccc tcttgaagtg gaggagggag atccaattgt
720


 


cctcccatgc aatcctccca aaggcctccc acctttacac atttattgga tgaatattga
780


 


attagaacac atcgaacaag atgaaagagt atacatgagc caaaagggag atctatactt
840


 


cgcaaacgtg gaagaaaagg acagtcgcaa tgactactgt tgctttgctg catttccaag
900


 


attaaggact attgtacaga aaatgccaat gaaactaaca gttaacagtt taaagcatgc
960


 


taatgactca agttcatcca cagaaattgg ttccaaggca aattccatca agcaaagaaa
1020


 


acccaaactg ctgttgcctc ccactgagag tggcagtgag tcttcaatta ccatcctcaa
1080


 


aggggaaatc ttgctgcttg agtgttttgc tgaaggcttg ccaactccac aggttgattg
1140


 


gaacaaaatt ggtggtgact taccaaaggg gagagaaaca aaagaaaatt atggcaagac
1200


 


tttgaagata gagaatgtct cctaccagga caaaggaaat tatcgctgca cagccagcaa
1260


 


tttcttggga acagccactc acgattttca cgttatagta gaagataaca tctctcatga
1320


 


gctcttcact ttacatccag agcctcctcg ctggacaaag aagcctcaga gtgctgtgta
1380


 


tagcaccgga agcaatggca tcttgttatg tgaggctgaa ggagaacctc aacccacaat
1440


 


caagtggaga gtcaatggct ccccagttga caatcatcca tttgctggtg atgttgtctt
1500


 


ccccagggaa atcagtttta ccaaccttca accaaatcat actgctgtgt accagtgtga
1560


 


agcctcaaat gtccatggaa ctatccttgc caatgccaat attgatgttg tggatgtccg
1620


 


tccattgata caaaccaaag atggagaaaa ttacgctaca gtggttgggt acagtgcttt
1680


 


cttacattgc gagttctttg cttcacctga ggcagtcgtg tcctggcaga aggtggaaga
1740


 


agtgaaaccc ctggagggca ggcggtatca tatctatgaa aatggcacat tgcagatcaa
1800


 


cagaaccacc gaagaagatg ctgggtctta ctcatgttgg gtagaaaatg ctataggaaa
1860


 


aactgcagtc acagccaatt tggatattag aaatgctaca aaacttagag tttctcctaa
1920


 


gaatcctcgt atccccaaat tgcatatgct tgaattacat tgtgaaagca aatgtgactc
1980


 


acatttgaaa cacagtttga agttgtcctg gagtaaagat ggagaagcct ttgaaattaa
2040


 


tggcacagaa gatggcagga taattattga tggagctaat ttgaccatat ctaatgtaac
2100


 


tttagaggac caaggtattt actgctgttc agctcatact gctctagaca gtgctgccga
2160


 


tataactcaa gtaactgttc ttgatgttcc ggatccacca gaaaaccttc acttgtctga
2220


 


aagacagaac aggagtgttc ggctgacctg ggaagctgga gctgaccaca acagcaatat
2280


 


tagcgagtat attgttgaat ttgaaggaaa caaagaagag cctggaaggt gggaggaact
2340


 


gaccagagtc caaggaaaga aaaccacagt tatcttacct ttggctccat ttgtgagata
2400


 


ccagttcagg gtcatagccg tgaacgaagt agggagaagt cagcctagcc agccgtcaga
2460


 


ccatcatgaa acaccaccag cagctccaga taggaatcca caaaacataa gggttcaagc
2520


 


ctctcaaccc aaggaaatga ttataaagtg ggagcctttg aaatccatgg agcagaatgg
2580


 


accaggccta gagtacagag tgacctggaa gccacaggga gccccagtgg agtgggaaga
2640


 


agaaacagtc acaaaccaca cattgcgggt gatgacgcct gctgtctatg ccccttatga
2700


 


tgtcaaggtc caggctatca atcaactagg atctgggcct gaccctcagt cagtgactct
2760


 


ctattctgga gaagactatc ctgatacagc tccagtgatc catggggtgg acgttataaa
2820


 


cagtacatta gttaaagtta cctggtcaac agttccaaag gacagagtac atggacgtct
2880


 


gaaaggctat cagataaatt ggtggaaaac aaaaagtctg ttggatggaa gaacacatcc
2940


 


caaagaagtg aacattctaa gattttcagg acaaagaaac tctggaatgg ttccttcctt
3000


 


agatgccttt agtgaatttc atttaacagt cttagcctat aactctaaag gagctggtcc
3060


 


tgaaagtgag ccttatatat ttcaaacacc agaaggagta cctgaacagc caacttttct
3120


 


aaaggtcatc aaagttgata aagacactgc cactttatct tggggactac ctaagaaatt
3180


 


aaatggaaac ttaactggct atcttttgca atatcagata ataaatgaca cctacgagat
3240


 


tggagaatta aatgatatta acattacaac tccatcaaag cccagctggc acctctcaaa
3300


 


cctgaatgca actaccaagt acaaattcta cttgagggct tgcacttcac agggctgtgg
3360


 


aaaaccgatc acggaggaaa gctccacctt aggagaaggg agtaaaggta tcgggaagat
3420


 


atcaggagta aatcttactc aaaagactca cccaatagag gtatttgagc cgggagctga
3480


 


acatatagtt cgcctaatga ctaagaattg gggcgataac gatagcattt ttcaagatgt
3540


 


aattgagaca agagggagag aatatgctgg tttatatgat gacatctcca ctcaaggctg
3600


 


gtttattgga ctgatgtgtg cgattgctct tctcacacta ctattattaa ctgtttgctt
3660


 


tgtgaagagg aatagaggtg gaaagtactc agttaaagaa aaggaagatt tgcatccaga
3720


 


cccagaaatt cagtcagtaa aagatgaaac ctttggtgaa tacagtgaca gtgatgaaaa
3780


 


gcctctcaaa ggaagccttc ggtcccttaa tagggatatg cagcctactg aaagtgctga
3840


 


cagcttagtc gaatacggag agggagacca tggtctcttc agtgaagatg gatcatttat
3900


 


tggtgcctac gctggatcta aggagaaggg atctgttgaa agcaatggaa gttctacagc
3960


 


aacttttccc cttcgggcat aaacacaaca tatgtaagca acgctactgg ttcaccccaa
4020


 


ccttccatat ttatctgttc aaaggagcaa gaactttcat ataggaatag aaacatgctg
4080


 


gccgaagatt tcatccagaa gtcaacatcc tgcaattatg ttgaaaagag tagtactttc
4140


 


ttcaaaatat aaaatgccaa gcacttcagg cctatgtttt gcttatattg ttttcaggtg
4200


 


ctcaaaatgc aaaacacaaa acaaatcctg catttagata cacctcaact aaatccaaag
4260


 


tccccattca gtatattcca tatttgcctg attttactat tcggtgtgtt tgcatagatg
4320


 


ttgctacttg gtgggttttt ctccgtatgc acattggtat acagtctctg agaactggct
4380


 


tggtgacttt gcttcactac aggttaaaag accataagca aactggttat ttaaaatgta
4440


 


aaaaggaata tgaaagtctt attaaaacac ttcattgaaa atatacagtc taaatttatt
4500


 


atttaaattt tactagcaaa agtcttaggt gaacaatcaa ctagtatttg ttgagctcct
4560


 


atttgcccag agatggtcat atttaaacag aagtatacgt ttttcagttt caacatgaat
4620


 


ttttttattt ctgtcagtta tgacatccac gagcatcact ttttgtgtct gttttttttt
4680


 


ttttcttgga ctaaattcaa ctgcatggaa gcggtggtca gaaggttgtt ttatacgaga
4740


 


acaggcagaa agtgcccatt gttcaggatt ctaatagcta catctactta atatcttcat
4800


 


ttctaaattg actgctttta cctttttctc atgtttatat aatggtatgc ttgcatatat
4860


 


ttcatgaata cattgtacat attatgttaa tatttacaca atttaaaata tagatgtgtt
4920


 


ttattttgaa gtgagaaaat gaacattaac aggcatgttt gtacagctag aatatattag
4980


 


taagatactg tttttcgtca ttccagagct acaactaata acacgaggtt ccaaagctga
5040


 


agactttgta taaagtattt gggttttgtt cttgtattgc tttctttcaa cagtttcaaa
5100


 


ataaaatatc atacaaatat tgagggaaat gttttcatat ttttcaaaat aggtttttat
5160


 


tgttgaatgt acatctaccc cagcccctca aaagaaaaac tgtttacata gaaattccta
5220


 


cacatacgtt tgcgtatatg ttattttaaa catctttgtg gtgagaattt tttccccgat
5280


 


attctccttc tgtcaaagtc agaacaaatt cagggaattt attttctggc agttgtgctc
5340


 


cagtcctttt aaaattgtac atgaacatgt tttagaaaca atatggagga tgatgcatac
5400


 


atgtcggtca agttcagcgc tcgacatttt atggaaagat ttttttaacc ttaccacgaa
5460


 


atacttaact actgtttaag tgaattgact tatttcactt tagtttttga actgtgatta
5520


 


ttggtatact gttatatcct caacttggat ttatggtaac cccttttagt tcatggagac
5580


 


caaaatttgg ggtatttata atagtcagcg caggaatgca catggaatat ctacttgtcc
5640


 


ttttgaacct cacgagtcat ccagaatgta tagacaggaa aagcatgtct tatttaaaac
5700


 


tgtaatttat gggctcagga tctgaccgca gtcccgggag taagcatttc aaagggggaa
5760


 


ggcagtgtgg tccctaccct gtgtgaatgt gaggatgtag acatccatca gtgcaactcg
5820


 


agctccatcc tcctccgatt tctaaggctc cagttttctg gagggacagt catcatgttt
5880


 


tgatttatct gggagaaaac tgtggtgcac agcttgtgag gagggcaagg ttgtgacgtt
5940


 


cgagcttagt tctggtgtta ttctgtctcc tcttctttgt catcagccaa aacgtggttt
6000


 


ttaaagagag tcatgcaggt tagaaataat gtcaaaaata tttaggaatt taataacctt
6060


 


taagtcagaa actaaaacaa atactgaaat attagctctt cctacacttc gtgttcccct
6120


 


ttagctgcct gaaaatcaag attgctccta ctcagatctt ctgagtggct aaaacttatg
6180


 


gatatgaaaa atgagattga atgatgacta tgctttgcta tcattgttac ctttcctcaa
6240


 


tactatttgg caactactgg gactcttcag cacaaaagga atagatctat gattgaccct
6300


 


gattttaatt gtgaaattat atgattcata tattttatga atcagaataa ccttcaaata
6360


 


aaataaatct aagtcggtta aaatggattt catgattttc cctcagaaaa tgagtaacgg
6420


 


agtccacggc gtgcaatggt aattataaat tggtgatgct tgtttgcaaa ttgcccactc
6480


 


gtgataagtc aacagccaat atttaaaact ttgttcgtta ctggctttac cctaactttc
6540


 


tctagtctac tgtcaatatc attttaatgt aattgattgt atatagtctc aagaatggtt
6600


 


ggtgggcatg agttcctaga gaactgtcca agggttggga aaatccaaat tctcttcctg
6660


 


gctccagcac tgattttgta cataaacatt aggcaggttg cttaaccttt ttatttcaaa
6720


 


ctctctcaac tctaaagtgc taataataat ctcagttacc ttatctttgt cacagggtgt
6780


 


tcttttttat gaagaaaaat ttgaaaatga taaaagctaa gatgccttct aacttcataa
6840


 


gcaaaccttt aactaattat gtatctgaaa gtcaccccca cataccaact caactttttt
6900


 


cctgtgaaca cataaatata tttttataga aaaacaaatc tacataaaat aaatctactg
6960


 


tttagtgagc agtatgactt gtacatgcca ttgaaaatta ttaatcagaa gaaaattaag
7020


 


cagggtcttt gctatacaaa agtgttttcc actaattttg catgcgtatt tataagaaaa
7080


 


atgtgaattt ggtggtttta ttctatcggt ataaaggcat cgatatttta gatgcacccg
7140


 


tgtttgtaaa aatgtagagc acaatggaat tatgctggaa gtctcaaata atattttttt
7200


 


cctattttat actcatggaa gagataagct aaagagggga caataatgag aaatgttggt
7260


 


gtgcttttct aagcatttaa aacataattg ccaattgaaa ccctaaatat gtttacatac
7320


 


cattaagata tgattcatgt aacaatgtta aattaattat aatgggattg ggtttgttat
7380


 


ctgtggtagt atatatccta gtgttcctat agtgaaataa gtagggttca gccaaagctt
7440


 


tctttgtttt gtaccttaaa ttgttcgatt acgtcatcaa aagagatgaa aggtatgtag
7500


 


aacaggttca cgtgattacc tttttctttt ggcttggatt aatattcata gtagaacttt
7560


 


ataaaacgtg tttgtattgt aggtggtgtt tgtattatgc ttatgactat gtatggtttg
7620


 


aaaatatttt cattatacat gaaattcaac tttccaaata aaagttctac ttcatgtaat
7680


 


ccaaaa
7686
















TABLE LIIIf





Nucleotide sequence alignment of 282P1G03 v.1 (SEQ ID NO: 182) and


282P1G03 v.7 (SEQ ID NO: 183)





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE LIVf





Peptide sequences of protein coded by 282P1G03 v.7 (SEQ ID NO: 184)
















MEPLLLGRGL IVYLMFLLLK FSKAIEIPSS VQQVPTIIKQ SKVQVAFPFD EYFQIECEAK
60


 


GNPEPTFSWT KDGNPFYFTD HRIIPSNNSG TFRIPNEGHI SHFQGKYRCF ASNKLGIAMS
120


 


EEIEFIVPSV PKLPKEKIDP LEVEEGDPIV LPCNPPKGLP PLHIYWMNIE LEHIEQDERV
180


 


YMSQKGDLYF ANVEEKDSRN DYCCFAAFPR LRTIVQKMPM KLTVNSLKHA NDSSSSTEIG
240


 


SKANSIKQRK PKLLLPPTES GSESSITILK GEILLLECFA EGLPTPQVDW NKIGGDLPKG
300


 


RETKENYGKT LKIENVSYQD KGNYRCTASN FLGTATHDFH VIVEDNISHE LFTLHPEPPR
360


 


WTKKPQSAVY STGSNGILLC EAEGEPQPTI KWRVNGSPVD NHPFAGDVVF PREISFTNLQ
420


 


PNHTAVYQCE ASNVHGTILA NANIDVVDVR PLIQTKDGEN YATVVGYSAF LHCEFFASPE
480


 


AVVSWQKVEE VKPLEGRRYH IYENGTLQIN RTTEEDAGSY SCWVENAIGK TAVTANLDIR
540


 


NATKLRVSPK NPRIPKLHML ELHCESKCDS HLKHSLKLSW SKDGEAFEIN GTEDGRIIID
600


 


GANLTISNVT LEDQGIYCCS AHTALDSAAD ITQVTVLDVP DPPENLHLSE RQNRSVRLTW
660


 


EAGADHNSNI SEYIVEFEGN KEEPGRWEEL TRVQGKKTTV ILPLAPFVRY QFRVIAVNEV
720


 


GRSQPSQPSD HHETPPAAPD RNPQNIRVQA SQPKEMIIKW EPLKSMEQNG PGLEYRVTWK
780


 


PQGAPVEWEE ETVTNHTLRV MTPAVYAPYD VKVQAINQLG SGPDPQSVTL YSGEDYPDTA
840


 


PVIHGVDVIN STLVKVTWST VPKDRVHGRL KGYQINWWKT KSLLDGRTHP KEVNILRFSG
900


 


QRNSGMVPSL DAFSEFHLTV LAYNSKGAGP ESEPYIFQTP EGVPEQPTFL KVIKVDKDTA
960


 


TLSWGLPKKL NGNLTGYLLQ YQIINDTYEI GELNDINITT PSKPSWHLSN LNATTKYKFY
1020


 


LRACTSQGCG KPITEESSTL GEGSKGIGKI SGVNLTQKTH PIEVFEPGAE HIVRLMTKNW
1080


 


GDNDSIFQDV IETRGREYAG LYDDISTQGW FIGLMCAIAL LTLLLLTVCF VKRNRGGKYS
1140


 


VKEKEDLHPD PEIQSVKDET FGEYSDSDEK PLKGSLRSLN RDMQPTESAD SLVEYGEGDH
1200


 


GLFSEDGSFI GAYAGSKEKG SVESNGSSTA TFPLRA
1236
















TABLE LVf





Amino acid sequence alignment of 282P1G03 v.1 (SEQ ID NO: 185) and


282P1G03 v.7 (SEQ ID NO: 186)





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE LIIg





Nucleotide sequence of transcript variant 282P1G03 v.8 (SEQ ID NO: 187)
















cggaccctgc gcgcccccgt cccggctccc ggccggctcg ggggagaagg cgcccgaggg
60


 


gaggcgccgg acagatcgcg tttcggaggc ggcgcaggtg ctgtaaactg caaaccataa
120


 


tcctgtctta atactgcaaa caaatcatag tggaactaag gggaacttaa tttactgttt
180


 


ccaggttaac taaggtctca gctgtaaacc aaaagtgaga ggagacatta agattttcat
240


 


tcttaccggg ttgtcttctt cctgaagagc aatggagccg cttttacttg gaagaggact
300


 


aatcgtatat ctaatgttcc tcctgttaaa attctcaaaa gcaattgaaa taccatcttc
360


 


agttcaacag gttccaacaa tcataaaaca gtcaaaagtc caagttgcct ttcccttcga
420


 


tgagtatttt caaattgaat gtgaagctaa aggaaatcca gaaccaacat tttcgtggac
480


 


taaggatggc aacccttttt atttcactga ccatcggata attccatcga acaattcagg
540


 


aacattcagg atcccaaacg aggggcacat atctcacttt caagggaaat accgctgctt
600


 


tgcttcaaat aaactgggaa tcgctatgtc agaagaaata gaatttatag ttccaaaatt
660


 


agaacacatc gaacaagatg aaagagtata catgagccaa aagggagatc tatacttcgc
720


 


aaacgtggaa gaaaaggaca gtcgcaatga ctactgttgc tttgctgcat ttccaagatt
780


 


aaggactatt gtacagaaaa tgccaatgaa actaacagtt aacagtttaa agcatgctaa
840


 


tgactcaagt tcatccacag aaattggttc caaggcaaat tccatcaagc aaagaaaacc
900


 


caaactgctg ttgcctccca ctgagagtgg cagtgagtct tcaattacca tcctcaaagg
960


 


ggaaatcttg ctgcttgagt gttttgctga aggcttgcca actccacagg ttgattggaa
1020


 


caaaattggt ggtgacttac caaaggggag agaaacaaaa gaaaattatg gcaagacttt
1080


 


gaagatagag aatgtctcct accaggacaa aggaaattat cgctgcacag ccagcaattt
1140


 


cttgggaaca gccactcacg attttcacgt tatagtagaa gataacatct ctcatgagct
1200


 


cttcacttta catccagagc ctcctcgctg gacaaagaag cctcagagtg ctgtgtatag
1260


 


caccggaagc aatggcatct tgttatgtga ggctgaagga gaacctcaac ccacaatcaa
1320


 


gtggagagtc aatggctccc cagttgacaa tcatccattt gctggtgatg ttgtcttccc
1380


 


cagggaaatc agttttacca accttcaacc aaatcatact gctgtgtacc agtgtgaagc
1440


 


ctcaaatgtc catggaacta tccttgccaa tgccaatatt gatgttgtgg atgtccgtcc
1500


 


attgatacaa accaaagatg gagaaaatta cgctacagtg gttgggtaca gtgctttctt
1560


 


acattgcgag ttctttgctt cacctgaggc agtcgtgtcc tggcagaagg tggaagaagt
1620


 


gaaacccctg gagggcaggc ggtatcatat ctatgaaaat ggcacattgc agatcaacag
1680


 


aaccaccgaa gaagatgctg ggtcttactc atgttgggta gaaaatgcta taggaaaaac
1740


 


tgcagtcaca gccaatttgg atattagaaa tgctacaaaa cttagagttt ctcctaagaa
1800


 


tcctcgtatc cccaaattgc atatgcttga attacattgt gaaagcaaat gtgactcaca
1860


 


tttgaaacac agtttgaagt tgtcctggag taaagatgga gaagcctttg aaattaatgg
1920


 


cacagaagat ggcaggataa ttattgatgg agctaatttg accatatcta atgtaacttt
1980


 


agaggaccaa ggtatttact gctgttcagc tcatactgct ctagacagtg ctgccgatat
2040


 


aactcaagta actgttcttg atgttccgga tccaccagaa aaccttcact tgtctgaaag
2100


 


acagaacagg agtgttcggc tgacctggga agctggagct gaccacaaca gcaatattag
2160


 


cgagtatatt gttgaatttg aaggaaacaa agaagagcct ggaaggtggg aggaactgac
2220


 


cagagtccaa ggaaagaaaa ccacagttat cttacctttg gctccatttg tgagatacca
2280


 


gttcagggtc atagccgtga acgaagtagg gagaagtcag cctagccagc cgtcagacca
2340


 


tcatgaaaca ccaccagcag ctccagatag gaatccacaa aacataaggg ttcaagcctc
2400


 


tcaacccaag gaaatgatta taaagtggga gcctttgaaa tccatggagc agaatggacc
2460


 


aggcctagag tacagagtga cctggaagcc acagggagcc ccagtggagt gggaagaaga
2520


 


aacagtcaca aaccacacat tgcgggtgat gacgcctgct gtctatgccc cttatgatgt
2580


 


caaggtccag gctatcaatc aactaggatc tgggcctgac cctcagtcag tgactctcta
2640


 


ttctggagaa gactatcctg atacagctcc agtgatccat ggggtggacg ttataaacag
2700


 


tacattagtt aaagttacct ggtcaacagt tccaaaggac agagtacatg gacgtctgaa
2760


 


aggctatcag ataaattggt ggaaaacaaa aagtctgttg gatggaagaa cacatcccaa
2820


 


agaagtgaac attctaagat tttcaggaca aagaaactct ggaatggttc cttccttaga
2880


 


tgcctttagt gaatttcatt taacagtctt agcctataac tctaaaggag ctggtcctga
2940


 


aagtgagcct tatatatttc aaacaccaga aggagtacct gaacagccaa cttttctaaa
3000


 


ggtcatcaaa gttgataaag acactgccac tttatcttgg ggactaccta agaaattaaa
3060


 


tggaaactta actggctatc ttttgcaata tcagataata aatgacacct acgagattgg
3120


 


agaattaaat gatattaaca ttacaactcc atcaaagccc agctggcacc tctcaaacct
3180


 


gaatgcaact accaagtaca aattctactt gagggcttgc acttcacagg gctgtggaaa
3240


 


accgatcacg gaggaaagct ccaccttagg agaagggagt aaaggtatcg ggaagatatc
3300


 


aggagtaaat cttactcaaa agactcaccc aatagaggta tttgagccgg gagctgaaca
3360


 


tatagttcgc ctaatgacta agaattgggg cgataacgat agcatttttc aagatgtaat
3420


 


tgagacaaga gggagagaat atgctggttt atatgatgac atctccactc aaggctggtt
3480


 


tattggactg atgtgtgcga ttgctcttct cacactacta ttattaactg tttgctttgt
3540


 


gaagaggaat agaggtggaa agtactcagt taaagaaaag gaagatttgc atccagaccc
3600


 


agaaattcag tcagtaaaag atgaaacctt tggtgaatac agtgacagtg atgaaaagcc
3660


 


tctcaaagga agccttcggt cccttaatag ggatatgcag cctactgaaa gtgctgacag
3720


 


cVtagtcgaa tacggagagg gagaccatgg tctcttcagt gaagatggat catttattgg
3780


 


tgcctacgct ggatctaagg agaagggatc tgttgaaagc aatggaagtt ctacagcaac
3840


 


ttttcccctt cgggcataaa cacaacatat gtaagcaacg ctactggttc accccaacct
3900


 


tccatattta tctgttcaaa ggagcaagaa ctttcatata ggaatagaaa catgctggcc
3960


 


gaagatttca tccagaagtc aacatcctgc aattatgttg aaaagagtag tactttcttc
4020


 


aaaatataaa atgccaagca cttcaggcct atgttttgct tatattgttt tcaggtgctc
4080


 


aaaatgcaaa acacaaaaca aatcctgcat ttagatacac ctcaactaaa tccaaagtcc
4140


 


ccattcagta tattccatat ttgcctgatt ttactattcg gtgtgtttgc atagatgttg
4200


 


ctacttggtg ggtttttctc cgtatgcaca ttggtataca gtctctgaga actggcttgg
4260


 


tgactttgct tcactacagg ttaaaagacc ataagcaaac tggttattta aaatgtaaaa
4320


 


aggaatatga aagtcttatt aaaacacttc attgaaaata tacagtctaa atttattatt
4380


 


taaattttac tagcaaaagt cttaggtgaa caatcaacta gtatttgttg agctcctatt
4440


 


tgcccagaga tggtcatatt taaacagaag tatacgtttt tcagtttcaa catgaatttt
4500


 


tttatttctg tcagttatga catccacgag catcactttt tgtgtctgtt tttttttttt
4560


 


tcttggacta aattcaactg catggaagcg gtggtcagaa ggttgtttta tacgagaaca
4620


 


ggcagaaagt gcccattgtt caggattcta atagctacat ctacttaata tcttcatttc
4680


 


taaattgact gcttttacct ttttctcatg tttatataat ggtatgcttg catatatttc
4740


 


atgaatacat tgtacatatt atgttaatat ttacacaatt taaaatatag atgtgtttta
4800


 


ttttgaagtg agaaaatgaa cattaacagg catgtttgta cagctagaat atattagtaa
4860


 


gatactgttt ttcgtcattc cagagctaca actaataaca cgaggttcca aagctgaaga
4920


 


ctttgtataa agtatttggg ttttgttctt gtattgcttt ctttcaacag tttcaaaata
4980


 


aaatatcata caaatattga gggaaatgtt ttcatatttt tcaaaatagg tttttattgt
5040


 


tgaatgtaca tctaccccag cccctcaaaa gaaaaactgt ttacatagaa attcctacac
5100


 


atacgtttgc gtatatgtta ttttaaacat ctttgtggtg agaatttttt ccccgatatt
5160


 


ctccttctgt caaagtcaga acaaattcag ggaatttatt ttctggcagt tgtgctccag
5220


 


tccttttaaa attgtacatg aacatgtttt agaaacaata tggaggatga tgcatacatg
5280


 


tcggtcaagt tcagcgctcg acattttatg gaaagatttt tttaacctta ccacgaaata
5340


 


cttaactact gtttaagtga attgacttat ttcactttag tttttgaact gtgattattg
5400


 


gtatactgtt atatcctcaa cttggattta tggtaacccc ttttagttca tggagaccaa
5460


 


aatttggggt atttataata gtcagcgcag gaatgcacat ggaatatcta cttgtccttt
5520


 


tgaacctcac gagtcatcca gaatgtatag acaggaaaag catgtcttat ttaaaactgt
5580


 


aatttatggg ctcaggatct gaccgcagtc ccgggagtaa gcatttcaaa gggggaaggc
5640


 


agtgtggtcc ctaccctgtg tgaatgtgag gatgtagaca tccatcagtg caactcgagc
5700


 


tccatcctcc tccgatttct aaggctccag ttttctggag ggacagtcat catgttttga
5760


 


tttatctggg agaaaactgt ggtgcacagc ttgtgaggag ggcaaggttg tgacgttcga
5820


 


gcttagttct ggtgttattc tgtctcctct tctttgtcat cagccaaaac gtggttttta
5880


 


aagagagtca tgcaggttag aaataatgtc aaaaatattt aggaatttaa taacctttaa
5940


 


gtcagaaact aaaacaaata ctgaaatatt agctcttcct acacttcgtg ttccccttta
6000


 


gctgcctgaa aatcaagatt gctcctactc agatcttctg agtggctaaa acttatggat
6060


 


atgaaaaatg agattgaatg atgactatgc tttgctatca ttgttacctt tcctcaatac
6120


 


tatttggcaa ctactgggac tcttcagcac aaaaggaata gatctatgat tgaccctgat
6180


 


tttaattgtg aaattatatg attcatatat tttatgaatc agaataacct tcaaataaaa
6240


 


taaatctaag tcggttaaaa tggatttcat gattttccct cagaaaatga gtaacggagt
6300


 


ccacggcgtg caatggtaat tataaattgg tgatgcttgt ttgcaaattg cccactcgtg
6360


 


ataagtcaac agccaatatt taaaactttg ttcgttactg gctttaccct aactttctct
6420


 


agtctactgt caatatcatt ttaatgtaat tgattgtata tagtctcaag aatggttggt
6480


 


gggcatgagt tcctagagaa ctgtccaagg gttgggaaaa tccaaattct cttcctggct
6540


 


ccagcactga ttttgtacat aaacattagg caggttgctt aaccttttta tttcaaactc
6600


 


tctcaactct aaagtgctaa taataatctc agttacctta tctttgtcac agggtgttct
6660


 


tttttatgaa gaaaaatttg aaaatgataa aagctaagat gccttctaac ttcataagca
6720


 


aacctttaac taattatgta tctgaaagtc acccccacat accaactcaa cttttttcct
6780


 


gtgaacacat aaatatattt ttatagaaaa acaaatctac ataaaataaa tctactgttt
6840


 


agtgagcagt atgacttgta catgccattg aaaattatta atcagaagaa aattaagcag
6900


 


ggtctttgct atacaaaagt gttttccact aattttgcat gcgtatttat aagaaaaatg
6960


 


tgaatttggt ggttttattc tatcggtata aaggcatcga tattttagat gcacccgtgt
7020


 


ttgtaaaaat gtagagcaca atggaattat gctggaagtc tcaaataata tttttttcct
7080


 


attttatact catggaagag ataagctaaa gaggggacaa taatgagaaa tgttggtgtg
7140


 


cttttctaag catttaaaac ataattgcca attgaaaccc taaatatgtt tacataccat
7200


 


taagatatga ttcatgtaac aatgttaaat taattataat gggattgggt ttgttatctg
7260


 


tggtagtata tatcctagtg ttcctatagt gaaataagta gggttcagcc aaagctttct
7320


 


ttgttttgta ccttaaattg ttcgattacg tcatcaaaag agatgaaagg tatgtagaac
7380


 


aggttcacgt gattaccttt ttcttttggc ttggattaat attcatagta gaactttata
7440


 


aaacgtgttt gtattgtagg tggtgtttgt attatgctta tgactatgta tggtttgaaa
7500


 


atattttcat tatacatgaa attcaacttt ccaaataaaa gttctacttc atgtaatcca
7560


 


aaa
7563
















TABLE LIIIg





Nucleotide sequence alignment of 282P1G03 v.1 (SEQ ID NO: 188) and


282P1G03 v.8 (SEQ ID NO: 189)





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE LIVg





Peptide sequences of protein coded by 282P1G03 v.8 (SEQ ID NO: 190)
















MEPLLLGRGL IVYLMFLLLK FSKAIEIPSS VQQVPTIIKQ SKVQVAFPFD EYFQIECEAK
60


 


GNPEPTFSWT KDGNPFYFTD HRIIPSNNSG TFRIPNEGHI SHFQGKYRCF ASNKLGIAMS
120


 


EEIEFIVPKL EHIEQDERVY MSQKGDLYFA NVEEKDSRND YCCFAAFPRL RTIVQKMPMK
180


 


LTVNSLKHAN DSSSSTEIGS KANSIKQRKP KLLLPPTESG SESSITILKG EILLLECFAE
240


 


GLPTPQVDWN KIGGDLPKGR ETKENYGKTL KIENVSYQDK GNYRCTASNF LGTATHDFHV
300


 


IVEDNISHEL FTLHPEPPRW TKKPQSAVYS TGSNGILLCE AEGEPQPTIK WRVNGSPVDN
360


 


HPFAGDVVFP REISFTNLQP NHTAVYQCEA SNVHGTILAN ANIDVVDVRP LIQTKDGENY
420


 


ATVVGYSAFL HCEFFASPEA VVSWQKVEEV KPLEGRRYHI YENGTLQINR TTEEDAGSYS
480


 


CWVENAIGKT AVTANLDIRN ATKLRVSPKN PRIPKLHMLE LHCESKCDSH LKHSLKLSWS
540


 


KDGEAFEING TEDGRIIIDG ANLTISNVTL EDQGIYCCSA HTALDSAADI TQVTVLDVPD
600


 


PPENLHLSER QNRSVRLTWE AGADHNSNIS EYIVEFEGNK EEPGRWEELT RVQGKKTTVI
660


 


LPLAPFVRYQ FRVIAVNEVG RSQPSQPSDH HETPPAAPDR NPQNIRVQAS QPKEMIIKWE
720


 


PLKSMEQNGP GLEYRVTWKP QGAPVEWEEE TVTNHTLRVM TPAVYAPYDV KVQAINQLGS
780


 


GPDPQSVTLY SGEDYPDTAP VIHGVDVINS TLVKVTWSTV PKDRVHGRLK GYQINWWKTK
840


 


SLLDGRTHPK EVNILRFSGQ RNSGMVPSLD AFSEFHLTVL AYNSKGAGPE SEPYIFQTPE
900


 


GVPEQPTFLK VIKVDKDTAT LSWGLPKKLN GNLTGYLLQY QIINDTYEIG ELNDINITTP
960


 


SKPSWHLSNL NATTKYKFYL RACTSQGCGK PITEESSTLG EGSKGIGKIS GVNLTQKTHP
1020


 


IEVFEPGAEH IVRLMTKNWG DNDSIFQDVI ETRGREYAGL YDDISTQGWF IGLMCAIALL
1080


 


TLLLLTVCFV KRNRGGKYSV KEKEDLHPDP EIQSVKDETF GEYSDSDEKP LKGSLRSLNR
1140


 


DMQPTESADS LVEYGEGDHG LFSEDGSFIG AYAGSKEKGS VESNGSSTAT FPLRA
1195
















TABLE LVg





Amino acid sequence alignment of 282P1G03 v.1 (SEQ ID NO: 191) and


282P1G03 v.8 (SEQ ID NO: 192)





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE LIIh





Nucleotide sequence of 282P1G03 v.28 (SEQ ID NO: 193)
















cggaccctgc gcgcccccgt cccggctccc ggccggctcg ggggagaagg cgcccgaggg
60


 


gaggcgccgg acagatcgcg tttcggaggc ggcgcagttt ccaggttaac taaggtctca
120


 


gctgtaaacc aaaagtgaga ggagacatta agattttcat tcttaccggg ttgtcttctt
180


 


cctgaagagc aatggagccg cttttacttg gaagaggact aatcgtatat ctaatgttcc
240


 


tcctgttaaa attctcaaaa gcaattgaaa taccatcttc agttcaacag gttccaacaa
300


 


tcataaaaca gtcaaaagtc caagttgcct ttcccttcga tgagtatttt caaattgaat
360


 


gtgaagctaa aggaaatcca gaaccaacat tttcgtggac taaggatggc aacccttttt
420


 


atttcactga ccatcggata attccatcga acaattcagg aacattcagg atcccaaacg
480


 


aggggcacat atctcacttt caagggaaat accgctgctt tgcttcaaat aaactgggaa
540


 


tcgctatgtc agaagaaata gaatttatag ttccaagtgt tccaaaactc ccaaaagaaa
600


 


aaattgaccc tcttgaagtg gaggagggag atccaattgt cctcccatgc aatcctccca
660


 


aaggcctccc acctttacac atttattgga tgaatattga attagaacac atcgaacaag
720


 


atgaaagagt atacatgagc caaaagggag atctatactt cgcaaacgtg gaagaaaagg
780


 


acagtcgcaa tgactactgt tgctttgctg catttccaag attaaggact attgtacaga
840


 


aaatgccaat gaaactaaca gttaacagtt taaagcatgc taatgactca agttcatcca
900


 


cagaaattgg ttccaaggca aattccatca agcaaagaaa acccaaactg ctgttgcctc
960


 


ccactgagag tggcagtgag tcttcaatta ccatcctcaa aggggaaatc ttgctgcttg
1020


 


agtgttttgc tgaaggcttg ccaactccac aggttgattg gaacaaaatt ggtggtgact
1080


 


taccaaaggg gagagaaaca aaagaaaatt atggcaagac tttgaagata gagaatgtct
1140


 


cctaccagga caaaggaaat tatcgctgca cagccagcaa tttcttggga acagccactc
1200


 


acgattttca cgttatagta gaagagcctc ctcgctggac aaagaagcct cagagtgctg
1260


 


tgtatagcac cggaagcaat ggcatcttgt tatgtgaggc tgaaggagaa cctcaaccca
1320


 


caatcaagtg gagagtcaat ggctccccag ttgacaatca tccatttgct ggtgatgttg
1380


 


tcttccccag ggaaatcagt tttaccaacc ttcaaccaaa tcatactgct gtgtaccagt
1440


 


gtgaagcctc aaatgtccat ggaactatcc ttgccaatgc caatattgat gttgtggatg
1500


 


tccgtccatt gatacaaacc aaagatggag aaaattacgc tacagtggtt gggtacagtg
1560


 


ctttcttaca ttgcgagttc tttgcttcac ctgaggcagt cgtgtcctgg cagaaggtgg
1620


 


aagaagtgaa acccctggag ggcaggcggt atcatatcta tgaaaatggc acattgcaga
1680


 


tcaacagaac caccgaagaa gatgctgggt cttactcatg ttgggtagaa aatgctatag
1740


 


gaaaaactgc agtcacagcc aatttggata ttagaaatgc tacaaaactt agagtttctc
1800


 


ctaagaatcc tcgtatcccc aaattgcata tgcttgaatt acattgtgaa agcaaatgtg
1860


 


actcacattt gaaacacagt ttgaagttgt cctggagtaa agatggagaa gcctttgaaa
1920


 


ttaatggcac agaagatggc aggataatta ttgatggagc taatttgacc atatctaatg
1980


 


taactttaga ggaccaaggt atttactgct gttcagctca tactgctcta gacagtgctg
2040


 


ccgatataac tcaagtaact gttcttgatg ttccggatcc accagaaaac cttcacttgt
2100


 


ctgaaagaca gaacaggagt gttcggctga cctgggaagc tggagctgac cacaacagca
2160


 


atattagcga gtatattgtt gaatttgaag gaaacaaaga agagcctgga aggtgggagg
2220


 


aactgaccag agtccaagga aagaaaacca cagttatctt acctttggct ccatttgtga
2280


 


gataccagtt cagggtcata gccgtgaacg aagtagggag aagtcagcct agccagccgt
2340


 


cagaccatca tgaaacacca ccagcagctc cagataggaa tccacaaaac ataagggttc
2400


 


aagcctctca acccaaggaa atgattataa agtgggagcc tttgaaatcc atggagcaga
2460


 


atggaccagg cctagagtac agagtgacct ggaagccaca gggagcccca gtggagtggg
2520


 


aagaagaaac agtcacaaac cacacattgc gggtgatgac gcctgctgtc tatgcccctt
2580


 


atgatgtcaa ggtccaggct atcaatcaac taggatctgg gcctgaccct cagtcagtga
2640


 


ctctctattc tggagaagac tatcctgata cagctccagt gatccatggg gtggacgtta
2700


 


taaacagtac attagttaaa gttacctggt caacagttcc aaaggacaga gtacatggac
2760


 


gtctgaaagg ctatcagata aattggtgga aaacaaaaag tctgttggat ggaagaacac
2820


 


atcccaaaga agtgaacatt ctaagatttt caggacaaag aaactctgga atggttcctt
2880


 


ccttagatgc ctttagtgaa tttcatttaa cagtcttagc ctataactct aaaggagctg
2940


 


gtcctgaaag tgagccttat atatttcaaa caccagaagg agtacctgaa cagccaactt
3000


 


ttctaaaggt catcaaagtt gataaagaca ctgccacttt atcttgggga ctacctaaga
3060


 


aattaaatgg aaacttaact ggctatcttt tgcaatatca gataataaat gacacctacg
3120


 


agattggaga attaaatgat attaacatta caactccatc aaagcccagc tggcacctct
3180


 


caaacctgaa tgcaactacc aagtacaaat tctacttgag ggcttgcact tcacagggct
3240


 


gtggaaaacc gatcacggag gaaagctcca ccttaggaga agggagtaaa ggtatcggga
3300


 


agatatcagg agtaaatctt actcaaaaga ctcacccaat agaggtattt gagccgggag
3360


 


ctgaacatat agttcgccta atgactaaga attggggcga taacgatagc atttttcaag
3420


 


atgtaattga gacaagaggg agagaatatg ctggtttata tgatgacatc tccactcaag
3480


 


gctggtttat tggactgatg tgtgcgattg ctcttctcac actactatta ttaactgttt
3540


 


gctttgtgaa gaggaataga ggtggaaagt actcagttaa agaaaaggaa gatttgcatc
3600


 


cagacccaga aattcagtca gtaaaagatg aaacctttgg tgaatacagt gacagtgatg
3660


 


aaaagcctct caaaggaagc cttcggtccc ttaataggga tatgcagcct actgaaagtg
3720


 


ctgacagctt agtcgaatac ggagagggag accatggtct cttcagtgaa gatggatcat
3780


 


ttattggtgc ctacgctgga tctaaggaga agggatctgt tgaaagcaat ggaagttcta
3840


 


cagcaacttt tccccttcgg gcataaacac aacatatgta agcaacgcta ctggttcacc
3900


 


ccaaccttcc atatttatct gttcaaagga gcaagaactt tcatatagga atagaaacat
3960


 


gctggccgaa gatttcatcc agaagtcaac atcctgcaat tatgttgaaa agagtagtac
4020


 


tttcttcaaa atataaaatg ccaagcactt caggcctatg ttttgcttat attgttttca
4080


 


ggtgctcaaa atgcaaaaca caaaacaaat cctgcattta gatacacctc aactaaatcc
4140


 


aaagtcccca ttcagtatat tccatatttg cctgatttta ctattcggtg tgtttgcata
4200


 


gatgttgcta cttggtgggt ttttctccgt atgcacattg gtatacagtc tctgagaact
4260


 


ggcttggtga ctttgcttca ctacaggtta aaagaccata agcaaactgg ttatttaaaa
4320


 


tgtaaaaagg aatatgaaag tcttattaaa acacttcatt gaaaatatac agtctaaatt
4380


 


tattatttaa attttactag caaaagtctt aggtgaacaa tcaactagta tttgttgagc
4440


 


tcctatttgc ccagagatgg tcatatttaa acagaagtat acgtttttca gtttcaacat
4500


 


gaattttttt atttctgtca gttatgacat ccacgagcat cactttttgt gtctgttttt
4560


 


ttttttttct tggactaaat tcaactgcat ggaagcggtg gtcagaaggt tgttttatac
4620


 


gagaacaggc agaaagtgcc cattgttcag gattctaata gctacatcta cttaatatct
4680


 


tcatttctaa attgactgct tttacctttt tctcatgttt atataatggt atgcttgcat
4740


 


atatttcatg aatacattgt acatattatg ttaatattta cacaatttaa aatatagatg
4800


 


tgttttattt tgaagtgaga aaatgaacat taacaggcat gtttgtacag ctagaatata
4860


 


ttagtaagat actgtttttc gtcattccag agctacaact aataacacga ggttccaaag
4920


 


ctgaagactt tgtataaagt atttgggttt tgttcttgta ttgctttctt tcaacagttt
4980


 


caaaataaaa tatcatacaa atattgaggg aaatgttttc atatttttca aaataggttt
5040


 


ttattgttga atgtacatct accccagccc ctcaaaagaa aaactgttta catagaaatt
5100


 


cctacacata cgtttgcgta tatgttattt taaacatctt tgtggtgaga attttttccc
5160


 


cgatattctc cttctgtcaa agtcagaaca aattcaggga atttattttc tggcagttgt
5220


 


gctccagtcc ttttaaaatt gtacatgaac atgttttaga aacaatatgg aggatgatgc
5280


 


atacatgtcg gtcaagttca gcgctcgaca ttttatggaa agattttttt aaccttacca
5340


 


cgaaatactt aactactgtt taagtgaatt gacttatttc actttagttt ttgaactgtg
5400


 


attattggta tactgttata tcctcaactt ggatttatgg taaccccttt tagttcatgg
5460


 


agaccaaaat ttggggtatt tataatagtc agcgcaggaa tgcacatgga atatctactt
5520


 


gtccttttga acctcacgag tcatccagaa tgtatagaca ggaaaagcat gtcttattta
5580


 


aaactgtaat ttatgggctc aggatctgac cgcagtcccg ggagtaagca tttcaaaggg
5640


 


ggaaggcagt gtggtcccta ccctgtgtga atgtgaggat gtagacatcc atcagtgcaa
5700


 


ctcgagctcc atcctcctcc gatttctaag gctccagttt tctggaggga cagtcatcat
5760


 


gttttgattt atctgggaga aaactgtggt gcacagcttg tgaggagggc aaggttgtga
5820


 


cgttcgagct tagttctggt gttattctgt ctcctcttct ttgtcatcag ccaaaacgtg
5880


 


gtttttaaag agagtcatgc aggttagaaa taatgtcaaa aatatttagg aatttaataa
5940


 


cctttaagtc agaaactaaa acaaatactg aaatattagc tcttcctaca cttcgtgttc
6000


 


ccctttagct gcctgaaaat caagattgct cctactcaga tcttctgagt ggctaaaact
6060


 


tatggatatg aaaaatgaga ttgaatgatg actatgcttt gctatcattg ttacctttcc
6120


 


tcaatactat ttggcaacta ctgggactct tcagcacaaa aggaatagat ctatgattga
6180


 


ccctgatttt aattgtgaaa ttatatgatt catatatttt atgaatcaga ataaccttca
6240


 


aataaaataa atctaagtcg gttaaaatgg atttcatgat tttccctcag aaaatgagta
6300


 


acggagtcca cggcgtgcaa tggtaattat aaattggtga tgcttgtttg caaattgccc
6360


 


actcgtgata agtcaacagc caatatttaa aactttgttc gttactggct ttaccctaac
6420


 


tttctctagt ctactgtcaa tatcatttta atgtaattga ttgtatatag tctcaagaat
6480


 


ggttggtggg catgagttcc tagagaactg tccaagggtt gggaaaatcc aaattctctt
6540


 


cctggctcca gcactgattt tgtacataaa cattaggcag gttgcttaac ctttttattt
6600


 


caaactctct caactctaaa gtgctaataa taatctcagt taccttatct ttgtcacagg
6660


 


gtgttctttt ttatgaagaa aaatttgaaa atgataaaag ctaagatgcc ttctaacttc
6720


 


ataagcaaac ctttaactaa ttatgtatct gaaagtcacc cccacatacc aactcaactt
6780


 


ttttcctgtg aacacataaa tatattttta tagaaaaaca aatctacata aaataaatct
6840


 


actgtttagt gagcagtatg acttgtacat gccattgaaa attattaatc agaagaaaat
6900


 


taagcagggt ctttgctata caaaagtgtt ttccactaat tttgcatgcg tatttataag
6960


 


aaaaatgtga atttggtggt tttattctat cggtataaag gcatcgatat tttagatgca
7020


 


cccgtgtttg taaaaatgta gagcacaatg gaattatgct ggaagtctca aataatattt
7080


 


ttttcctatt ttatactcat ggaagagata agctaaagag gggacaataa tgagaaatgt
7140


 


tggtgtgctt ttctaagcat ttaaaacata attgccaatt gaaaccctaa atatgtttac
7200


 


ataccattaa gatatgattc atgtaacaat gttaaattaa ttataatggg attgggtttg
7260


 


ttatctgtgg tagtatatat cctagtgttc ctatagtgaa ataagtaggg ttcagccaaa
7320


 


gctttctttg ttttgtacct taaattgttc gattacgtca tcaaaagaga tgaaaggtat
7380


 


gtagaacagg ttcacgtgat tacctttttc ttttggcttg gattaatatt catagtagaa
7440


 


ctttataaaa cgtgtttgta ttgtaggtgg tgtttgtatt atgcttatga ctatgtatgg
7500


 


tttgaaaata ttttcattat acatgaaatt caactttcca aataaaagtt ctacttcatg
7560


 


taatccaaaa
7570
















TABLE LIIIh





Nucleotide sequence alignment of 282P1G03 v.1 (SEQ ID NO: 194) and


282P1G03 v.28 (SEQ ID NO: 195)





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE LIVh





Peptide sequences of protein coded by 282P1G03 v.28 (SEQ ID NO: 196)
















MEPLLLGRGL IVYLMFLLLK FSKAIEIPSS VQQVPTIIKQ SKVQVAFPFD EYFQIECEAK
60


 


GNPEPTFSWT KDGNPFYFTD HRIIPSNNSG TFRIPNEGHI SHFQGKYRCF ASNKLGIAMS
120


 


EEIEFIVPSV PKLPKEKIDP LEVEEGDPIV LPCNPPKGLP PLHIYWMNIE LEHIEQDERV
180


 


YMSQKGDLYF ANVEEKDSRN DYCCFAAFPR LRTIVQKMPM KLTVNSLKHA NDSSSSTEIG
240


 


SKANSIKQRK PKLLLPPTES GSESSITILK GEILLLECFA EGLPTPQVDW NKIGGDLPKG
300


 


RETKENYGKT LKIENVSYQD KGNYRCTASN FLGTATHDFH VIVEEPPRWT KKPQSAVYST
360


 


GSNGILLCEA EGEPQPTIKW RVNGSPVDNH PFAGDVVFPR EISFTNLQPN HTAVYQCEAS
420


 


NVHGTILANA NIDVVDVRPL IQTKDGENYA TVVGYSAFLH CEFFASPEAV VSWQKVEEVK
480


 


PLEGRRYHIY ENGTLQINRT TEEDAGSYSC WVENAIGKTA VTANLDIRNA TKLRVSPKNP
540


 


RIPKLHMLEL HCESKCDSHL KHSLKLSWSK DGEAFEINGT EDGRIIIDGA NLTISNVTLE
600


 


DQGIYCCSAH TALDSAADIT QVTVLDVPDP PENLHLSERQ NRSVRLTWEA GADHNSNISE
660


 


YIVEFEGNKE EPGRWEELTR VQGKKTTVIL PLAPFVRYQF RVIAVNEVGR SQPSQPSDHH
720


 


ETPPAAPDRN PQNIRVQASQ PKEMIIKWEP LKSMEQNGPG LEYRVTWKPQ GAPVEWEEET
780


 


VTNHTLRVMT PAVYAPYDVK VQAINQLGSG PDPQSVTLYS GEDYPDTAPV IHGVDVINST
840


 


LVKVTWSTVP KDRVHGRLKG YQINWWKTKS LLDGRTHPKE VNILRFSGQR NSGMVPSLDA
900


 


FSEFHLTVLA YNSKGAGPES EPYIFQTPEG VPEQPTFLKV IKVDKDTATL SWGLPKKLNG
960


 


NLTGYLLQYQ IINDTYEIGE LNDINITTPS KPSWHLSNLN ATTKYKFYLR ACTSQGCGKP
1020


 


ITEESSTLGE GSKGIGKISG VNLTQKTHPI EVFEPGAEHI VRLMTKNWGD NDSIFQDVIE
1080


 


TRGREYAGLY DDISTQGWFI GLMCAIALLT LLLLTVCFVK RNRGGKYSVK EKEDLHPDPE
1140


 


IQSVKDETFG EYSDSDEKPL KGSLRSLNRD MQPTESADSL VEYGEGDHGL FSEDGSFIGA
1200


 


YAGSKEKGSV ESNGSSTATF PLRA
1224
















TABLE LVh





Amino acid sequence alignment of 282P1G03 v.1 (SEQ ID NO: 197)


and 282P1G03 v.28 (SEQ ID NO: 198)

## Claims

1. An isolated protein comprising the amino acid sequence of SEQ ID NO:3.

2. A composition comprising an excipient and the protein of claim 1.

3. A method of generating a mammalian immune response directed to a protein, comprising: exposing cells of the mammal's immune system to the protein of claim 1, whereby the mammalian immune response to the protein is elicited, wherein the immune response is activation of a B cell, whereby the induced B cell generates antibodies that specifically bind to the protein.