# Patent

## Title
Nucleic acid and corresponding protein entitled 193P1E1B useful in treatment and detection of cancer

## Abstract

A novel gene 0193P1E1B (also designated 193P1E1B) and its encoded protein, and variants thereof, are described wherein 193P1E1B exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, 193P1E1B provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The 193P1E1B gene or fragment thereof, or its encoded protein, or variants thereof, or a fragment thereof, can be used to elicit a humoral or cellular immune response; antibodies or T cells reactive with 193P1E1B can be used in active or passive immunization.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority from pending U.S. patent application Ser. No. 10/013,312, filed 7 Dec. 2001. The contents of the application listed in this paragraph is 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 a gene and its encoded protein, termed 193P1E1B, expressed in certain cancers, and to diagnostic and therapeutic methods and compositions useful in the management of cancers that express 193P1E1B.
BACKGROUND OF THE INVENTION
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 Sep. 2, 1996 (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 193P1 E1 B, that has now been found to be over-expressed in the cancer(s) listed in Table I. Northern blot expression analysis of 193P1E1B gene expression in normal tissues shows a restricted expression pattern in adult tissues. The nucleotide (FIG. 2) and amino acid (FIG. 2, and FIG. 3) sequences of 193P1E1B are provided. The tissue-related profile of 193P1 E1B in normal adult tissues, combined with the over-expression observed in the tissues listed in Table I, shows that 193P1 E1B is aberrantly over-expressed in at least some cancers, and thus serves as a useful diagnostic, prophylactic, prognostic, and/or therapeutic target for cancers of the tissue(s) such as those listed in Table I.
The invention provides polynucleotides corresponding or complementary to all or part of the 193P1E1B genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding 193P1E1B-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100 or more than 100 contiguous amino acids of a 193P1E1B-related protein, as well as the peptides/proteins themselves; DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides or oligonucleotides complementary or having at least a 90% homology to the 193P1E1B genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the 193P1E1B genes, mRNAs, or to 193P1E1B-encoding polynucleotides. Also provided are means for isolating cDNAs and the genes encoding 193P1E1B. Recombinant DNA molecules containing 193P1E1B polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 193P1E1B gene products are also provided. The invention further provides antibodies that bind to 193P1E1B proteins and polypeptide fragments thereof, including polyclonal and monoclonal antibodies, murine and other mammalian antibodies, chimeric antibodies, humanized and fully human antibodies, and antibodies labeled with a detectable marker or therapeutic agent. In certain embodiments, there is a proviso that the entire nucleic acid sequence of FIG. 2 is not encoded and/or the entire amino acid sequence of FIG. 2 is not prepared. In certain embodiments, the entire nucleic acid sequence of FIG. 2 is encoded and/or the entire amino acid sequence of FIG. 2 is prepared, either of which are in respective human unit dose forms.
The invention further provides methods for detecting the presence and status of 193P1E1B polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 193P1E1B. A typical embodiment of this invention provides methods for monitoring 193P1E1B gene products in a issue 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 193P1E1B such as cancers of tissues listed in Table I, including therapies aimed at inhibiting the transcription, translation, processing or function of 193P1E1B as well as cancer vaccines. In one aspect, the invention provides compositions, and methods comprising them, for treating a cancer that expresses 193P1E1B in a human subject wherein the composition comprises a carrier suitable for human use and a human unit dose of one or more than one agent that inhibits the production or function of 193P1E1B. Preferably, the carrier is a uniquely human carrier. In another aspect of the invention, the agent is a moiety that is immunoreactive with 193P1E1B protein. Non-limiting examples of such moieties include, but are not limited to, antibodies (such as single chain, monoclonal, polyclonal, humanized, chimeric, or human antibodies), functional equivalents thereof (whether naturally occurring or synthetic), and combinations thereof. The antibodies can be conjugated to a diagnostic or therapeutic moiety. In another aspect, the agent is a small molecule as defined herein.
In another aspect, the agent comprises one or more than one peptide which comprises a cytotoxic T lymphocyte (CTL) epitope that binds an HLA class I molecule in a human to elicit a CTL response to 193P1E1B and/or one or more than one peptide which comprises a helper T lymphocyte (HTL) epitope which binds an HLA class II molecule in a human to elicit an HTL response. The peptides of the invention may be on the same or on one or more separate polypeptide molecules. In a further aspect of the invention, the agent comprises one or more than one nucleic acid molecule that expresses one or more than one of the CTL or HTL response stimulating peptides as described above. In yet another aspect of the invention, the one or more than one nucleic acid molecule may express a moiety that is immunologically reactive with 193P1E1B as described above. The one or more than one nucleic acid molecule may also be, or encodes, a molecule that inhibits production of 193P1E1B. Non-limiting examples of such molecules include, but are not limited to, those complementary to a nucleotide sequence essential for production of 193P1E1B (e.g. antisense sequences or molecules that form a triple helix with a nucleotide double helix essential for 193P1E1B production) or a ribozyme effective to lyse 193P1E1B mRNA.
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 193P1E1B SSH sequence of 227 nucleotides.
FIG. 2. A) The cDNA and amino acid sequence of 193P1E1B variant 1 (also called “193P1E1B v.1” or “193P1E1B variant 1”) is shown in FIG. 2A. The start methionine is underlined. The open reading frame extends from nucleic acid 805-2043 including the stop codon.
B) The cDNA and amino acid sequence of 193P1E1B variant 2 (also called “193P1E1B v.2”) is shown in FIG. 2B. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 805-2043 including the stop codon.
C) The cDNA and amino acid sequence of 193P1E1B variant 3 (also called “193P1E1B v.3”) is shown in FIG. 2C. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 805-2043 including the stop codon.
D) The cDNA and amino acid sequence of 193P1E1B variant 4 (also called “193P1E1B v.4”) is shown in FIG. 2D. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 805-2043 including the stop codon.
E) The cDNA and amino acid sequence of 193P1E1B variant 5 (also called “193P1E1B v.5”) is shown in FIG. 2E. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 805-2043 including the stop codon.
F) The cDNA and amino acid sequence of 193P1E1B variant 6 (also called “193P1E1B v.6”) is shown in FIG. 2F. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 805-2043 including the stop codon.
G) The cDNA and amino acid sequence of 193P1E1B variant 7 (also called “193P1E1B v.7”) is shown in FIG. 2G. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 805-2043 including the stop codon.
H) The cDNA and amino acid sequence of 193P1E1B variant 8 (also called “193P1E1B v.8”) is shown in FIG. 2H. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 805-2043 including the stop codon.
I) The cDNA and amino acid sequence of 193P1E1B variant 9 (also called “193P1E1B v.9”) is shown in FIG. 21. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 989-1981 including the stop codon.
J) The cDNA and amino acid sequence of 193P1E1B variant 10 (also called “193P1E1B v.10”) is shown in FIG. 2J. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 805-1971 including the stop codon.
K) The cDNA and amino acid sequence of 193P1E1B variant 11 (also called “193P1E1B v.11”) is shown in FIG. 2K. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 989-1909 including the stop codon.
L) The cDNA and amino acid sequence of 193P1E1B variant 12 (also called “193P1E1B v.12”) is shown in FIG. 2L. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 805-1026 including the stop codon.
M) The cDNA and amino acid sequence of 193P1E1B variant 13 (also called “193P1E1B v.13”) is shown in FIG. 2M. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 952-2070 including the stop codon.
FIG. 3.
A) Amino acid sequence of 193P1E1B v.1 is shown in FIG. 3A; it has 412 amino acids.
B) The amino acid sequence of 193P1E1B v.5 is shown in FIG. 3B; it has 412 amino acids.
C) The amino acid sequence of 193P1E1B v.6 is shown in FIG. 3C; it has 412 amino acids.
D) The amino acid sequence of 193P1E1B v.9 is shown in FIG. 3D; it has 330 amino acids.
E) The amino acid sequence of 193P1E1B v.10 is shown in FIG. 3E; it has 388 amino acids.
F) The amino acid sequence of 193P1E1B v.11 is shown in FIG. 3F; it has 308 amino acids.
G) The amino acid sequence of 193P1E1B v.12 is shown in FIG. 3G; it has 73 amino acids.
H) The amino acid sequence of 193P1E1B v.13 is shown in FIG. 3H; it has 372 amino acids. As used herein, a reference to 193P1E1B includes all variants thereof, including those shown in FIGS. 2, 3, 10, and 11, unless the context clearly indicates otherwise.
FIG. 4. FIG. 4A shows the alignment of 193P1E1B v.1 with gi 2178775. FIG. 4B shows the alignment of 193P1E1B v.5 with gi 2178775. FIG. 4C shows the alignment of 193P1E1B v.11 with gi 2178775. FIG. 4D shows the alignment of 193P1E1B v.12 with gi 2178775. FIG. 4E shows the alignment of 193P1E1B v.1 with E. coli arginine repressor. FIG. 4F shows the Alignment of 193P1E1B v.1 with human adenosine deaminase. FIG. 4G shows the Clustal alignment of 193P1E1B protein variants.
FIG. 5. Hydrophilicity amino acid profile of 193P1E1B determined by computer algorithm sequence analysis using the method of Hopp and Woods (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828) accessed on the Protscale website located on the World Wide Web through the ExPasy molecular biology server.
FIG. 6. Hydropathicity amino acid profile of 193P1E1B determined by computer algorithm sequence analysis using the method of Kyte and Doolittle (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132) accessed on the ProtScale website located on the World Wide Web through the ExPasy molecular biology server.
FIG. 7. Percent accessible residues amino acid profile of 193P1E1B determined by computer algorithm sequence analysis using the method of Janin (Janin J., 1979 Nature 277:491-492) accessed on the ProtScale website located on the World Wide Web through the ExPasy molecular biology server.
FIG. 8. Average flexibility amino acid profile of 193P1E1B determined by computer algorithm sequence analysis using the method of Bhaskaran and Ponnuswamy (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255) accessed on the ProtScale website located on the World Wide Web through the ExPasy molecular biology server.
FIG. 9. Beta-turn amino acid profile of 193P1E1B determined by computer algorithm sequence analysis using the method of Deleage and Roux (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294) accessed on the ProtScale website located on the World Wide Web through the ExPasy molecular biology server.
FIG. 10. Schematic alignment of SNP variants of 193P1E1B. Variants 193P1E1B v.2 through v.8 are variants with single nucleotide differences. Though these SNP variants are shown separately, they could also occur in any combinations and in any transcript variants that contains the base pairs. Numbers correspond to those of 193P1E1B v.1. Black box shows the same sequence as 193P1E1B v. 1. SNPs are indicated above the box.
FIG. 11. Schematic alignment of protein variants of 193P1E1B. Protein variants correspond to nucleotide variants. Nucleotide variants 193P1E1B v.2, v.3, v.4, v.7, and v.8 in FIG. 10 code for the same protein as 193P1E1B v.1. Nucleotide variants 193P1E1B v.9 through v.13 are splice variants of v.1. Single amino acid differences were indicated above the boxes. Black boxes represent the same sequence as 193P1E1B v.1. Numbers underneath the box correspond to amino acid positions in 193P1E1B v.1.
FIG. 12. Intentionally Omitted.
FIG. 13. Secondary structure prediction for 193P1E1B (SEQ ID NO: 123). The secondary structure of 193P1E1B protein was predicted using the HNN-Hierarchical Neural Network method , accessed from the ExPasy molecular biology server. This method predicts the presence and location of alpha helices, extended strands, and random coils from the primary protein sequence. The percent of the protein in a given secondary structure is as follows: h: Alpha helix 29.13%; c: Random coil 60.92%; e: Extended strand 9.95%.
FIG. 14. Expression of 193P1E1B by RT-PCR. (A) The schematic diagram depicts the location of PCR primers Set A and set B on the sequences of the 3 variants of 193P1E1B. (B and C) First strand cDNA was prepared from vital pool 1 (VP1: liver, lung and kidney), vital pool 2 (VP2, pancreas, colon and stomach), prostate xenograft pool (LAPC-4AD, LAPC-4AI, LAPC-9AD, LAPC-9AI), normal thymus, prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, metastasis cancer pool, pancreas cancer pool, and from prostate cancer metastasis to lymph node from 2 different patients. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primer Set A (B) or primer Set B (C) to 193P1E1B, was performed at 30 cycles of amplification. Strong expression of 193P1E1B was observed in prostate cancer xenograft pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, metastasis pool, pancreas cancer pool, and in the 2 different prostate cancer metastasis to lymph node. Low expression was observed in prostate cancer pool, but no expression was detected in VP1 and VP2. FIG. 14C shows that the transcipt encoding encoding 193P1E1B v. 1 through v.8, is expressed ate higher levels that the transcript encoding 193P1E1B v.9. But both transcripts are expressed at similar proportion in all tissues tested.
FIG. 15. Expression of 193P1E1B in normal human tissues. Two multiple tissue northern blots, with 2 μg of mRNA/lane were probed with 193P1E1B sequence. Size standards in kilobases (kb) are indicated on the side. The results show expression of two 193P1E1B transcripts, approximately 3.5 kb and 2 kb, in testis and thymus.
FIG. 16. Expression of 193P1E1B in prostate cancer xenografts. RNA was extracted from normal prostate, and from prostate cancer xenografts, LAPC-4AD, LAPC4AI, LAPC-9AD, and LAPC-9AI. Northern blot with 10 μg of total RNA/lane was probed with 193P1E1B sequence. Size standards in kilobases (kb) are indicated on the side. The results show expression of 193P1E1B in all 4 xenografts but not in normal prostate.
FIG. 17. Expression of 193P1E1B in patient cancer specimens. RNA was extracted from a pool of three patients for each of the following, bladder cancer, colon cancer, ovary cancer and metastasis cancer, as well as from normal prostate (NP), normal bladder (NB), normal kidney (NK), normal colon (NC). Northern blots with 10 μg of total RNA/lane were probed with 193P1E1B sequence. Size standards in kilobases (kb) are indicated on the side. The results show expression of 193P1E1B in bladder cancer pool, colon cancer pool, ovary cancer pool and metastasis cancer pool, but not in any of the normal tissues tested.
FIG. 18. Expression of 193P1E1B in bladder cancer patient specimens. RNA was extracted from bladder cancer cell lines (CL), normal bladder (N), bladder tumors (T) and matched normal adjacent tissue (NAT) isolated from bladder cancer patients. Northern blots with 10 μg of total RNA/lane were probed with 193P1E1B sequence. Size standards in kilobases (kb) are indicated on the side. The results show expression of 193P1E1B in the two bladder cancer cell lines, and in 3 patient bladder tumors tested but not in normal bladder tissues.
FIG. 19. Expression of 193P1E1B in cancer metastasis patient specimens. RNA was extracted from the following cancer metastasis tissues, colon metastasis to lung, lung metastasis to lymph node, lung metastasis to skin, and breast metastasis to lymph node, as well as from normal bladder (NB), normal lung (NL), normal breast (NBr), and normal ovary (NO). Northern blots with 10 μg of total RNA/lane were probed with 193P1E1B sequence. Size standards in kilobases (kb) are indicated on the side. The results show expression of 193P1E1B in all four different cancer metastasis samples but not in normal tissues.
FIG. 20. Expression of 193P1E1B in pancreas, ovary and testis cancer patient specimens. RNA was extracted from pancreatic cancer (P1), ovarian cancer (P2, P3), and testicular cancer (P4, P5) isolated from cancer patients, as well as from normal pancreas (NPa). Northern blots with 10 μg of total RNA/lane were probed with 193P1E1B sequence. Size standards in kilobases (kb) are indicated on the side. The results show expression of 193P1E1B in pancreatic, ovarian and testicular cancer specimens but not in normal pancreas.
FIG. 21. Expression of 193P1E1B in Normal versus Patient Cancer Specimens. First strand cDNA was prepared from a panel of normal tissues (stomach, brain, heart, liver, spleen, skeletal muscle, tests prostate, bladder, kidney, colon, lung and pancreas) and from a panel of patient cancer pools (prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, pancreas cancer pool, ovary cancer pool, breast cancer pool, metastasis cancer pool, LAPC prostate xenograft pool (XP), and from prostate cancer metastasis to lymph node from 2 different patients (PMLN2). Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primer Set A as described in FIG. 14, was performed was performed at 26 and 30 cycles of amplification. Samples were run on an agarose gel, and PCR products were quantitated using the Alphalmager software. Relative expression was calculated by normalizing to signal obtained using actin primers. Results show restricted 193P1E1B expression in normal testis amongst all normal tissues tested. 193P1E1B expression was strongly upregulated in cancers of the bladder, colon, lung, pancreas, ovary, breast, and to a lesser extent in prostate and kidney cancers.
FIG. 22. Expression of 193P1E1B in Normal versus Patient Cancer Specimens. First strand cDNA was prepared from a panel of normal tissues (stomach, brain, heart, liver, spleen, skeletal muscle, testis prostate, bladder, kidney, colon, lung and pancreas) and from a panel of patient cancer pools (prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, pancreas cancer pool, ovary cancer pool, breast cancer pool, metastasis cancer pool, LAPC prostate xenograft pool (XP), and from prostate cancer metastasis to lymph node from 2 different patients (PMLN2). Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primer Set A as described in FIG. 14, was performed was performed at 26 and 30 cycles of amplification. Samples were run on an agarose gel, and PCR products were quantitated using the Alphalmager software. Relative expression was calculated by normalizing to signal obtained using actin primers. Results show restricted 193P1E1B expression in normal testis amongst all normal tissues tested. 193P1E1B expression was strongly upregulated in cancers of the bladder, colon, lung, pancreas, ovary, breast, and to a lesser extent in prostate and kidney cancers.



DETAILED DESCRIPTION OF THE INVENTION
Outline of Sections
I.) Definitions
II.) 193P1E1B Polynucleotides
II.A.) Uses of 193P1E1B 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 193P1E1B-Encoding Nucleic Acid Molecules
II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems
III.) 193P1E1B-related Proteins

    
    
        III.A.) Motif-bearing Protein Embodiments
        III.B.) Expression of 193P1E1B-related Proteins
        III.C.) Modifications of 193P1E1B-related Proteins
        III.D.) Uses of 193P1E1B-related Proteins
    
    


IV.) 193P1E1B Antibodies
V.) 193P1E1B Cellular Immune Responses
VI.) 193P1E1B Transgenic Animals
VII.) Methods for the Detection of 193P1E1B
VIII.) Methods for Monitoring the Status of 193P1E1B-related Genes and Their Products
IX.) Identification of Molecules That Interact With 193P1E1B
X.) Therapeutic Methods and Compositions
X.A.) Anti-Cancer Vaccines
X.B.) 193P1E1B as a Target for Antibody-Based Therapy
X.C.) 193P1E1B 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 193P1E1B.
XII.) Inhibition of 193P1E1B Protein Function

    
    
        XII.A.) Inhibition of 193P1E1B With Intracellular Antibodies
        XII.B.) Inhibition of 193P1E1B with Recombinant Proteins
        XII.C.) Inhibition of 193P1E1B Transcription or Translation
        XII.D.) General Considerations for Therapeutic Strategies
    
    


XIII.) Identification, Characterization and Use of Modulators of 193P1E1B
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-Jewell 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 193P1E1B (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence 193P1E1B. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.
The term “analog” refers to a molecule which is structurally similar or shares similar or corresponding attributes with another molecule (e.g. a 193P1E1B-related protein). For example, an analog of a 193P1E1B protein can be specifically bound by an antibody or T cell that specifically binds to 193P1E1B.
The term “antibody” is used in the broadest sense. Therefore, an “antibody” can be naturally occurring or man-made such as monoclonal antibodies produced by conventional hybridoma technology. Anti-193P1E1B antibodies comprise monoclonal and polyclonal antibodies as well as fragments containing the antigen-binding domain and/or one or more complementarity determining regions of these antibodies.
An “antibody fragment” is defined as at least a portion of the variable region of the immunoglobulin molecule that binds to its target, i.e., the antigen-binding region. In one embodiment it specifically covers single anti-193P1E1B antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-193P1E1B antibody compositions with polyepitopic specificity.
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)), oligocarbarnates (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/US96110287), 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, Jan 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, Mass.). A number of well-known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations such as the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate H, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.), which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art. In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J.; Asinex, Moscow, RU; 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, Sapaonana 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 193P1E1B genes or that encode polypeptides other than 193P1E1B gene product or fragments thereof. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated 193P1E1B polynucleotide. A protein is said to be “isolated,” for example, when physical, mechanical or chemical methods are employed to remove the 193P1E1B proteins from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated 193P1E1B protein. Alternatively, an isolated protein can be prepared by chemical means.
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 TxNxM+ under the TNM system. As is the case with locally advanced prostate cancer, surgery is generally not indicated for patients with metastatic disease, and hormonal (androgen ablation) therapy is a preferred treatment modality. Patients with metastatic prostate cancer eventually develop an androgen-refractory state within 12 to 18 months of treatment initiation. Approximately half of these androgen-refractory patients die within 6 months after developing that status. The most common site for prostate cancer metastasis is bone. Prostate cancer bone metastases are often osteoblastic rather than osteolytic (i.e., resulting in net bone formation). Bone metastases are found most frequently in the spine, followed by the femur, pelvis, rib cage, skull and humerus. Other common sites for metastasis include lymph nodes, lung, liver and brain. Metastatic prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body radionuclide scans, skeletal radiography, and/or bone lesion biopsy.
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 193P1E1B-related protein, refers to any pattern of amino acids forming part of the primary sequence of a protein, that is associated with a particular function (e.g. protein-protein interaction, protein-DNA interaction, etc) or modification (e.g. that is phosphorylated, glycosylated or amidated), or localization (e.g. secretory sequence, nuclear localization sequence, etc.) or a sequence that is correlated with being immunogenic, either humorally or cellularly. A motif can be either contiguous or capable of being aligned to certain positions that are generally correlated with a certain function or property. In the context of HLA motifs, “motif” refers to the pattern of residues in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino acids for a class II HLA motif, which is recognized by a particular HLA molecule. Peptide motifs for HLA binding are typically different for each protein encoded by each human HLA allele and differ in the patter 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

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

Actinium-227

    (AC-227)
    Parent of Radium-223 (Ra-223) which is an alpha emitter used to treat metastases in the skeleton resulting from cancer (i.e., breast and prostate cancers), and cancer radioimmunotherapy

Bismuth-212

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

Bismuth-213

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

Cadmium-109

    (Cd-109)
    Cancer detection

Cobalt-60

    (Co-60)
    Radiation source for radiotherapy of cancer, for food irradiators, and for sterilization of medical supplies

Copper-64

    (Cu-64)
    A positron emitter used for cancer therapy and SPECT imaging

Copper-67

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

Dysprosium-166

    (Dy-166)
    Cancer radioimmunotherapy

Erbium-169

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

Europium-152

    (Eu-152)
    Radiation source for food irradiation and for sterilization of medical supplies

Europium-154

    (Eu-154)
    Radiation source for food irradiation and for sterilization of medical supplies

Gadolinium-153

    (Gd-153)
    Osteoporosis detection and nuclear medical quality assurance devices

Gold-198

    (Au-198)
    Implant and intracavity therapy of ovarian, prostate, and brain cancers

Holmium-166

    (Ho-166)
    Multiple myeloma treatment in targeted skeletal therapy, cancer radioimmunotherapy, bone marrow ablation, and rheumatoid arthritis treatment

Iodine-125

    (I-125)
    Osteoporosis detection, diagnostic imaging, tracer drugs, brain cancer treatment, radiolabeling, tumor imaging, mapping of receptors in the brain, interstitial radiation therapy, brachytherapy for treatment of prostate cancer, determination of glomerular filtration rate (GFR), determination of plasma volume, detection of deep vein thrombosis of the legs

Iodine-131

    (I-131)
    Thyroid function evaluation, thyroid disease detection, treatment of thyroid cancer as well as other non-malignant thyroid diseases (i.e., Graves disease, goiters, and hyperthyroidism), treatment of leukemia, lymphoma, and other forms of cancer (e.g., breast cancer) using radioimmunotherapy

Iridium-192

    (Ir-192)
    Brachytherapy, brain and spinal cord tumor treatment, treatment of blocked arteries (i.e., arteriosclerosis and restenosis), and implants for breast and prostate tumors

Lutetium-177

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

Molybdenum-99

    (Mo-99)
    Parent of Technetium-99m (Tc-99m) which is used for imaging the brain, liver, lungs, heart, and other organs. Currently, Tc-99m is the most widely used radioisotope used for diagnostic imaging of various cancers and diseases involving the brain, heart, liver, lungs; also used in detection of deep vein thrombosis of the legs

Osmium-194

    (Os-194)
    Cancer radioimmunotherapy

Palladium-103

    (Pd-103)
    Prostate cancer treatment

Platinum-195m

    (Pt-195m)
    Studies on biodistribution and metabolism of cisplatin, a chemotherapeutic drug

Phosphorus-32

    (P-32)
    Polycythemia rubra vera (blood cell disease) and leukemia treatment, bone cancer diagnosis/treatment; colon, pancreatic, and liver cancer treatment; radiolabeling nucleic acids for in vitro research, diagnosis of superficial tumors, treatment of blocked arteries (i.e., arteriosclerosis and restenosis), and intracavity therapy

Phosphorus-33

    (P-33)
    Leukemia treatment, bone disease diagnosis/treatment, radiolabeling, and treatment of blocked arteries (i.e., arteriosclerosis and restenosis)

Radium-223

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

Rhenium-186

    (Re-186)
    Bone cancer pain relief, rheumatoid arthritis treatment, and diagnosis and treatment of lymphoma and bone, breast, colon, and liver cancers using radioimmunotherapy

Rhenium-188

    (Re-188)
    Cancer diagnosis and treatment using radioimmunotherapy, bone cancer pain relief, treatment of rheumatoid arthritis, and treatment of prostate cancer

Rhodium-105

    (Rh-105)
    Cancer radioimmunotherapy

Samarium-145

    (Sm-145)
    Ocular cancer treatment

Samarium-153

    (Sm-153)
    Cancer radioimmunotherapy and bone cancer pain relief

Scandium-47

    (Sc-47)
    Cancer radioimmunotherapy and bone cancer pain relief

Selenium-75

    (Se-75)
    Radiotracer used in brain studies, imaging of adrenal cortex by gamma-scintigraphy, lateral locations of steroid secreting tumors, pancreatic scanning, detection of hyperactive parathyroid glands, measure rate of bile acid loss from the endogenous pool

Strontium-85

    (Sr-85)
    Bone cancer detection and brain scans

Strontium-89

    (Sr-89)
    Bone cancer pain relief, multiple myeloma treatment, and osteoblastic therapy

Technetium-99m

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

Thorium-228

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

Thorium-229

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

Thulium-170

    (Tm-170)
    Gamma source for blood irradiators, energy source for implanted medical devices

Tin-117m

    (Sn-117m)
    Cancer immunotherapy and bone cancer pain relief

Tungsten-188

    (W-188)
    Parent for Rhenium-188 (Re-188) which is used for cancer diagnostics/treatment, bone cancer pain relief, rheumatoid arthritis treatment, and treatment of blocked arteries (i.e., arteriosclerosis and restenosis)

Xenon-127

    (Xe-127)
    Neuroimaging of brain disorders, high resolution SPECT studies, pulmonary function tests, and cerebral blood flow studies

Ytterbium-175

    (Yb-175)
    Cancer radioimmunotherapy

Yttrium-90

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

Yttrium-91

    (Y-91)
    A gamma-emitting label for Yttrium-90 (Y-90) which is used for cancer radioimmunotherapy (i.e., lymphoma, breast, colon, kidney, lung, ovarian, prostate, pancreatic, and inoperable liver cancers)


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 193P1E1B, ligands including hormones, neuropeptides, chemokines, odorants, phospholipids, and functional equivalents thereof that bind and preferably inhibit 193P1E1B protein function. Such non-limiting small molecules preferably have a molecular weight of less than about 10 kDa, more preferably below about 9, about 8, about 7, about 6, about 5 or about 4 kDa. In certain embodiments, small molecules physically associate with, or bind, 193P1E1B protein; are not found in naturally occurring metabolic pathways; and/or are more soluble in aqueous than non-aqueous solutions
“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 5° 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 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x 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 x SSC (sodium chloride/sodium. citrate) and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1 x 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 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1 x 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, 652, 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 193P1E1B protein shown in FIG. 2 or FIG. 3. An analog is an example of a variant protein. Splice isoforms and single nucleotides polymorphisms (SNPs) are further examples of variants.
The “193P1E1B-related proteins” of the invention include those specifically identified herein, as well as allelic variants, conservative substitution variants, analogs and homologs that can be isolated/generated and characterized without undue experimentation following the methods outlined herein or readily available in the art. Fusion proteins that combine parts of different 193P1E1B proteins or fragments thereof, as well as fusion proteins of a 193P1E1B protein and a heterologous polypeptide are also included. Such 193P1E1B proteins are collectively referred to as the 193P1E1B-related proteins, the proteins of the invention, or 193P1E1B. The term “193P1E1B-related protein” refers to a polypeptide fragment or a 193P1E1B protein sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, or 664 or more amino acids.
II.) 193P1E1B Polynucleotides
One aspect of the invention provides polynucleotides corresponding or complementary to all or part of a 193P1E1B gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding a 193P1E1B-related protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to a 193P1E1B gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides that hybridize to a 193P1E1B gene, mRNA, or to a 193P1E1B encoding polynucleotide (collectively, “193P1E1B polynucleotides”). In all instances when referred to in this section, T can also be U in FIG. 2.
Embodiments of a 193P1E1B polynucleotide include: a 193P1E1B polynucleotide having the sequence shown in FIG. 2, the nucleotide sequence of 193P1E1B as shown in FIG. 2 wherein T is U; at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2; or, at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2 where T is U. For example, embodiments of 193P1E1B nucleotides comprise, without limitation:

    
    
        (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 805 through nucleotide residue number 2043, 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 805 through nucleotide residue number 2043, 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 805 through nucleotide residue number 2043, 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 805 through nucleotide residue number 2043, 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 805 through nucleotide residue number 2043, 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 805 through nucleotide residue number 2043, 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 805 through nucleotide residue number 2043, 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 805 through nucleotide residue number 2043, 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 989 through nucleotide residue number 1981, including the stop codon, wherein T can also be U;
        (XI) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2J, from nucleotide residue number 805 through nucleotide residue number 1971, including the stop codon, wherein T can also be U;
        (XII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2K, from nucleotide residue number 989 through nucleotide residue number 1909, including the stop codon, wherein T can also be U;
        (XIII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2L, from nucleotide residue number 805 through nucleotide residue number 1026, including the stop codon, wherein T can also be U;
        (XIV) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2M, from nucleotide residue number 952 through nucleotide residue number 2070, including the stop codon, wherein T can also be U;
        (XV) a polynucleotide that encodes a 193P1E1B-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homologous to an entire amino acid sequence shown in FIGS. 2A-M;
        (XVI) a polynucleotide that encodes a 193P1E1B-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to an entire amino acid sequence shown in FIGS. 2A-M;
        (XVII) a polynucleotide that encodes at least one peptide set forth in Tables VIII-XXI and XXII-XLIX;
        (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-C in any whole number increment up to 412 that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
        (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 FIGS. 3A-C in any whole number increment up to 412 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
        (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 FIGS. 3A-C in any whole number increment up to 412 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
        (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 FIGS. 3A-C in any whole number increment up to 412 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
        (XXII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIGS. 3A-C in any whole number increment up to 412 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;
        (XXIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3D in any whole number increment up to 330 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
        (XXIV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3D in any whole number increment up to 330 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
        (XXV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3D in any whole number increment up to 330 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
        (XXVI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3D in any whole number increment up to 330 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
        (XXVII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3D in any whole number increment up to 330 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9
        (XXVIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3E in any whole number increment up to 388 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
        (XXIX) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3E in any whole number increment up to 388 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
        (XXX) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3E in any whole number increment up to 388 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
        (XXXI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3E in any whole number increment up to 388 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
        (XXXII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3E in any whole number increment up to 388 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9
        (XXXIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3F in any whole number increment up to 308 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
        (XXXIV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3F in any whole number increment up to 308 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
        (XXXV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3F in any whole number increment up to 308 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
        (XXXVI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3F in any whole number increment up to 308 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,,34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
        (XXXVII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3F in any whole number increment up to 308 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9
        (XXXVIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3G in any whole number increment up to 73 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
        (XXXIX) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3G in any whole number increment up to 73 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
        (XL) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3G in any whole number increment up to 73 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
        (XLI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3G in any whole number increment up to 73 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
        (XLII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3G in any whole number increment up to 73 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9
        (XLIII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3H in any whole number increment up to 372 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
        (XLIV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3H in any whole number increment up to 372 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
        (XLV) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3H in any whole number increment up to 372 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
        (XLVI) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3H in any whole number increment up to 372 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
        (XLVII) a polynucleotide that encodes a peptide region of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a peptide of FIG. 3H in any whole number increment up to 372 that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9
        (XLVIII) a polynucleotide that is fully complementary to a polynucleotide of any one of (I)-(XLVII). (XLIX) a peptide that is encoded by any of (I) to (XLVIII); and
        (L) a composition comprising a polynucleotide of any of (I)-(XLVIII) or peptide of (XLIX) together with a pharmaceutical excipient and/or in a human unit dose form.
        (LI) a method of using a polynucleotide of any (I)-(XLVIII) or peptide of (XLIX) or a composition of (L) in a method to modulate a cell expressing 193P1E1B,
        (LII) a method of using a polynucleotide of any (I)-(XLVIII) or peptide of (XLIX) or a composition of (L) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 193P1E1B
        (LIII) a method of using a polynucleotide of any (I)-(XLVIII) or peptide of (XLIX) or a composition of (L) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 193P1E1B, said cell from a cancer of a tissue listed in Table I;
        (LIV) a method of using a polynucleotide of any (I)-(XLVIII) or peptide of (XLIX) or a composition of (L) in a method to diagnose, prophylax, prognose, or treat a a cancer;
        (LV) a method of using a polynucleotide of any (I)-(XLVIII) or peptide of (XLIX) or a composition of (L) in a method to diagnose, prophylax, prognose, or treat a a cancer of a tissue listed in Table I; and,
        (LVI) a method of using a polynucleotide of any (I)-(XLVIII) or peptide of (XLIX) or a composition of (L) in a method to identify or characterize a modulator of a cell expressing 193P1E1B.
    
    


As used herein, a range is understood to disclose specifically all whole unit positions thereof.
Typical embodiments of the invention disclosed herein include 193P1E1B polynucleotides that encode specific portions of 193P1E1B mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example:
(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 410, 412 or more contiguous amino acids of 193P1E1B variant 1; the maximal lengths relevant for other variants are: variant 5, 412 amino acids; variant 6, 412 amino acids, variant 9, 330 amino acids, variant 10, 388 amino acids, variant 11, 308 amino acids, variant 12, 73 amino acids, and variant 13, 372 amino acids.
For example, representative embodiments of the invention disclosed herein include: polynucleotides and their encoded peptides themselves encoding about amino acid 1 to about amino acid 10 of the 193P1E1B protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the 193P1E1B protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the 193P1E1B protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 30 to about amino acid 40 of the 193P1E1B protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 40 to about amino acid 50 of the 193P1E1B protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 50 to about amino acid 60 of the 193P1E1B protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 60 to about amino acid 70 of the 193P1E1B protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 70 to about amino acid 80 of the 193P1E1B protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 80 to about amino acid 90 of the 193P1E1B protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 90 to about amino acid 100 of the 193P1E1B protein shown in FIG. 2 or FIG. 3, in increments of about 10 amino acids, ending at the carboxyl terminal amino acid set forth in FIG. 2 or FIG. 3. Accordingly, polynucleotides encoding portions of the amino acid sequence (of about 10 amino acids), of amino acids, 100 through the carboxyl terminal amino acid of the 193P1E1B protein are embodiments of the invention. Wherein it is understood that each particular amino acid position discloses that position plus or minus five amino acid residues.
Polynucleotides encoding relatively long portions of a 193P1E1B protein are also within the scope of the invention. For example, polynucleotides encoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 30, or 40 or 50 etc.) of the 193P1E1B protein “or variant” shown in FIG. 2 or FIG. 3 can be generated by a variety of techniques well known in the art. These polynucleotide fragments can include any portion of the 193P1E1B sequence as shown in FIG. 2.
Additional illustrative embodiments of the invention disclosed herein include 193P1E1B polynucleotide fragments encoding one or more of the biological motifs contained within a 193P1E1B protein “or variant” sequence, including one or more of the motif-bearing subsequences of a 193P1E1B protein “or variant” set forth in Tables VIII-XXI and XXII-XLIX. In another embodiment, typical polynucleotide fragments of the invention encode one or more of the regions of 193P1E1B protein or variant that exhibit homology to a known molecule. In another embodiment of the invention, typical polynucleotide fragments can encode one or more of the 193P1E1B protein or variant N-glycosylation sites, cAMP and cGMP-dependent protein kinase phosphorylation sites, casein kinase II phosphorylation sites or N-myristoylation site and amidation sites.
Note that to determine the starting position of any peptide set forth in Tables VIII-XXI and Tables XXII to XLIX (collectively HLA Peptide Tables) respective to its parental protein, e.g., variant 1, variant 2, etc., reference is made to three factors: the particular variant, the length of the peptide in an HLA Peptide Table, and the Search Peptides listed in Table LVII. Generally, a unique Search Peptide is used to obtain HLA peptides for a particular variant. The position of each Search Peptide relative to its respective parent molecule is listed in Table VII. Accordingly, if a Search Peptide begins at position “X”, one must add the value “X minus 1” to each position in Tables VIII-XXI and Tables XXII-IL to obtain the actual position of the HLA peptides in their parental molecule. For example if a particular Search Peptide begins at position 150 of its parental molecule, one must add 150 -1, i.e., 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.
II.A.) Uses of 193P1E1B Polynucleotides
II.A.1.) Monitoring of Genetic Abnormalities
The polynucleotides of the preceding paragraphs have a number of different specific uses. The human 193P1E1B gene maps to the chromosomal location set forth in the Example entitled “Chromosomal Mapping of 193P1E1B.” For example, because the 193P1E1B gene maps to this chromosome, polynucleotides that encode different regions of the 193P1E1B proteins are used to characterize cytogenetic abnormalities of this chromosomal locale, such as abnormalities that are identified as being associated with various cancers. In certain genes, a variety of chromosomal abnormalities including rearrangements have been identified as frequent cytogenetic abnormalities in a number of different cancers (see e.g. Krajinovic et al., Mutat. Res. 382(3-4): 81-83 (1998); Johansson et al., Blood 86(10): 3905-3914 (1995) and Finger et al., P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotides encoding specific regions of the 193P1E1B proteins provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the chromosomal region that encodes 193P1E1B that may contribute to the malignant phenotype. In this context, these polynucleotides satisfy a need in the art for expanding the sensitivity of chromosomal screening in order to identify more subtle and less common chromosomal abnormalities (see e.g. Evans et al., Am. J. Obstet. Gynecol 171(4): 1055-1057 (1994)).
Furthermore, as 193P1E1B was shown to be highly expressed in bladder and other cancers, 193P1E1B polynucleotides are used in methods assessing the status of 193P1E1B gene products in normal versus cancerous tissues. Typically, polynucleotides that encode specific regions of the 193P1E1B proteins are used to assess the presence of perturbations (such as deletions, insertions, point mutations, or alterations resulting in a loss of an antigen etc.) in specific regions of the 193P1E1B gene, such as regions containing one or more motifs. Exemplary assays include both RT-PCR assays as well as single-strand conformation polymorphism (SSCP) analysis (see, e.g., Marrogi et al., J. Cutan. Pathol. 26(8): 369-378 (1999), both of which utilize polynucleotides encoding specific regions of a protein to examine these regions within the protein.
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 193P1E1B. For example, antisense molecules can be RNAs or other molecules, including peptide nucleic acids (PNAs) or non-nucleic acid molecules such as phosphorothioate derivatives that specifically bind DNA or RNA in a base pair-dependent manner. A skilled artisan can readily obtain these classes of nucleic acid molecules using the 193P1E1B polynucleotides and polynucleotide sequences disclosed herein.
Antisense technology entails the administration of exogenous oligonucleotides that bind to a target polynucleotide located within the cells. The term “antisense” refers to the fact that such oligonucleotides are complementary to their intracellular targets, e.g., 193P1E1B. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The 193P1E1B antisense oligonucleotides of the present invention include derivatives such as S-oligonucleotides (phosphorothioate derivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhanced cancer cell growth inhibitory action. S-oligos (nucleoside phosphorothioates) are isoelectronic analogs of an oligonucleotide (O-oligo) in which a nonbridging oxygen atom of the phosphate group is replaced by a sulfur atom. The S-oligos of the present invention can be prepared by treatment of the corresponding O-oligos with 3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transfer reagent. See, e.g., Iyer, R. P. et al., J. Org. Chem. 55:4693-4698 (1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254(1990). Additional 193P1E1B antisense oligonucleotides of the present invention include morpholino antisense oligonucleotides known in the art (see, e.g., Partridge et al., 1996, Antisense & Nucleic Acid Drug Development 6: 169-175).
The 193P1E1B antisense oligonucleotides of the present invention typically can be RNA or DNA that is complementary to and stably hybridizes with the first 100 5′ codons or last 100 3′ codons of a 193P1E1B genomic sequence or the corresponding mRNA. Absolute complementarity is not required, although high degrees of complementarity are preferred. Use of an oligonucleotide complementary to this region allows for the selective hybridization to 193P1E1B mRNA and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, 193P1E1B antisense oligonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to 193P1E1B mRNA. Optionally, 193P1E1B antisense oligonucleotide is a 30-mer oligonucleotide that is complementary to a region in the first 10 5′ codons or last 10 3′ codons of 193P1E1B. Alternatively, the antisense molecules are modified to employ ribozymes in the inhibition of 193P1E1B expression, see, e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).
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 193P1E1B polynucleotide in a sample and as a means for detecting a cell expressing a 193P1E1B protein.
Examples of such probes include polypeptides comprising all or part of the human 193P1E1B cDNA sequence shown in FIG. 2. Examples of primer pairs capable of specifically amplifying 193P1E1B mRNAs are also described in the Examples. As will be understood by the skilled artisan, a great many different primers and probes can be prepared based on the sequences provided herein and used effectively to amplify and/or detect a 193P1E1B mRNA.
The 193P1E1B polynucleotides of the invention are useful for a variety of purposes, including but not limited to their use as probes and primers for the amplification and/or detection of the 193P1E1B gene(s), mRNA(s), or fragments thereof; as reagents for the diagnosis and/or prognosis of prostate cancer and other cancers; as coding sequences capable of directing the expression of 193P1E1B polypeptides; as tools for modulating or inhibiting the expression of the 193P1E1B gene(s) and/or translation of the 193P1E1B transcript(s); and as therapeutic agents.
The present invention includes the use of any probe as described herein to identify and isolate a 193P1E1B or 193P1E1B related nucleic acid sequence from a naturally occurring source, such as humans or other mammals, as well as the isolated nucleic acid sequence per se, which would comprise all or most of the sequences found in the probe used.
II.A.4.) Isolation of 193P1E1B-Encoding Nucleic Acid Molecules
The 193P1E1B cDNA sequences described herein enable the isolation of other polynucleotides encoding 193P1E1B gene product(s), as well as the isolation of polynucleotides encoding 193P1E1B gene product homologs, alternatively spliced isoforms, allelic variants, and mutant forms of a 193P1E1B gene product as well as polynucleotides that encode analogs of 193P1E1B-related proteins. Various molecular cloning methods that can be employed to isolate full length cDNAs encoding a 193P1E1B gene are well known (see, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989; Current Protocols in Molecular Biology. Ausubel et al., Eds., Wiley and Sons, 1995). For example, lambda phage cloning methodologies can be conveniently employed, using commercially available cloning systems (e.g., Lambda ZAP Express, Stratagene). Phage clones containing 193P1E1B gene cDNAs can be identified by probing with a labeled 193P1E1B cDNA or a fragment thereof. For example, in one embodiment, a 193P1E1B cDNA (e.g., FIG. 2) or a portion thereof can be synthesized and used as a probe to retrieve overlapping and full-length cDNAs corresponding to a 193P1E1B gene. A 193P1E1B gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with 193P1E1B DNA probes or primers.
II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems
The invention also provides recombinant DNA or RNA molecules containing a 193P1E1B polynucleotide, a fragment, analog or homologue thereof, including but not limited to phages, plasmids, phagemids, cosmids, YACs, BACs, as well as various viral and non-viral vectors well known in the art and cells transformed or transfected with such recombinant DNA or RNA molecules. Methods for generating such molecules are well known (see, for example, Sambrook et al., 1989, supra).
The invention further provides a host-vector system comprising a recombinant DNA molecule containing a 193P1E1B polynucleotide, fragment, analog or homologue thereof within a suitable prokaryotic or eukaryotic host cell. Examples of suitable eukaryotic host cells include a yeast cell, a plant cell, or an animal cell, such as a mammalian cell or an insect cell (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 TsuPrl, other transfectable or transducible prostate cancer cell lines, primary cells (PrEC), as well as a number of mammalian cells routinely used for the expression of recombinant proteins (e.g., COS, CHO, 293, 293T cells). More particularly, a polynucleotide comprising the coding sequence of 193P1E1B or a fragment, analog or homolog thereof can be used to generate 193P1E1B proteins or fragments thereof using any number of host-vector systems routinely used and widely known in the art.
A wide range of host-vector systems suitable for the expression of 193P1E1B proteins or fragments thereof are available, see for example, Sambrook et al., 1989, supra; Current Protocols in Molecular Biology, 1995, supra). Preferred vectors for mammalian expression include but are not limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Using these expression vectors, 193P1E1B can be expressed in several prostate cancer and non-prostate cell lines, including for example 293, 293T, rat-1, NIH 3T3 and TsuPrl. The host-vector systems of the invention are useful for the production of a 193P1E1B protein or fragment thereof. Such host-vector systems can be employed to study the functional properties of 193P1E1B and 193P1E1B mutations or analogs.
Recombinant human 193P1E1B protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a 193P1E1B-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding 193P1E1B or fragment, analog or homolog thereof, a 193P1E1B-related protein is expressed in the 293T cells, and the recombinant 193P1E1B protein is isolated using standard purification methods (e.g., affinity purification using anti-193P1E1B antibodies). In another embodiment, a 193P1E1B coding sequence is subcloned into the retroviral vector pSRαMSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPr1,293 and rat-1 in order to establish 193P1E1B expressing cell lines. Various other expression systems well known in the art can also be employed. Expression constructs encoding a leader peptide joined in frame to a 193P1E1B coding sequence can be used for the generation of a secreted form of recombinant 193P1E1B protein.
As discussed herein, redundancy in the genetic code permits variation in 193P1E1B gene sequences. In particular, it is known in the art that specific host species often have specific codon preferences, and thus one can adapt the disclosed sequence as preferred for a desired host. For example, preferred analog codon sequences typically have rare codons (i.e., codons having a usage frequency of less than about 20% in known sequences of the desired host) replaced with higher frequency codons. Codon preferences for a specific species are calculated, for example, by utilizing codon usage tables available on the INTERNET.
Additional sequence modifications are known to enhance protein expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon/intron splice site signals, transposon-like repeats, and/or other such well-characterized sequences that are deleterious to gene expression. The GC content of the sequence is adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Where possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures. Other useful modifications include the addition of a translational initiation consensus sequence at the start of the open reading frame, as described in Kozak, Mol. Cell Biol., 9:5073-5080 (1989). Skilled artisans understand that the general rule that eukaryotic ribosomes initiate translation exclusively at the 5′ proximal AUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS 92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).
III.) 193P1E1B-Related Proteins
Another aspect of the present invention provides 193P1E1B-related proteins. Specific embodiments of 193P1E1B proteins comprise a polypeptide having all or part of the amino acid sequence of human 193P1E1B as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of 193P1E1B proteins comprise variant, homolog or analog polypeptides that have alterations in the amino acid sequence of 193P1E1B shown in FIG. 2 or FIG. 3.
Embodiments of a 193P1E1B polypeptide include: a 193P1E1B polypeptide having a sequence shown in FIG. 2, a peptide sequence of a 193P1E1B as shown in FIG. 2 wherein T is U; at least 10 contiguous nucleotides of a polypeptide having the sequence as shown in FIG. 2; or, at least 10 contiguous peptides of a polypeptide having the sequence as shown in FIG. 2 where T is U. For example, embodiments of 193P1E1B peptides comprise, without limitation:

    
    
        (I) a protein comprising, consisting essentially of, or consisting of an amino acid sequence as shown in FIGS. 2A-M or FIGS. 3A-H;
        (II) a 193P1E1B-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% homologous to an entire amino acid sequence shown in FIGS. 2A-M;
        (III) a 193P1E1B-related protein that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% identical to an entire amino acid sequence shown in FIGS. 2A-M or 3A-H;
        (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 FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G, or 3H in any whole number increment up to 412, 412, 412, 330, 388, 308, 73, or 372 respectively that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
        (X) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G, or 3H in any whole number increment up to 412, 412, 412, 330, 388, 308, 73, or 372 respectively, that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
        (XI) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G, or 3H in any whole number increment up to 412, 412, 412, 330, 388, 308, 73, or 372 respectively, that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
        (XII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G, or 3H in any whole number increment up to 412, 412, 412, 330, 388, 308, 73, or 372 respectively, that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
        (XIII) a polypeptide comprising at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acids of a protein of FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G, or 3H in any whole number increment up to 412, 412, 412, 330, 388, 308, 73, or 372 respectively, that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 amino acid position(s) having a value greater than 0.5 in the Beta-turn profile of FIG. 9;
        (XIV) a peptide that occurs at least twice in Tables VIII-XXI and XXII to XLIX, collectively;
        (XV) a peptide that occurs at least three times in Tables VIII-XXI and XXII to XLIX, collectively;
        (XVI) a peptide that occurs at least four times in Tables VIII-XXI and XXII to XLIX, collectively;
        (XVII) a peptide that occurs at least five times in Tables VIII-XXI and XXII to XLIX, collectively;
        (XVIII) a peptide that occurs at least once in Tables VIII-XXI, and at least once in tables XXII to XLIX;
        (XIX) a peptide that occurs at least once in Tables VIII-XXI, and at least twice in tables XXII to XLIX;
        (XX) a peptide that occurs at least twice in Tables VIII-XXI, and at least once in tables XXII to XLIX;
        (XXI) a peptide that occurs at least twice in Tables VIII-XXI, and at least twice in tables XXII to XLIX;
        (XXII) 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;
        
        
        (XXIII) a composition comprising a peptide of (I)-(XXII) or an antibody or binding region thereof together with a pharmaceutical excipient and/or in a human unit dose form.
        (XXIV) a method of using a peptide of (I)-(XXII), or an antibody or binding region thereof or a composition of (XXIII) in a method to modulate a cell expressing 193P1E1B,
        (XXV) a method of using a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition of (XXIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 193P1E1B
        (XXVI) a method of using a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition (XXIII) in a method to diagnose, prophylax, prognose, or treat an individual who bears a cell expressing 193P1E1B, said cell from a cancer of a tissue listed in Table I;
        (XXVII) a method of using a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition of (XXIII) in a method to diagnose, prophylax, prognose, or treat a a cancer;
        (XXVIII) a method of using a peptide of (l)-(XXII) or an antibody or binding region thereof or a composition of (XXIII) in a method to diagnose, prophylax, prognose, or treat a a cancer of a tissue listed in Table I; and,
        (XXIX) a method of using a a peptide of (I)-(XXII) or an antibody or binding region thereof or a composition (XXIII) in a method to identify or characterize a modulator of a cell expressing 193P1E1B.
    
    


As used herein, a range is understood to specifically disclose all whole unit positions thereof.
Typical embodiments of the invention disclosed herein include 193P1E1B polynucleotides that encode specific portions of 193P1E1B mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example:
(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 410, 412 or more contiguous amino acids of 193P1E1B variant 1; the maximal lengths relevant for other variants are: variant 5, 412 amino acids; variant 6, 412 amino acids, variant 9, 330, variant 10, 388 amino acids, variant 11, 308 amino acids, variant 12, 73, and variant 13, 372 amino acids.
In general, naturally occurring allelic variants of human 193P1E1B share a high degree of structural identity and homology (e.g., 90% or more homology). Typically, allelic variants of a 193P1E1B protein contain conservative amino acid substitutions within the 193P1E1B sequences described herein or contain a substitution of an amino acid from a corresponding position in a homologue of 193P1E1B. One class of 193P1E1B allelic variants are proteins that share a high degree of homology with at least a small region of a particular 193P1E1B amino acid sequence, but further contain a radical departure from the sequence, such as a non-conservative substitution, truncation, insertion or frame shift. In comparisons of protein sequences, the terms, similarity, identity, and homology each have a distinct meaning as appreciated in the field of genetics. Moreover, orthology and paralogy can be important concepts describing the relationship of members of a given protein family in one organism to the members of the same family in other organisms.
Amino acid abbreviations are provided in Table II. Conservative amino acid substitutions can frequently be made in a protein without altering either the conformation or the function of the protein. Proteins of the invention can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 conservative substitutions. Such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa. Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered “conservative” in particular environments (see, e.g. Table III herein; pages 13-15 “Biochemistry” 2nd ED. Lubert Stryer ed (Stanford University); Henikoff et al., PNAS 1992 Vol 89 10915-10919; Lei et al., J Biol Chem May 19, 1995; 270(20):11882-6).
Embodiments of the invention disclosed herein include a wide variety of art-accepted variants or analogs of 193P1E1B proteins such as polypeptides having amino acid insertions, deletions and substitutions. 193P1E1B variants can be made using methods known in the art such as site-directed mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)) or other known techniques can be performed on the cloned DNA to produce the 193P1E1B variant DNA.
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, 193P1E1B variants, analogs or homologs, have the distinguishing attribute of having at least one epitope that is “cross reactive” with a 193P1E1B protein having an amino acid sequence of FIG. 3. As used in this sentence, “cross reactive” means that an antibody or T cell that specifically binds to a 193P1E1B variant also specifically binds to a 193P1E1B protein having an amino acid sequence set forth in FIG. 3. A polypeptide ceases to be a variant of a protein shown in FIG. 3, when it no longer contains any epitope capable of being recognized by an antibody or T cell that specifically binds to the starting 193P1E1B protein. Those skilled in the art understand that antibodies that recognize proteins bind to epitopes of varying size, and a grouping of the order of about four or five amino acids, contiguous or not, is regarded as a typical number of amino acids in a minimal epitope. See, e.g., Nair et al., J. Immunol 2000 165(12): 6949-6955; Hebbes et al., Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Immunol (1985) 135(4):2598-608.
Other classes of 193P1E1B-related protein variants share 70%, 75%, 80%, 85% or 90% or more similarity with an amino acid sequence of FIG. 3, or a fragment thereof. Another specific class of 193P1E1B protein variants or analogs comprises one or more of the 193P1E1B biological motifs described herein or presently known in the art. Thus, encompassed by the present invention are analogs of 193P1E1B fragments (nucleic or amino acid) that have altered functional (e.g. immunogenic) properties relative to the starting fragment. It is to be appreciated that motifs now or which become part of the art are to be applied to the nucleic or amino acid sequences of FIG. 2 or FIG. 3.
As discussed herein, embodiments of the claimed invention include polypeptides containing less than the full amino acid sequence of a 193P1E1B protein shown in FIG. 2 or FIG. 3. For example, representative embodiments of the invention comprise peptides/proteins having any 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of a 193P1E1B protein shown in FIG. 2 or FIG. 3.
Moreover, representative embodiments of the invention disclosed herein include polypeptides consisting of about amino acid 1 to about amino acid 10 of a 193P1E1B protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 10 to about amino acid 20 of a 193P1E1B protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 20 to about amino acid 30 of a 193P1E1B protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 30 to about amino acid 40 of a 193P1E1B protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 40 to about amino acid 50 of a 193P1E1B protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 50 to about amino acid 60 of a 193P1E1B protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 60 to about amino acid 70 of a 193P1E1B protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 70 to about amino acid 80 of a 193P1E1B protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 80 to about amino acid 90 of a 193P1E1B protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 90 to about amino acid 100 of a 193P1E1B protein shown in FIG. 2 or FIG. 3, etc. throughout the entirety of a 193P1E1B amino acid sequence. Moreover, polypeptides consisting of about amino acid 1 (or 20or 30 or 40 etc.) to about amino acid 20, (or 130, or 140 or 150 etc.) of a 193P1E1B protein shown in FIG. 2 or FIG. 3 are embodiments of the invention. It is to be appreciated that the starting and stopping positions in this paragraph refer to the specified position as well as that position plus or minus 5 residues.
193P1E1B-related proteins are generated using standard peptide synthesis technology or using chemical cleavage methods well known in the art. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode a 193P1E1B-related protein. In one embodiment, nucleic acid molecules provide a means to generate defined fragments of a 193P1E1B protein (or variants, homologs or analogs thereof).
III.A.) Motif-Bearing Protein Embodiments
Additional illustrative embodiments of the invention disclosed herein include 193P1E1B polypeptides comprising the amino acid residues of one or more of the biological motifs contained within a 193P1E1B polypeptide sequence set forth in FIG. 2 or FIG. 3. Various motifs are known in the art, and a protein can be evaluated for the presence of such motifs by a number of publicly available Internet sites.
Motif bearing subsequences of all 193P1E1B 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. 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 193P1E1B motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 193P1E1B motifs discussed above are associated with growth dysregulation and because 193P1E1B is overexpressed in certain cancers (See, e.g., Table I). Casein kinase 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 193P1E1B protein that are capable of optimally binding to specified HLA alleles. Moreover, processes for identifying peptides that have sufficient binding affinity for HLA molecules and which are correlated with being immunogenic epitopes, are well known in the art, and are carried out without undue experimentation. In addition, processes for identifying peptides that are immunogenic epitopes, are well known in the art, and are carried out without undue experimentation either in vitro or in vivo.
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(34): 201-212; Sette et al., J. Immunol. 2001 166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20; Kondo et al., Immunogenetics 1997 45(4): 249-258; Sidney et 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.
193P1E1B-related proteins are embodied in many forms, preferably in isolated form. A purified 193P1E1B protein molecule will be substantially free of other proteins or molecules that impair the binding of 193P1E1B to antibody, T cell or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a 193P1E1B-related proteins include purified 193P1E1B-related proteins and functional, soluble 193P1E1B-related proteins. In one embodiment, a functional, soluble 193P1E1B protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.
The invention also provides 193P1E1B proteins comprising biologically active fragments of a 193P1E1B amino acid sequence shown in FIG. 2 or FIG. 3. Such proteins exhibit properties of the starting 193P1E1B protein, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the starting 193P1E1B protein; to be bound by such antibodies; to elicit the activation of HTL or CTL; and/or, to be recognized by HTL or CTL that also specifically bind to the starting protein.
193P1E1B-related polypeptides that contain particularly interesting structures can be predicted and/or identified using various analytical techniques well known in the art, including, for example, the methods of Chou-Fasman, Gamier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis, or based on immunogenicity. Fragments that contain such structures are particularly useful in generating subunit-specific anti-193P1E1B antibodies or T cells or in identifying cellular factors that bind to 193P1E1B. For example, hydrophilicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can be generated, and immunogenic peptide fragments identified, using the method of Janin J., 1979, Nature 277:491492. 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 193P1E1B protein that are capable of optimally binding to specified HLA alleles (e.g., by using the SYFPEITHI site at World Wide Web; EpimatrixTM and EpimerTM, Brown University, URL and BIMAS. Illustrating this, peptide epitopes from 193P1E1B that are presented in the context of human MHC Class I molecules, e.g., HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (see, e.g., Tables VIII-XXI, XXII-XLIX). Specifically, the complete amino acid sequence of the 193P1E1B protein and relevant portions of other variants, i.e., for HLA Class I predictions 9 flanking residues on either side of a point mutation or exon junction, and for HLA Class II predictions 14 flanking residues on either side of a point mutation or exon junction corresponding to that variant, were entered into the HLA Peptide Motif Search algorithm found in the Bioinformatics and Molecular Analysis Section (BIMAS) web site listed above; in addition to the site SYFPEITHI.
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 at, J. Immunol. 152:163-75 (1994)). This algorithm allows location and ranking of 8-mer, 9-mer, and 10-mer peptides from a complete protein sequence for predicted binding to HLA-A2 as well as numerous other HLA Class I molecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-mers. For example, for Class I HLA-A2, the epitopes preferably contain a leucine (L) or methionine (M) at position 2 and a valine (V) or leucine (L) at the C-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7 (1992)). Selected results of 193P1E1B predicted binding peptides are shown in Tables VIII-XXI and XXII-XLIX herein. In Tables VIII-XXI and XXII-XLVII, selected candidates, 9-mers and 10-mers, for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score. In Tables XLVI-XLIX, selected candidates, 15-mers, for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score. The binding score corresponds to the estimated half time of dissociation of complexes containing the peptide at 37° 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 SYFPETHI or BIMAS, are to be “applied” to a 193P1E1B protein in accordance with the invention. As used in this context “applied” means that a 193P1E1B protein is evaluated, e.g., visually or by computer-based patterns finding methods, as appreciated by those of skill in the relevant art. Every subsequence of a 193P1E1B protein of 8, 9, 10, or 11 amino acid residues that bears an HLA Class I motif, or a subsequence of 9 or more amino acid residues that bear an HLA Class II motif are within the scope of the invention.
III.B.) Expression of 193P1E1B-related Proteins
In an embodiment described in the examples that follow, 193P1E1B can be conveniently expressed in cells (such as 293T cells) transfected with a commercially available expression vector such as a CMV-driven expression vector encoding 193P1E1B with a C-terminal 6×His and MYC tag (pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, Nashville Tenn.). The Tag5 vector provides an IgGK secretion signal that can be used to facilitate the production of a secreted 193P1E1B protein in transfected cells. The secreted HIS-tagged 193P1E1B in the culture media can be purified, e.g., using a nickel column using standard techniques.
III.C.) Modifications of 193P1E1B-Related Proteins
Modifications of 193P1E1B-related proteins such as covalent modifications are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a 193P1E1B polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of a 193P1E1B protein. Another type of covalent modification of a 193P1E1B polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of a protein of the invention. Another type of covalent modification of 193P1E1B comprises linking a 193P1E1B polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
The 193P1E1B-related proteins of the present invention can also be modified to form a chimeric molecule comprising 193P1E1B fused to another, heterologous polypeptide or amino acid sequence. Such a chimeric molecule can be synthesized chemically or recombinantly. A chimeric molecule can have a protein of the invention fused to another tumor-associated antigen or fragment thereof. Alternatively, a protein in accordance with the invention can comprise a fusion of fragments of a 193P1E1B sequence (amino or nucleic acid) such that a molecule is created that is not, through its length, directly homologous to the amino or nucleic acid sequences shown in FIG. 2 or FIG. 3. Such a chimeric molecule can comprise multiples of the same subsequence of 193P1E1B. A chimeric molecule can comprise a fusion of a 193P1E1B-related protein with a polyhistidine epitope tag, which provides an epitope to which immobilized nickel can selectively bind, with cytokines or with growth factors. The epitope tag is generally placed at the amino- or carboxyl-terminus of a 193P1E1B protein. In an alternative embodiment, the chimeric molecule can comprise a fusion of a 193P1E1B-related protein with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an “immunoadhesin”), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a 193P1E1B polypeptide in place of at least one variable region within an Ig molecule. In a preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgGI molecule. For the production of immunoglobulin fusions see, e.g., U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
III.D.) Uses of 193P1E1B-Related Proteins
The proteins of the invention have a number of different specific uses. As 193P1E1B is highly expressed in prostate and other cancers, 193P1E1B-related proteins are used in methods that assess the status of 193P1E1B gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of a 193P1E1B protein are used to assess the presence of perturbations (such as deletions, insertions, point mutations etc.) in those regions (such as regions containing one or more motifs). Exemplary assays utilize antibodies or T cells targeting 193P1E1B-related proteins comprising the amino acid residues of one or more of the biological motifs contained within a 193P1E1B polypeptide sequence in order to evaluate the characteristics of this region in normal versus cancerous tissues or to elicit an immune response to the epitope. Alternatively, 193P1E1B-related proteins that contain the amino acid residues of one or more of the biological motifs in a 193P1E1B protein are used to screen for factors that interact with that region of 193P1E1B.
193P1E1B protein fragments/subsequences are particularly useful in generating and characterizing domain-specific antibodies (e.g., antibodies recognizing an extracellular or intracellular epitope of a 193P1E1B protein), for identifying agents or cellular factors that bind to 193P1E1B or a particular structural domain thereof, and in various therapeutic and diagnostic contexts, including but not limited to diagnostic assays, cancer vaccines and methods of preparing such vaccines.
Proteins encoded by the 193P1E1B genes, or by analogs, homologs or fragments thereof, have a variety of uses, including but not limited to generating antibodies and in methods for identifying ligands and other agents and cellular constituents that bind to a 193P1E1B gene product. Antibodies raised against a 193P1E1B protein or fragment thereof are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of 193P1E1B protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers. 193P1E1B-related nucleic acids or proteins are also used in generating HTL or CTL responses.
Various immunological assays useful for the detection of 193P1E1B proteins are used, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), immunocytochemical methods, and the like. Antibodies can be labeled and used as immunological imaging reagents capable of detecting 193P1E1B-expressing cells (e.g., in radioscintigraphic imaging methods). 193P1E1B proteins are also particularly useful in generating cancer vaccines, as further described herein.
IV.) 193P1E1B Antibodies
Another aspect of the invention provides antibodies that bind to 193P1E1B-related proteins. Preferred antibodies specifically bind to a 193P1E1B-related protein and do not bind (or bind weakly) to peptides or proteins that are not 193P1E1B-related proteins under physiological conditions. In this context, examples of physiological conditions include: 1) phosphate buffered saline; 2) Tris-buffered saline containing 25mM Tris and 150 mM NaCl; or saline (0.9% NaCl); 4) animal serum such as human serum; or 5) a combination of any of 1) through 4); these reactions preferably taking place at pH 7.5, alternatively in a range of pH 7.0 to 8.0, or alternatively in a range of pH 6.5 to 8.5; also, these reactions taking place at a temperature between 4° C. to 37° C. For example, antibodies that bind 193P1E1B can bind 193P1E1B-related proteins such as the homologs or analogs thereof.
193P1E1B antibodies of the invention are particularly useful in cancer (see, e.g., Table I) diagnostic and prognostic assays, and imaging methodologies. Similarly, such antibodies are useful in the treatment, diagnosis, and/or prognosis of other cancers, to the extent 193P1E1B is also expressed or overexpressed in these other cancers. Moreover, intracellularly expressed antibodies (e.g., single chain antibodies) are therapeutically useful in treating cancers in which the expression of 193P1E1B is involved, such as advanced or metastatic prostate cancers.
The invention also provides various immunological assays useful for the detection and quantification of 193P1E1B and mutant 193P1E1B-related proteins. Such assays can comprise one or more 193P1E1B antibodies capable of recognizing and binding a 193P1E1B-related protein, as appropriate. These assays are performed within various immunological assay formats well known in the art, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.
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 193P1E1B are also provided by the invention, including but not limited to radioscintigraphic imaging methods using labeled 193P1E1B antibodies. Such assays are clinically useful in the detection, monitoring, and prognosis of 193P1E1B expressing cancers such as prostate cancer.
193P1E1B antibodies are also used in methods for purifying a 193P1E1B-related protein and for isolating 193P1E1B homologues and related molecules. For example, a method of purifying a 193P1E1B-related protein comprises incubating a 193P1E1B antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a 193P1E1B-related protein under conditions that permit the 193P1E1B antibody to bind to the 193P1E1B-related protein; washing the solid matrix to eliminate impurities; and eluting the 193P1E1B-related protein from the coupled antibody. Other uses of 193P1E1B antibodies in accordance with the invention include generating ant-idiotypic antibodies that mimic a 193P1E1B 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 193P1E1B-related protein, peptide, or fragment, in isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, N.Y. (1989)). In addition, fusion proteins of 193P1E1B can also be used, such as a 193P1E1B GST-fusion protein. In a particular embodiment, a GST fusion protein comprising all or most of the amino add sequence of FIG. 2 or FIG. 3 is produced, then used as an immunogen to generate appropriate antibodies. In another embodiment, a 193P1E1B-related protein is synthesized and used as an immunogen.
In addition, naked DNA immunization techniques known in the art are used (with or without purified 193P1E1B-related protein or 193P1E1B expressing cells) to generate an immune response to the encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).
The amino acid sequence of a 193P1E1B protein as shown in FIG. 2 or FIG. 3 can be analyzed to select specific regions of the 193P1E1B protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of a 193P1E1B amino acid sequence are used to identify hydrophilic regions in the 193P1E1B structure. Regions of a 193P1E1B protein that show immunogenic structure, as well as other regions and domains, can readily be identified using various other methods known in the art, such as Chou-Fasman, Gamier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles can be generated using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can be generated using the method of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can be generated using the method of Janin J., 1979, Nature 277:491-492. Average Flexibility profiles can be generated-using the method of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-turn profiles can be generated using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294. Thus, each region identified by any of these programs or methods is within the scope of the present invention. Methods for the generation of 193P1E1B antibodies are further illustrated by way of the examples provided herein. Methods for preparing a protein or polypeptide for use as an immunogen are well known in the art. Also well known in the art are methods for preparing immunogenic conjugates of a protein with a carrier, such as BSA, KLH or other carrier protein. In some circumstances, direct conjugation using, for example, carbodiimide reagents are used; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, Ill., are effective. Administration of a 193P1E1B immunogen is often conducted by injection over a suitable time period and with use of a suitable adjuvant, as is understood in the art During the immunization schedule, titers of antibodies can be taken to determine adequacy of antibody formation.
193P1E1B monoclonal antibodies can be produced by various means well known in the art For example, immortalized cell lines that secrete a desired monoclonal antibody are prepared using the standard hybridoma technology of Kohler and Milstein or modifications that immortalize antibody-producing B cells, as is generally known. Immortalized cell lines that secrete the desired antibodies are screened by immunoassay in which the antigen is a 193P1E1B-related protein. When the appropriate immortalized cell culture is identified, the cells can be expanded and antibodies produced either from in vitro cultures or from ascites fluid.
The antibodies or fragments of the invention can also be produced, by recombinant means. Regions that bind specifically to the desired regions of a 193P1E1B protein can also be produced in the context of chimeric or complementarity-determining region (CDR) grafted antibodies of multiple species origin. Humanized or human 193P1E1B antibodies can also be produced, and are preferred for use in therapeutic contexts. Methods for humanizing murine and other non-human antibodies, by substituting one or more of the non-human antibody CDRs for corresponding human antibody sequences, are well known (see for example, Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen et al., 1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151: 2296.
Methods for producing fully human monoclonal antibodies include phage display and transgenic methods (for review, see Vaughan et al., 1998, Nature Biotechnology 16: 535-539). Fully human 193P1E1B monoclonal antibodies can be generated using cloning technologies employing large human Ig gene combinatorial libraries (i.e., phage display) (Griffiths and Hoogenboom, Building an in vitro immune system: human antibodies from phage display libraries. In: Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man, Clark, M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatorial libraries. Id., pp 65-82). Fully human 193P1E1B monoclonal antibodies can also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application WO98/24893, Kuchelapati and Jakobovits et at, published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614; U.S. Pat. Nos. 6,162,963 issued Dec. 19, 2000; 6,150,584 issued Nov. 12, 2000; and, 6,114,598 issued Sep. 5, 2000). This method avoids the in vitro manipulation required with phage display technology and efficiently produces high affinity authentic human antibodies.
Reactivity of 193P1E1B antibodies with a 193P1E1B-related protein can be established by a number of well known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses using, as appropriate, 193P1E1B-related proteins, 193P1E1B-expressing cells or extracts thereof. A 193P1E1B antibody or fragment thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator or an enzyme. Further, bi-specific antibodies specific for two or more 193P1E1B epitopes are generated using methods generally known in the art. Homodimeric antibodies can also be generated by cross-linking techniques known in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).
V.) 193Pi E1B 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 World Wide Web; Sette, A. and Sidney, J. Curr. Opin. Immunol 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et al., Cell 74:929-937, 1993; Kondo et al., J. Immunol. 155:4307-4312, 1995; Sidney et al., J. Immunol 157:3480-3490, 1996; Sidney et al., Human Immunol. 45:79-93, 1996; Selle, A. and Sidney, J. Immunogenetics 1999 Nov; 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 at, 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 51 Cr release involving peptide-sensitized targets, T cell proliferation, or lymphokine release.
VI.) 193P1E1B Transgenic Animals
Nucleic acids that encode a 193P1E1B-related protein can also be used to generate either transgenic animals or “knock out” animals that, in turn, are useful in the development and screening of therapeutically useful reagents. In accordance with established techniques, cDNA encoding 193P1E1B can be used to clone genomic DNA that encodes 193P1E1B. The cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode 193P1E1B. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 issued Apr. 12, 1988, and 4,870,009 issued Sep. 26, 1989. Typically, particular cells would be targeted for 193P1E1B transgene incorporation with tissue-specific enhancers.
Transgenic animals that include a copy of a transgene encoding 193P1E1B can be used to examine the effect of increased expression of DNA that encodes 193P1E1B. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression. In accordance with this aspect of the invention, an animal is treated with a reagent and a reduced incidence of a pathological condition, compared to untreated animals that bear the transgene, would indicate a potential therapeutic intervention for the pathological condition.
Alternatively, non-human homologues of 193P1E1B can be used to construct a 193P1E1B “knockout” animal that has a defective or altered gene encoding 193P1E1B as a result of homologous recombination between the endogenous gene encoding 193P1E1B and altered genomic DNA encoding 193P1E1B introduced into an embryonic cell of the animal. For example, cDNA that encodes 193P1E1B can be used to clone genomic DNA encoding 193P1E1B in accordance with established techniques. A portion of the genomic DNA encoding 193P1E1B can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g., Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected (see, e.g., Li et al., Cell, 69:915 (1992)). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras (see, e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal, and the embryo brought to term to create a “knock out” animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knock out animals can be characterized, for example, for their ability to defend against certain pathological conditions or for their development of pathological conditions due to absence of a 193P1E1B polypeptide.
VIII.) Methods for the Detection of 193P1E1B
Another aspect of the present invention relates to methods for detecting 193P1E1B polynucleotides and 193P1E1B-related proteins, as well as methods for identifying a cell that expresses 193P1E1B. The expression profile of 193P1E1B makes it a diagnostic marker for metastasized disease. Accordingly, the status of 193P1E1B gene products provides information useful for predicting a variety of factors including susceptibility to advanced stage disease, rate of progression, and/or tumor aggressiveness. As discussed in detail herein, the status of 193P1E1B gene products in patient samples can be analyzed by a variety protocols that are well known in the art including immunohistochemical analysis, the variety of Northern blotting techniques including in situ hybridization, RT-PCR analysis (for example on laser capture micro-dissected samples), Western blot analysis and tissue array analysis.
More particularly, the invention provides assays for the detection of 193P1E1B polynucleotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, semen, cell preparations, and the like. Detectable 193P1E1B polynucleotides include, for example, a 193P1E1B gene or fragment thereof, 193P1E1B mRNA, alternative splice variant 193P1E1B mRNAs, and recombinant DNA or RNA molecules that contain a 193P1E1B polynucleotide. A number of methods for amplifying and/or detecting the presence of 193P1E1B polynucleotides are well known in the art and can be employed in the practice of this aspect of the invention.
In one embodiment, a method for detecting a 193P1E1B mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using a 193P1E1B polynucleotides as sense and antisense primers to amplify 193P1E1B cDNAs therein; and detecting the presence of the amplified 193P1E1B cDNA. Optionally, the sequence of the amplified 193P1E1B cDNA can be determined.
In another embodiment, a method of detecting a 193P1E1B gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using 193P1E1B polynucleotides as sense and antisense primers; and detecting the presence of the amplified 193P1E1B gene. Any number of appropriate sense and antisense probe combinations can be designed from a 193P1E1B nucleotide sequence (see, e.g., FIG. 2) and used for this purpose.
The invention also provides assays for detecting the presence of a 193P1E1B protein in a tissue or other biological sample such as serum, semen, bone, prostate, urine, cell preparations, and the like. Methods for detecting a 193P1E1B-related protein are also well known and include, for example, immunoprecipitation, immunohistochemical analysis, Western blot analysis, molecular binding assays, ELISA, ELIFA and the like. For example, a method of detecting the presence of a 193P1E1B-related protein in a biological sample comprises first contacting the sample with a 193P1E1B antibody, a 193P1E1B-reactive fragment thereof, or a recombinant protein containing an antigen-binding region of a 193P1E1B antibody; and then detecting the binding of 193P1E1B-related protein in the sample.
Methods for identifying a cell that expresses 193P1E1B are also within the scope of the invention. In one embodiment, an assay for identifying a cell that expresses a 193P1E1B gene comprises detecting the presence of 193P1E1B mRNA in the cell. Methods for the detection of particular mRNAs in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled 193P1E1B riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 193P1E1B, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like). Alternatively, an assay for identifying a cell that expresses a 193P1E1B gene comprises detecting the presence of 193P1E1B-related protein in the cell or secreted by the cell. Various methods for the detection of proteins are well known in the art and are employed for the detection of 193P1E1B-related proteins and cells that express 193P1E1B-related proteins.
193P1E1B expression analysis is also useful as a tool for identifying and evaluating agents that modulate 193P1E1B gene expression. For example, 193P1E1B expression is significantly upregulated in prostate cancer, and is expressed in cancers of the tissues listed in Table I. Identification of a molecule or biological agent that inhibits 193P1E1B expression or over-expression in cancer cells is of therapeutic value. For example, such an agent can be identified by using a screen that quantifies 193P1E1B expression by RT-PCR, nucleic acid hybridization or antibody binding.
VIII.) Methods for Monitoring the Status of 193P1E1B-Related Genes and Their Products
Oncogenesis is known to be a multistep process where cellular growth becomes progressively dysregulated and cells progress from a normal physiological state to precancerous and then cancerous states (see, e.g., Alers et al., Lab Invest. 77(5): 437-438 (1997) and Isaacs et al., Cancer Surv. 23: 19-32 (1995)). In this context, examining a biological sample for evidence of dysregulated cell growth (such as aberrant 193P1E1B expression in cancers) allows for early detection of such aberrant physiology, before a pathologic state such as cancer has progressed to a stage that therapeutic options are more limited and or the prognosis is worse. In such examinations, the status of 193P1E1B in a biological sample of interest can be compared, for example, to the status of 193P1E1B in a corresponding normal sample (e.g. a sample from that individual or alternatively another individual that is not affected by a pathology). An alteration in the status of 193P1E1B in the biological sample (as compared to the normal sample) provides evidence of dysregulated cellular growth. In addition to using a biological sample that is not affected by a pathology as a normal sample, one can also use a predetermined normative value such as a predetermined normal level of mRNA expression (see, e.g., Grever et al., J. Comp. Neurol. Dec. 9, 1996; 376(2): 306-14 and U.S. Pat. No. 5,837,501) to compare 193P1E1B 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 193P1E1B expressing cells) as well as the level, and biological activity of expressed gene products (such as 193P1E1B mRNA, polynucleotides and polypeptides). Typically, an alteration in the status of 193P1E1B comprises a change in the location of 193P1E1B and/or 193P1E1B expressing cells and/or an increase in 193P1E1B mRNA and/or protein expression.
193P1E1B status in a sample can be analyzed by a number of means well known in the art, including without limitation, immunohistochemical analysis, in situ hybridization, RT-PCR analysis on laser capture micro-dissected samples, Western blot analysis, and tissue array analysis. Typical protocols for evaluating the status of a 193P1E1B gene and gene products are found, for example in Ausubel et al. eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus, the status of 193P1E1B in a biological sample is evaluated by various methods utilized by skilled artisans including, but not limited to genomic Southern analysis (to examine, for example perturbations in a 193P1E1B gene), Northern analysis and/or PCR analysis of 193P1E1B mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of 193P1E1B mRNAs), and, Western and/or immunohistochemical analysis (to examine, for example alterations in polypeptide sequences, alterations in polypeptide localization within a sample, alterations in expression levels of 193P1E1B proteins and/or associations of 193P1E1B proteins with polypeptide binding partners). Detectable 193P1E1B polynucleotides include, for example, a 193P1E1B gene or fragment thereof, 193P1E1B mRNA, alternative splice variants, 193P1E1B mRNAs, and recombinant DNA or RNA molecules containing a 193P1E1B polynucleotide.
The expression profile of 193P1E1B makes it a diagnostic marker for local and/or metastasized disease, and provides information on the growth or oncogenic potential of a biological sample. In particular, the status of 193P1E1B provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The invention provides methods and assays for determining 193P1E1B status and diagnosing cancers that express 193P1E1B, such as cancers of the tissues listed in Table I. For example, because 193P1E1B mRNA is so highly expressed in prostate and other cancers relative to normal prostate tissue, assays that evaluate the levels of 193P1E1B mRNA transcripts or proteins in a biological sample can be used to diagnose a disease associated with 193P1E1B dysregulation, and can provide prognostic information useful in defining appropriate therapeutic options.
The expression status of 193P1E1B provides information including the presence, stage and location of dysplastic, precancerous and cancerous cells, predicting susceptibility to various stages of disease, and/or for gauging tumor aggressiveness. Moreover, the expression profile makes it useful as an imaging reagent for metastasized disease. Consequently, an aspect of the invention is directed to the various molecular prognostic and diagnostic methods for examining the status of 193P1E1B in biological samples such as those from individuals suffering from, or suspected of suffering from a pathology characterized by dysregulated cellular growth, such as cancer.
As described above, the status of 193P1E1B in a biological sample can be examined by a number of well-known procedures in the art. For example, the status of 193P1E1B in a biological sample taken from a specific location in the body can be examined by evaluating the sample for the presence or absence of 193P1E1B expressing cells (e.g. those that express 193P1E1B mRNAs or proteins). This examination can provide evidence of dysregulated cellular growth, for example, when 193P1E1B-expressing cells are found in a biological sample that does not normally contain such cells (such as a lymph node), because such alterations in the status of 193P1E1B in a biological sample are often associated with dysregulated cellular growth. Specifically, one indicator of dysregulated cellular growth is the metastases of cancer cells from an organ of origin (such as the prostate) to a different area of the body (such as a lymph node). In this context, evidence of dysregulated cellular growth is important for example because occult lymph node metastases can be detected in a substantial proportion of patients with prostate cancer, and such metastases are associated with known predictors of disease progression (see, e.g., Murphy et al., Prostate 42(4): 315-317 (2000);Su et al., Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al., J Urol August 1995 154(2 Pt 1):474-8).
In one aspect, the invention provides methods for monitoring 193P1E1B gene products by determining the status of 193P1E1B gene products expressed by cells from an individual suspected of having a disease associated with dysregulated cell growth (such as hyperplasia or cancer) and then comparing the status so determined to the status of 193P1E1B gene products in a corresponding normal sample. The presence of aberrant 193P1E1B gene products in the test sample relative to the normal sample provides an indication of the presence of dysregulated cell growth within the cells of the individual.
In another aspect, the invention provides assays useful in determining the presence of cancer in an individual, comprising detecting a significant increase in 193P1E1B mRNA or protein expression in a test cell or tissue sample relative to expression levels in the corresponding normal cell or tissue. The presence of 193P1E1B mRNA can, for example, be evaluated in tissues including but not limited to those listed in Table I. The presence of significant 193P1E1B expression in any of these tissues is useful to indicate the emergence, presence and/or severity of a cancer, since the corresponding normal tissues do not express 193P1E1B mRNA or express it at lower levels.
In a related embodiment, 193P1E1B status is determined at the protein level rather than at the nucleic acid level. For example, such a method comprises determining the level of 193P1E1B protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 193P1E1B expressed in a corresponding normal sample. In one embodiment, the presence of 193P1E1B protein is evaluated, for example, using immunohistochemical methods. 193P1E1B antibodies or binding partners capable of detecting 193P1E1B protein expression are used in a variety of assay formats well known in the art for this purpose.
In a further embodiment, one can evaluate the status of 193P1E1B nucleotide and amino add sequences in a biological sample in order to identify perturbations in the structure of these molecules. These perturbations can include insertions, deletions, substitutions and the like. Such evaluations are useful because perturbations in the nucleotide and amino acid sequences are observed in a large number of proteins associated with a growth dysregulated phenotype (see, e.g., Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For example, a mutation in the sequence of 193P1E1B may be indicative of the presence or promotion of a tumor. Such assays therefore have diagnostic and predictive value where a mutation in 193P1E1B indicates a potential loss of function or increase in tumor growth.
A wide variety of assays for observing perturbations in nucleotide and amino acid sequences are well known in the art. For example, the size and structure of nucleic acid or amino acid sequences of 193P1E1B gene products are observed by the Northern, Southern, Western, PCR and DNA sequencing protocols discussed herein. In addition, other methods for observing perturbations in nucleotide and amino acid sequences such as single strand conformation polymorphism analysis are well known in the art (see, e.g., U.S. Pat. Nos. 5,382,510 issued Sep. 7, 1999, and 5,952,170 issued Jan. 17, 1995).
Additionally, one can examine the methylation status of a 193P1E1B gene in a biological sample. Aberrant demethylation and/or hypermethylation of CpG islands in gene 5′ regulatory regions frequency 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 at. eds., 1995.
Gene amplification is an additional method for assessing the status of 193P1E1B. Gene amplification is measured in a sample directly, for example, by conventional Southern blotting or Northern blotting to quantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies are employed that recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn are labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
Biopsied tissue or peripheral blood can be conveniently assayed for the presence of cancer cells using for example, Northern, dot blot or RT-PCR analysis to detect 193P1E1B expression. The presence of RT-PCR amplifiable 193P1E1B mRNA provides an indication of the presence of cancer. RT-PCR assays are well known in the art. RT-PCR detection assays for tumor cells in peripheral blood are currently being evaluated for use in the diagnosis and management of a number of human solid tumors. In the prostate cancer field, these include RT-PCR assays for the detection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000; Heston et al., 1995, Clin. Chem. 41:1687-1688).
A further aspect of the invention is an assessment of the susceptibility that an individual has for developing cancer. In one embodiment, a method for predicting susceptibility to cancer comprises detecting 193P1E1B mRNA or 193P1E1B protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of 193P1E1B mRNA expression correlates to the degree of susceptibility. In a specific embodiment, the presence of 193P1E1B in prostate or other tissue is examined, with the presence of 193P1E1B in the sample providing an indication of prostate cancer susceptibility (or the emergence or existence of a prostate tumor). Similarly, one can evaluate the integrity 193P1E1B nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations in 193P1E1B gene products in the sample is an indication of cancer susceptibility (or the emergence or existence of a tumor).
The invention also comprises methods for gauging tumor aggressiveness. In one embodiment, a method for gauging aggressiveness of a tumor comprises determining the level of 193P1E1B mRNA or 193P1E1B protein expressed by tumor cells, comparing the level so determined to the level of 193P1E1B mRNA or 193P1E1B protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of 193P1E1B mRNA or 193P1E1B protein expression in the tumor sample relative to the normal sample indicates the degree of aggressiveness. In a specific embodiment, aggressiveness of a tumor is evaluated by determining the extent to which 193P1E1B is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors. Another embodiment is the evaluation of the integrity of 193P1E1B nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations indicates more aggressive tumors.
Another embodiment of the invention is directed to methods for observing the progression of a malignancy in an individual over time. In one embodiment, methods for observing the progression of a malignancy in an individual over time comprise determining the level of 193P1E1B mRNA or 193P1E1B protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of 193P1E1B mRNA or 193P1E1B protein expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of 193P1E1B mRNA or 193P1E1B protein expression in the tumor sample over time provides information on the progression of the cancer. In a specific embodiment, the progression of a cancer is evaluated by determining 193P1E1B expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer. Also, one can evaluate the integrity 193P1E1B nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like, where the presence of one or more perturbations indicates a progression of the cancer.
The above diagnostic approaches can be combined with any one of a wide variety of prognostic and diagnostic protocols known in the art. For example, another embodiment of the invention is directed to methods for observing a coincidence between the expression of 193P1E1B gene and 193P1E1B gene products (or perturbations in 193P1E1B gene and 193P1E1B gene products) and a factor that is associated with malignancy, as a means for diagnosing and prognosticating the status of a tissue sample. A wide variety of factors associated with malignancy can be utilized, such as the expression of genes associated with malignancy (e.g. PSA, PSCA and PSM expression for prostate cancer etc.) as well as gross cytological observations (see, e.g., Bocking et al., 1984, Anal. Quant. Cytol. 6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al., 1998, Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods for observing a coincidence between the expression of 193P1E1B gene and 193P1E1B gene products (or perturbations in 193P1E1B gene and 193P1E1B gene products) and another factor that is associated with malignancy are useful, for example, because the presence of a set of specific factors that coincide with disease provides information crucial for diagnosing and prognosticating the status of a tissue sample.
In one embodiment, methods for observing a coincidence between the expression of 193P1E1B gene and 193P1E1B gene products (or perturbations in 193P1E1B gene and 193P1E1B gene products) and another factor associated with malignancy entails detecting the overexpression of 193P1E1B mRNA or protein in a tissue sample, detecting the overexpression of PSA mRNA or protein in a tissue sample (or PSCA or PSM expression), and observing a coincidence of 193P1E1B mRNA or protein and PSA mRNA or protein overexpression (or PSCA or PSM expression). In a specific embodiment, the expression of 193P1E1B and PSA mRNA in prostate tissue is examined, where the coincidence of 193P1E1B and PSA mRNA overexpression in the sample indicates the existence of prostate cancer, prostate cancer susceptibility or the emergence or status of a prostate tumor.
Methods for detecting and quantifying the expression of 193P1E1B mRNA or protein are described herein, and standard nucleic acid and protein detection and quantification technologies are well known in the art. Standard methods for the detection and quantification of 193P1E1B mRNA include in situ hybridization using labeled 193P1E1B riboprobes, Northern blot and related techniques using 193P1E1B polynucleotide probes, RT-PCR analysis using primers specific for 193P1E1B, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like. In a specific embodiment, semi-quantitative RT-PCR is used to detect and quantify 193P1E1B mRNA expression. Any number of primers capable of amplifying 193P1E1B can be used for this purpose, including but not limited to the various primer sets specifically described herein. In a specific embodiment, polyclonal or monoclonal antibodies specifically reactive with the wild-type 193P1E1B protein can be used in an immunohistochemical assay of biopsied tissue.
IX.) Identification of Molecules That Interact With 193P1E1B
The 193P1E1B protein and nucleic acid sequences disclosed herein allow a skilled artisan to identify proteins, small molecules and other agents that interact with 193P1E1B, as well as pathways activated by 193P1E1B via anyone of a variety of art accepted protocols. For example, one can utilize one of the so-called interaction trap systems (also referred to as the “two-hybrid assay”). In such systems, molecules interact and reconstitute a transcription factor which directs expression of a reporter gene, whereupon the expression of the reporter gene is assayed. Other systems identify protein-protein interactions in vivo through reconstitution of a eukaryotic transcriptional activator, see, e.g., U.S. Pat. Nos. 5,955,280 issued Sep. 21, 1999, 5,925,523 issued Jul. 20, 1999, 5,846,722 issued Dec. 8, 1998 and 6,004,746 issued Dec. 21, 1999. Algorithms are also available in the art for genome-based predictions of protein function (see, e.g., Marcotte, et al., Nature 402: Nov. 4, 1999, 83-86).
Alternatively one can screen peptide libraries to identify molecules that interact with 193P1E1B protein sequences. In such methods, peptides that bind to 193P1E1B are identified by screening libraries that encode a random or controlled collection of amino acids. Peptides encoded by the libraries are expressed as fusion proteins of bacteriophage coat proteins, the bacteriophage particles are then screened against the 193P1E1B protein(s).
Accordingly, peptides having a wide variety of uses, such as therapeutic, prognostic or diagnostic reagents, are thus identified without any prior information on the structure of the expected ligand or receptor molecule. Typical peptide libraries and screening methods that can be used to identify molecules that interact with 193P1E1B protein sequences are disclosed for example in U.S. Pat. Nos. 5,723,286 issued Mar. 3, 1998 and 5,733,731 issued Mar. 31, 1998.
Alternatively, cell lines that express 193P1E1B are used to identify protein-protein interactions mediated by 193P1E1B. Such interactions can be examined using immunoprecipitation techniques (see, e.g., Hamilton B. J., et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51). 193P1E1B protein can be immunoprecipitated from 193P1E1B-expressing cell lines using anti-193P1E1B antibodies. Alternatively, antibodies against His-tag can be used in a cell line engineered to express fusions of 193P1E1B and a His-tag (vectors mentioned above). The immunoprecipitated complex can be examined for protein association by procedures such as Western blotting, 35S-methionine labeling of proteins, protein microsequencing, silver staining and two-dimensional gel electrophoresis.
Small molecules and ligands that interact with 193P1E1B can be identified through related embodiments of such screening assays. For example, small molecules can be identified that interfere with protein function, including molecules that interfere with 193P1E1B's ability to mediate phosphorylation and de-phosphorylation, interaction with DNA or RNA molecules as an indication of regulation of cell cycles, second messenger signaling or tumorigenesis. Similarly, small molecules that modulate 193P1E1B-related ion channel, protein pump, or cell communication functions are identified and used to treat patients that have a cancer that expresses 193P1E1B (see, e.g., Hille, B., Ionic Channels of Excitable Membranes 2nd Ed., Sinauer Assoc., Sunderland, Mass., 1992). Moreover, ligands that regulate 193P1E1B function can be identified based on their ability to bind 193P1E1B and activate a reporter construct. Typical methods are discussed for example in U.S. Pat. No. 5,928,868 issued Jul. 27, 1999, and include methods for forming hybrid ligands in which at least one ligand is a small molecule. In an illustrative embodiment, cells engineered to express a fusion protein of 193P1E1B and a DNA-binding protein are used to co-express a fusion protein of a hybrid ligand/small molecule and a cDNA library transcriptional activator protein. The cells further contain a reporter gene, the expression of which is conditioned on the proximity of the first and second fusion proteins to each other, an event that occurs only if the hybrid ligand binds to target sites on both hybrid proteins. Those cells that express the reporter gene are selected and the unknown small molecule or the unknown ligand is identified. This method provides a means of identifying modulators, which activate or inhibit 193P1E1B.
An embodiment of this invention comprises a method of screening for a molecule that interacts with a 193P1E1B amino acid sequence shown in FIG. 2 or FIG. 3, comprising the steps of contacting a population of molecules with a 193P1E1B amino acid sequence, allowing the population of molecules and the 193P1E1B amino acid sequence to interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the 193P1E1B amino acid sequence, and then separating molecules that do not interact with the 193P1E1B amino acid sequence from molecules that do. In a specific embodiment, the method further comprises purifying, characterizing and identifying a molecule that interacts with the 193P1E1B amino acid sequence. The identified molecule can be used to modulate a function performed by 193P1E1B. In a preferred embodiment, the 193P1E1B amino acid sequence is contacted with a library of peptides.
X.) Therapeutic Methods and Compositions
The identification of 193P1E1B as a protein that is normally expressed in a restricted set of tissues, but which is also expressed in prostate and other cancers, opens a number of therapeutic approaches to the treatment of such cancers. As contemplated herein, 193P1E1B functions as a transcription factor involved in activating tumor-promoting genes or repressing genes that block tumorigenesis.
Accordingly, therapeutic approaches that inhibit the activity of a 193P1E1B protein are useful for patients suffering from a cancer that expresses 193P1E1B. These therapeutic approaches generally fall into two classes. One class comprises various methods for inhibiting the binding or association of a 193P1E1B protein with its binding partner or with other proteins. Another class comprises a variety of methods for inhibiting the transcription of a 193P1E1B gene or translation of 193P1E1B mRNA.
X.A.) Anti-Cancer Vaccines
The invention provides cancer vaccines comprising a 193P1E1B-related protein or 193P1E1B related nucleic acid. In view of the expression of 193P1E1B, cancer vaccines prevent and/or treat 193P1E1B-expressing cancers with minimal or no effects on non-target tissues. The use of a tumor antigen in a vaccine that generates humoral and/or cell-mediated immune responses as anti-cancer therapy is well known in the art and has been employed in prostate cancer using human PSMA and rodent PAP immunogens (Hodge et al., 1995, Int J. Cancer 63:231-237; Fong et al., 1997, J. Immunol. 159:3113-3117).
Such methods can be readily practiced by employing a 193P1E1B-related protein, or a 193P1E1B-encoding nucleic acid molecule and recombinant vectors capable of expressing and presenting the 193P1E1B immunogen (which typically comprises a number of antibody or T cell epitopes). Skilled artisans understand that a wide variety of vaccine systems for delivery of immunoreactive epitopes are known in the art (see, e.g., Heryln et al., Ann Med 1999 Feb 31(1):66-78; Maruyama et al., Cancer Immunol Immunother June 2000 49(3):123-32) Briefly, such methods of generating an immune response (e.g. humoral and/or cell-mediated) in a mammal, comprise the steps of: exposing the mammal's immune system to an immunoreactive epitope (e.g. an epitope present in a 193P1E1B protein shown in FIG. 3 or analog or homolog thereof) so that the mammal generates an immune response that is specific for that epitope (e.g. generates antibodies that specifically recognize that epitope). In a preferred method, a 193P1E1B immunogen contains a biological motif, see e.g., Tables VIII-XXI and XXII-XLIX, or a peptide of a size range from 193P1E1B indicated in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.
The entire 193P1E1B protein, immunogenic regions or epitopes thereof can be combined and delivered by various means. Such vaccine compositions can include, for example, lipopeptides (e.g., Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge, et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine 12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995), peptide compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin Exp Immunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J. P., J. Immunol. Methods 196:17-32, 1996), peptides formulated as multivalent peptides; peptides for use in ballistic delivery systems, typically crystallized peptides, viral delivery vectors (Perkus, M. E. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. et al., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology 4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al., Virology 175:535, 1990), particles of viral or synthetic origin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996; Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. et al., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R., and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al., Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol. 148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked or particle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993; Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993; Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev. Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993). Toxin-targeted delivery technologies, also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Mass.) may also be used.
In patients with 193P1E1B-associated cancer, the vaccine compositions of the invention can also be used in conjunction with other treatments used for cancer, e.g., surgery, chemotherapy, drug therapies, radiation therapies, etc. including use in combination with immune adjuvants such as IL-2, IL-12, GM-CSF, and the like.
Cellular Vaccines:
CTL epitopes can be determined using specific algorithms to identify peptides within 193P1E1B protein that bind corresponding HLA alleles (see e.g., Table IV; EpimerTM and EpimatrixTM, Brown University; and, BIMAS, and SYFPEITHI. In a preferred embodiment, a 193P1E1B immunogen contains one or more amino acid sequences identified using techniques well known in the art, such as the sequences shown in Tables VIII-XXI and XXII-XLIX or a peptide of 8, 9, 10 or 11 amino acids specified by an HLA Class I motif/supermotif (e.g., Table IV (A), Table IV (D), or Table IV (E)) and/or a peptide of at least 9 amino acids that comprises an HLA Class II motif/supermotif (e.g., Table IV (B) or Table IV (C)). As is appreciated in the art, the HLA Class I binding groove is essentially closed ended so that peptides of only a particular size range can fit into the groove and be bound, generally HLA Class I epitopes are 8, 9, 10, or 11 amino acids long. In contrast, the HLA Class II binding groove is essentially open ended; therefore a peptide of about 9 or more amino acids can be bound by an HLA Class 11 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, i.e., additional amino acids can be attached to the amino and/or carboxyl termini of a motif-bearing sequence. HLA Class II epitopes are often 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long, or longer than 25 amino acids.
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 193P1E1B protein) so that an immune response is generated. A typical embodiment consists of a method for generating an immune response to 193P1E1B in a host, by contacting the host with a sufficient amount of at least one 193P1E1B B cell or cytotoxic T-cell epitope or analog thereof; and at least one periodic interval thereafter re-contacting the host with the 193P1E1B B cell or cytotoxic T-cell epitope or analog thereof. A specific embodiment consists of a method of generating an immune response against a 193P1E1B-related protein or a man-made multiepitopic peptide comprising: administering 193P1E1B immunogen (e.g. a 193P1E1B protein or a peptide fragment thereof, a 193P1E1B fusion protein or analog etc.) in a vaccine preparation to a human or another mammal. Typically, such vaccine preparations further contain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or a universal helper epitope such as a 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 193P1E1B immunogen by: administering in vivo to muscle or skin of the individual's body a DNA molecule that comprises a DNA sequence that encodes a 193P1E1B immunogen, the DNA sequence operatively linked to regulatory sequences which control the expression of the DNA sequence; wherein the DNA molecule is taken up by cells, the DNA sequence is expressed in the cells and an immune response is generated against the immunogen (see, e.g., U.S. Pat. No. 5,962,428). Optionally a genetic vaccine facilitator such as anionic lipids; saponins; lectins; estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; and urea is also administered. In addition, an antiidiotypic antibody can be administered that mimics 193P1E1B, in order to generate a response to the target antigen.
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 193P1E1B. Constructs comprising DNA encoding a 193P1E1B-related protein/immunogen and appropriate regulatory sequences can be injected directly into muscle or skin of an individual, such that the cells of the muscle or skin take-up the construct and express the encoded 193P1E1B protein/immunogen. Alternatively, a vaccine comprises a 193P1E1B-related protein. Expression of the 193P1E1B-related protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells that bear a 193P1E1B protein. Various prophylactic and therapeutic genetic immunization techniques known in the art can be used (for review, see information and references published at Internet address genweb.com). Nucleic acid-based delivery is described, for instance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720. Examples of DNA-based delivery technologies include “naked DNA”, facilitated (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, lentivirus, and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol. 8:658-663; Tsang et al. J. Natl. Cancer Inst. 87:982-990 (1995)). Non-viral delivery systems can also be employed by introducing naked DNA encoding a 193P1E1B-related protein into the patent (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 Calmelte 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 193P1E1B-related nucleic add molecule. In one embodiment, the full-length human 193P1E1B cDNA is employed. In another embodiment, 193P1E1B nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopes are employed.
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 193P1E1B antigen to a patient's immune system. Dendritic cells express MHC class I and II molecules, B7 co-stimulator, and IL-12, and are thus highly specialized antigen presenting cells. In prostate cancer, autologous dendritic cells pulsed with peptides of the prostate-specific membrane antigen (PSMA) are being used in a Phase I clinical trial to stimulate prostate cancer patients' immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphy et al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used to present 193P1E1B peptides to T cells in the context of MHC class I or II molecules. In one embodiment, autologous dendritic cells are pulsed with 193P1E1B peptides capable of binding to MHC class I and/or class II molecules. In another embodiment, dendritic cells are pulsed with the complete 193P1E1B protein. Yet another embodiment involves engineering the overexpression of a 193P1E1B gene in dendritic cells using various implementing vectors known in the art, such as adenovirus (Arthur et al., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al., 1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNA transfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), or tumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med. 186:1177-1182). Cells that express 193P1E1B can also be engineered to express immune modulators, such as GM-CSF, and used as immunizing agents.
X.B.) 193P1E1B as a Target for Antibody-Based Therapy
193P1E1B is an attractive target for antibody-based therapeutic strategies. A number of antibody strategies are known in the art for targeting both extracellular and intracellular molecules (see, e.g., complement and ADCC mediated killing as well as the use of intrabodies). Because 193P1E1B is expressed by cancer cells of various lineages relative to corresponding normal cells, systemic administration of 193P1E1B-immunoreactive compositions are prepared that exhibit excellent sensitivity without toxic, non-specific and/or non-target effects caused by binding of the immunoreactive composition to non-target organs and tissues. Antibodies specifically reactive with domains of 193P1E1B are useful to treat 193P1E1B-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.
193P1E1B antibodies can be introduced into a patient such that the antibody binds to 193P1E1B and modulates a function, such as an interaction with a binding partner, and consequently mediates destruction of the tumor cells and/or inhibits the growth of the tumor cells. Mechanisms by which such antibodies exert a therapeutic effect can include complement-mediated cytolysis, antibody-dependent cellular cytotoxicity, modulation of the physiological function of 193P1E1B, inhibition of ligand binding or signal transduction pathways, modulation of tumor cell differentiation, alteration of tumor angiogenesis factor profiles, and/or apoptosis.
Those skilled in the art understand that antibodies can be used to specifically target and bind immunogenic molecules such as an immunogenic region of a 193P1E1B sequence shown in FIG. 2 or FIG. 3. In addition, skilled artisans understand that it is routine to conjugate antibodies to cytotoxic agents (see, e.g., 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. 193P1E1B), 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-193P1E1B antibody) that binds to a marker (e.g. 193P1E1B) expressed, accessible to binding or localized on the cell surfaces. A typical embodiment is a method of delivering a cytotoxic and/or therapeutic agent to a cell expressing 193P1E1B, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 193P1E1B epitope, and, exposing the cell to the antibody-agent conjugate. Another illustrative embodiment is a method of treating an individual suspected of suffering from metastasized cancer, comprising a step of administering parenterally to said individual a pharmaceutical composition comprising a therapeutically effective amount of an antibody conjugated to a cytotoxic and/or therapeutic agent.
Cancer immunotherapy using anti-193P1E1B antibodies can be done in accordance with various approaches that have been successfully employed in the treatment of other types of cancer, including but not limited to colon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari et al., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al., 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al., 1994, Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res. 55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin. Immunol. 11:117-127). Some therapeutic approaches involve conjugation of naked antibody to a toxin or radioisotope, such as the conjugation of 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, 193P1E1B antibodies can be administered in conjunction with radiation, chemotherapy or hormone ablation. Also, antibodies can be conjugated to a toxin such as calicheamicin (e.g., 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 193P1E1B antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well. Fan et al. (Cancer Res. 53:4637-4642, 1993), Prewett et al. (International J. of Onco. 9:217-224, 1996), and Hancock et al. (Cancer Res. 51:4575-4580, 1991) describe the use of various antibodies together with chemotherapeutic agents.
Although 193P1E1B antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well.
Cancer patients can be evaluated for the presence and level of 193P1E1B expression, preferably using immunohistochemical assessments of tumor tissue, quantitative 193P1E1B imaging, or other techniques that reliably indicate the presence and degree of 193P1E1B expression. Immunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this purpose. Methods for immunohistochemical analysis of tumor tissues are well known in the art.
Anti-193P1E1B monoclonal antibodies that treat prostate and other cancers include those that initiate a potent immune response against the tumor or those that are directly cytotoxic. In this regard, anti-193P1E1B monoclonal antibodies (mAbs) can elicit tumor cell lysis by either complement-mediated or antibody-dependent cell cytotoxicity (ADCC) mechanisms, both of which require an intact Fc portion of the immunoglobulin molecule for interaction with effector cell Fc receptor sites on complement proteins. In addition, anti-193P1E1B mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express 193P1E1B. Mechanisms by which directly cytotoxic mAbs act include: inhibition of cell growth, modulation of cellular differentiation, modulation of tumor angiogenesis factor profiles, and the induction of apoptosis. The mechanism(s) by which a particular anti-193P1E1B mAb exerts an anti-tumor effect is evaluated using any number of in vitro assays that evaluate cell death such as ADCC, ADMMC, complement-mediated cell lysis, and so forth, as is generally known in the art.
In some patients, the use of murine or other non-human monoclonal antibodies, or human/mouse chimeric mAbs can induce moderate to strong immune responses against the non-human antibody. This can result in clearance of the antibody from circulation and reduced efficacy. In the most severe cases, such an immune response can lead to the extensive formation of immune complexes which, potentially, can cause renal failure. Accordingly, preferred monoclonal antibodies used in the therapeutic methods of the invention are those that are either fully human or humanized and that bind specifically to the target 193P1E1B antigen with high affinity but exhibit low or no antigenicity in the patient.
Therapeutic methods of the invention contemplate the administration of single anti-193P1E1B mAbs as well as combinations, or cocktails, of different mAbs. Such mAb cocktails can have certain advantages inasmuch as they contain mAbs that target different epitopes, exploit different effector mechanisms or combine directly cytotoxic mAbs with mAbs that rely on immune effector functionality. Such mAbs in combination can exhibit synergistic therapeutic effects. In addition, anti-193P1E1B mAbs can be administered concomitantly with other therapeutic modalities, including but not limited to various chemotherapeutic agents, androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery or radiation. The anti-193P1E1B mAbs are administered in their “naked” or unconjugated form, or can have a therapeutic agent(s) conjugated to them.
Anti-193P1E1B antibody formulations are administered via any route capable of delivering the antibodies to a tumor cell. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumor, intradermal, and the like. Treatment generally involves repeated administration of the anti-193P1E1B antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg/kg body weight. In general, doses in the range of 10-1000 mg mAb per week are effective and well tolerated.
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-193P1E1B mAb preparation represents an acceptable dosing regimen. Preferably, the initial loading dose is administered as a 90-minute or longer infusion. The periodic maintenance dose is administered as a 30 minute or longer infusion, provided the initial dose was well tolerated. As appreciated by those of skill in the art, various factors can influence the ideal dose regimen in a particular case. Such factors include, for example, the binding affinity and half life of the Ab or mAbs used, the degree of 193P1E1B expression in the patient, the extent of circulating shed 193P1E1B antigen, the desired steady-state antibody concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient.
Optionally, patients should be evaluated for the levels of 193P1E1B in a given sample (e.g. the levels of circulating 193P1E1B antigen and/or 193P1E1B expressing cells) in order to assist in the determination of the most effective dosing regimen, etc. Such evaluations are also used for monitoring purposes throughout therapy, and are useful to gauge therapeutic success in combination with the evaluation of other parameters (for example, urine cytology and/or ImmunoCyt levels in bladder cancer therapy, or by analogy, serum PSA levels in prostate cancer therapy).
Anti-idiotypic anti-193P1E1B antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a 193P1E1B-related protein. In particular, the generation of anti-idiotypic antibodies is well known in the art; this methodology can readily be adapted to generate anti-idiotypic anti-193P1E1B antibodies that mimic an epitope on a 193P1E1B-related protein (see, for example, Wagner et al., 1997, Hybridoma 16: 33-40; Foon et al., 1995, J. Clin. Invest. 96:334-342; Herlyn et al., 1996, Cancer Immunol. Immunother. 43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccine strategies.
X.C.) 193P1E1B 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-S-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 193P1E1B 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 (TM). For HLA Class II a similar rationale is employed; again 34 epitopes are selected from at least one TAA (see, e.g., Rosenberg et al., Science 278:1447-1450). Epitopes from one TAA may be used in combination with epitopes from one or more additional TAAs to produce a vaccine that targets tumors with varying expression patterns of frequently-expressed TAAs.
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 193P1E1B, the PADRE® universal helper T cell epitope or multiple HTL epitopes from 193P1E1B (see e.g., Tables VIII-XXI and XXII to XLIX), and an endoplasmic reticulum-translocating signal sequence can be engineered. A vaccine may also comprise epitopes that are derived from other TAAs.
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., LeIF), 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 Feigner, et al., Proc. Nat'l. Acad. Sci. USA 84:7413 (1987). In addition, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds (PINC) could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.
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.
XC.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: 48), Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 49), and Streptococcus 18 kD protein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO: 50). Other examples include peptides bearing a DR 14-7 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: AKXVAAWTLKAAA (SEQ ID NO: 51), where a 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 193P1E1B. Optionally, a helper T cell (HTL) peptide, such as a natural or artificial loosely restricted HLA Class II peptide, can be included to facilitate the CTL response. Thus, a vaccine in accordance with the invention is used to treat a cancer which expresses or overexpresses 193P1E1B.
X.D. Adoptive Immunotherapy
Antigenic 193P1E1B-related peptides are used to elicit a CTL and/or HTL response ex vivo, as well. The resulting CTL or HTL cells, can be used to treat tumors in patients that do not respond to other conventional forms of therapy, or will not respond to a therapeutic vaccine peptide or nucleic acid in accordance with the invention. Ex vivo CTL or HTL responses to a particular antigen are induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of antigen-presenting cells (APC), such as dendritic cells, and the appropriate immunogenic peptide. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cell (e.g., a tumor cell). Transfected dendritic cells may also be used as antigen presenting cells.
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 193P1E1B. In therapeutic applications, peptide and/or nucleic acid compositions are administered to a patient in an amount sufficient to elicit an effective B cell, CTL and/or HTL response to the antigen and to cure or at least partially arrest or slow symptoms and/or complications. An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition administered, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.
For pharmaceutical compositions, the immunogenic peptides of the invention, or DNA encoding them, are generally administered to an individual already bearing a tumor that expresses 193P1E1B. The peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences. Patients can be treated with the immunogenic peptides separately or in conjunction with other treatments, such as surgery, as appropriate.
For therapeutic use, administration should generally begin at the first diagnosis of 193P1E1B-associated cancer. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. The embodiment of the vaccine composition (i.e., including, but not limited to embodiments such as peptide cocktails, polyepitopic polypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells) delivered to the patient may vary according to the stage of the disease or the patient's health status. For example, in a patient with a tumor that expresses 193P1E1B, a vaccine comprising 193P1E1B-specific CTL may be more efficacious in killing tumor cells in patient with advanced disease than alternative embodiments.
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 μ, 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-193P1E1B antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1 to about 10 mg/kg body weight. In general, doses in the range of 10-500 mg mAb per week are effective and well tolerated. Moreover, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti-193P1E1B mAb preparation represents an acceptable dosing regimen. As appreciated by those of skill in the art, various factors can influence the ideal dose in a particular case. Such factors include, for example, half life of a composition, the binding affinity of an Ab, the immunogenicity of a substance, the degree of 193P1E1B expression in the patient, the extent of circulating shed 193P1E1B antigen, the desired steady-state concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient. Non-limiting preferred human unit doses are, for example, 500 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 193P1E1B.
As disclosed herein, 193P1E1B polynucleotides, polypeptides, reactive cytotoxic T cells (CTL), reactive helper T cells (HTL) and anti-polypeptide antibodies are used in well known diagnostic, prognostic and therapeutic assays that examine conditions associated with dysregulated cell growth such as cancer, in particular the cancers listed in Table I (see, e.g., both its specific pattern of tissue expression as well as its overexpression in certain cancers as described for example in the Example entitled “Expression analysis of 193P1E1B in normal tissues, and patient specimens”).
193P1E1B can be analogized to a prostate associated antigen PSA, the archetypal marker that has been used by medical practitioners for years to identify and monitor the presence of prostate cancer (see, e.g., Merrill et al., J. Urol. 163(2): 503-5120 (2000); Polascik et al., J. Urol. Aug; 162(2):293-306 (1999) and Fortier et al., J. Nat. Cancer Inst. 91(19): 1635-1640(1999)). A variety of other diagnostic markers are also used in similar contexts including p53 and K-ras (see, e.g., Tulchinsky et al., Int J Mol Med Jul. 4, 1999 (1):99-102 and Minimoto et al., Cancer Detect Prev 2000;24(1):1-12). Therefore, this disclosure of 193P1E1B polynucleotides and polypeptides (as well as 193P1E1B polynucleotide probes and anti-193P1E1B antibodies used to identify the presence of these molecules) and their properties allows skilled artisans to utilize these molecules in methods that are analogous to those used, for example, in a variety of diagnostic assays directed to examining conditions associated with cancer.
Typical embodiments of diagnostic methods which utilize the 193P1E1B polynucleotides, polypeptides, reactive T cells and antibodies are analogous to those methods from well-established diagnostic assays, which employ, e.g., PSA polynucleotides, polypeptides, reactive T cells and antibodies. For example, just as PSA polynucleotides are used as probes (for example in Northern analysis, see, e.g., Sharief et al., Biochem. Mol. Biol. Int. 33(3):567-74(1994)) and primers (for example in PCR analysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189-1190 (2000)) to observe the presence and/or the level of PSA mRNAs in methods of monitoring PSA overexpression or the metastasis of prostate cancers, the 193P1E1B polynucleotides described herein can be utilized in the same way to detect 193P1E1B overexpression or the metastasis of prostate and other cancers expressing this gene. Alternatively, just as PSA polypeptides are used to generate antibodies specific for PSA which can then be used to observe the presence and/or the level of PSA proteins in methods to monitor PSA protein overexpression (see, e.g., Stephan et al., Urology 55(4):560-3 (2000)) or the metastasis of prostate cells (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the 193P1E1B polypeptides described herein can be utilized to generate antibodies for use in detecting 193P1E1B overexpression or the metastasis of prostate cells and cells of other cancers expressing this gene.
Specifically, because metastases involves the movement of cancer cells from an organ of origin (such as the lung or prostate gland etc.) to a different area of the body (such as a lymph node), assays which examine a biological sample for the presence of cells expressing 193P1E1B polynucleotides and/or polypeptides can be used to provide evidence of metastasis. For example, when a biological sample from tissue that does not normally contain 193P1E1B-expressing cells (lymph node) is found to contain 193P1E1B-expressing cells such as the 193P1E1B expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis.
Alternatively 193P1E1B polynucleotides and/or polypeptides can be used to provide evidence of cancer, for example, when cells in a biological sample that do not normally express 193P1E1B or express 193P1E1B at a different level are found to express 193P1E1B or have an increased expression of 193P1E1B (see, e.g., the 193P1E1B expression in the cancers listed in Table I and in patient samples etc. shown in the accompanying Figures). In such assays, artisans may further wish to generate supplementary evidence of metastasis by testing the biological sample for the presence of a second tissue restricted marker (in addition to 193P1E1B) such as PSA, PSCA etc. (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)).
Just as PSA polynucleotide fragments and polynucleotide variants are employed by skilled artisans for use in methods of monitoring PSA, 193P1E1B polynucleotide fragments and polynucleotide variants are used in an analogous manner. In particular, typical PSA polynucleotides used in methods of monitoring PSA are probes or primers which consist of fragments of the PSA cDNA sequence. Illustrating this, primers used to PCR amplify a PSA polynucleotide must include less than the whole PSA sequence to function in the polymerase chain reaction. In the context of such PCR reactions, skilled artisans generally create a variety of different polynucleotide fragments that can be used as primers in order to amplify different portions of a polynucleotide of interest or to optimize amplification reactions (see, e.g., Caetano-Anolles, G. Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., Methods Mol. Biol. 98:121-154 (1998)). An additional illustration of the use of such fragments is provided in the Example entitled “Expression analysis of 193P1E1B in normal tissues, and patient specimens,” where a 193P1E1B polynucleotide fragment is used as a probe to show the expression of 193P1E1B RNAs in cancer cells. In addition, variant polynucleotide sequences are typically used as primers and probes for the corresponding mRNAs in PCR and Northern analyses (see, e.g., Sawai et al., Fetal Diagn. Ther. November-December 1996 11 (6):407-13 and Current Protocols In Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubel et al. eds., 1995)). Polynucleotide fragments and variants are useful in this context where they are capable of binding to a target polynucleotide sequence (e.g., a 193P1E1B 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. 193P1E1B polypeptide fragments and polypeptide analogs or variants can also be used in an analogous manner. This practice of using polypeptide fragments or polypeptide variants to generate antibodies (such as anti-PSA antibodies or T cells) is typical in the art with a wide variety of systems such as fusion proteins being used by practitioners (see, e.g., Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubel et al. eds., 1995). In this context, each epitope(s) functions to provide the architecture with which an antibody or T cell is reactive. Typically, skilled artisans create a variety of different polypeptide fragments that can be used in order to generate immune responses specific for different portions of a polypeptide of interest (see, e.g., U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it may be preferable to utilize a polypeptide comprising one of the 193P1E1B biological motifs discussed herein or a motif-bearing subsequence which is readily identified by one of skill in the art based on motifs available in the art. Polypeptide fragments, variants or analogs are typically useful in this context as long as they comprise an epitope capable of generating an antibody or T cell specific for a target polypeptide sequence (e.g. a 193P1E1B polypeptide shown in FIG. 3).
As shown herein, the 193P1E1B polynucleotides and polypeptides (as well as the 193P1E1B polynucleotide probes and anti-193P1E1B antibodies or T cells used to identify the presence of these molecules) exhibit specific properties that make them useful in diagnosing cancers such as those listed in Table I. Diagnostic assays that measure the presence of 193P1E1B gene products, in order to evaluate the presence or onset of a disease condition described herein, such as prostate cancer, are used to identify patients for preventive measures or further monitoring, as has been done so successfully with PSA. Moreover, these materials satisfy a need in the art for molecules having similar or complementary characteristics to PSA in situations where, for example, a definite diagnosis of metastasis of prostatic origin cannot be made on the basis of a test for PSA alone (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)), and consequently, materials such as 193P1E1B polynucleotides and polypeptides (as well as the 193P1E1B polynucleotide probes and anti-193P1E1B antibodies used to identify the presence of these molecules) need to be employed to confirm a metastases of prostatic origin.
Finally, in addition to their use in diagnostic assays, the 193P1E1B polynucleotides disclosed herein have a number of other utilities such as their use in the identification of oncogenetic associated chromosomal abnormalities in the chromosomal region to which the 193P1E1B gene maps (see the Example entitled “Chromosomal Mapping of 193P1E1B” below). Moreover, in addition to their use in diagnostic assays, the 193P1E1B-related proteins and polynucleotides disclosed herein have other utilities such as their use in the forensic analysis of tissues of unknown origin (see, e.g., Takahama K Forensic Sci Int Jun. 28, 1996;80(1-2): 63-9).
Additionally, 193P1E1B-related proteins or polynucleotides of the invention can be used to treat a pathologic condition characterized by the over-expression of 193P1E1B. For example, the amino acid or nucleic acid sequence of FIG. 2 or FIG. 3, or fragments of either, can be used to generate an immune response to a 193P1E1B antigen. Antibodies or other molecules that react with 193P1E1B can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.
XII.) Inhibition of 193P1E1B Protein Function
The invention includes various methods and compositions for inhibiting the binding of 193P1E1B to its binding partner or its association with other protein(s) as well as methods for inhibiting 193P1E1B function.
XII.A.) Inhibition of 193P1E1B With Intracellular Antibodies
In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to 193P1E1B are introduced into 193P1E1B expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti-193P1E1B antibody is expressed intracellularly, binds to 193P1E1B protein, and thereby inhibits its function. Methods for engineering such intracellular single chain antibodies are well known. Such intracellular antibodies, also known as “intrabodies”, are specifically targeted to a particular compartment within the cell, providing control over where the inhibitory activity of the treatment is focused. This technology has been successfully applied in the art (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodies have been shown to virtually eliminate the expression of otherwise abundant cell surface receptors (see, e.g., Richardson et al., 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337).
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 193P1E1B in the nucleus, thereby preventing its activity within the nucleus. Nuclear targeting signals are engineered into such 193P1E1B intrabodies in order to achieve the desired targeting. Such 193P1E1B intrabodies are designed to bind specifically to a particular 193P1E1B domain. In another embodiment, cytosolic intrabodies that specifically bind to a 193P1E1B protein are used to prevent 193P1E1B from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus (e.g., preventing 193P1E1B from forming transcription complexes with other factors).
In order to specifically direct the expression of such intrabodies to particular cells, the transcription of the intrabody is placed under the regulatory control of an appropriate tumor-specific promoter and/or enhancer. In order to target intrabody expression specifically to prostate, for example, the PSA promoter and/or promoter/enhancer can be utilized (See, for example, U.S. Pat. No. 5,919,652 issued Jul. 6, 1999).
XII.B.) Inhibition of 193P1E1B with Recombinant Proteins
In another approach, recombinant molecules bind to 193P1E1B and thereby inhibit 193P1E1B function. For example, these recombinant molecules prevent or inhibit 193P1E1B from accessing/binding to its binding partner(s) or associating with other protein(s). Such recombinant molecules can, for example, contain the reactive part(s) of a 193P1E1B specific antibody molecule. In a particular embodiment, the 193P1E1B binding domain of a 193P1E1B binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two 193P1E1B ligand binding domains linked to the Fc portion of a human IgG, such as human IgG1. Such IgG portion can contain, for example, the 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 193P1E1B, whereby the dimeric fusion protein specifically binds to 193P1E1B and blocks 193P1E1B interaction with a binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking technologies.
XII.C.) Inhibition of 193P1E1B Transcription or Translation
The present invention also comprises various methods and compositions for inhibiting the transcription of the 193P1E1B gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of 193P1E1B mRNA into protein.
In one approach, a method of inhibiting the transcription of the 193P1E1B gene comprises contacting the 193P1E1B gene with a 193P1E1B antisense polynucleotide. In another approach, a method of inhibiting 193P1E1B mRNA translation comprises contacting a 193P1E1B mRNA with an antisense polynucleotide. In another approach, a 193P1E1B specific ribozyme is used to cleave a 193P1E1B message, thereby inhibiting translation. Such antisense and ribozyme based methods can also be directed to the regulatory regions of the 193P1E1B gene, such as 193P1E1B promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a 193P1E1B gene transcription factor are used to inhibit 193P1E1B mRNA transcription. The various polynucleotides and compositions useful in the aforementioned methods have been described above. The use of antisense and ribozyme molecules to inhibit transcription and translation is well known in the art.
Other factors that inhibit the transcription of 193P1E1B by interfering with 193P1E1B transcriptional activation are also useful to treat cancers expressing 193P1E1B. Similarly, factors that interfere with 193P1E1B processing are useful to treat cancers that express 193P1E1B. Cancer treatment methods utilizing such factors are also within the scope of the invention.
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 193P1E1B (i.e., antisense, ribozyme, polynucleotides encoding intrabodies and other 193P1E1B inhibitory molecules). A number of gene therapy approaches are known in the art. Recombinant vectors encoding 193P1E1B antisense polynucleotides, ribozymes, factors capable of interfering with 193P1E1B transcription, and so forth, can be delivered to target tumor cells using such gene therapy approaches.
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 193P1E1B to a binding partner, etc.
In vivo, the effect of a 193P1E1B 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 193P1E1B
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 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 Apr. 2, 2002; U.S. Pat. No. 6,107,540 issued Aug. 22, 2000; Hogan et al., Manipulating the Mouse Embryo: A laboratory Manual, Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington, D.C., (1987).
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 thymectomized 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 oligonudeotides. 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. Natl. Acad. Sci. USA 90:6340-6344 (1993); Yamada et al., Human Gene Therapy 1:39-45 (1994); Leavitt et al., Proc. Natl. Acad Sci. USA 92:699-703 (1995); Leavitt et al., Human Gene Therapy 5: 1151-120 (1994); and Yamada et al., Virology 205: 121-126 (1994)).
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, Besffit, 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 diagnostic and therapeutic applications described herein, kits are also within the scope of the invention. Such kits can comprise a carrier, package or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method. For example, the container(s) can comprise a probe that is or can be detectably labeled. Such probe can be an antibody or polynucleotide specific for a FIG. 2-related protein or a FIG. 2 gene or message, respectively. Where the method utilizes nucleic acid hybridization to detect the target nucleic acid, the kit can also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radioisotope label. The kit can include all or part of the amino acid sequences in FIG. 2 or FIG. 3 or analogs thereof, or a nucleic acid molecules that encodes such amino acid sequences.
The kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes; carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package inserts with instructions for use.
A label can be present on the container to indicate that the composition is used for a specific therapy or non-therapeutic application, such as a diagnostic or laboratory application, and can also indicate directions for either in vivo or in viro 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 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 or plastic. The container can hold amino acid sequence(s), small molecule(s), nucleic acid sequence(s), and/or antibody(s), in one embodiment the container holds a polynucleotide for use in examining the mRNA expression profile of a cell, together with reagents used for this purpose.
The container can alternatively hold a composition which is effective for treating, diagnosis, prognosing or prophylaxing a condition and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agents in the composition can be an antibody capable of specifically binding 193P1E1B and modulating the function of 193P1E1B.
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 issue set forth in Table I. The article of manufacture can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and/ordextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, stirrers, needles, syringes, and/or package inserts with indications and/or instructions for use.
EXAMPLES
Various aspects of the invention are further described and illustrated by way of the several examples that follow, none of which are intended to limit the scope of the invention.
Example 1
SSH-Generated Isolation of cDNA Fragment of the 193P1E1B Gene
To isolate genes that are over-expressed in prostate cancer we used the Suppression Subtractive Hybridization (SSH) procedure using cDNA derived from prostate cancer xenograft tissues. LAPC-9AD xenograft was obtained from Dr. Charles Sawyers (UCLA) and was generated as described (Klein et al., 1997, Nature Med. 3:402-408; Craft et al., 1999, Cancer Res. 59:5030-5036). LAPC-9AD2 was generated from LAPC-9AD xenograft by growing LAPC-9AD xenograft tissues within a piece of human bone implanted in SCID mice. Tumors were then harvested and subsequently passaged subcutaneously into other SCID animals to generate LAPC-9AD2.
The 193P1E1B SSH cDNA sequence was derived from a subtraction consisting of a prostate cancer xenograft LAPC-9AD2 minus prostate cancer xenograft LAPC-9AD. By RT-PCR, the 193P1E1B cDNA was identified as highly expressed in the prostate cancer xenograft pool (LAPC4-AD, LAPC4-AI, LAPC9-AD, LAPC9-AI), bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, metastasis cancer pool, pancreas cancer pool, with low expression observed in the prostate cancer pool, and no expression observed in vital pool 1 (kidney, liver, lung), and in vital pool 2 (stomach, colon, pancreas) (FIG. 14).
The 193P1E1B SSH cDNA of 227 bp is listed in FIG. 1. The full length 193P1E1B cDNAs and ORFs are described in FIG. 2 with the protein sequences listed in FIG. 3. 193P1E1B v.1, v.2, v.3, v.4, v.5, v.6, v.7, v.8, v.11, v.12 and v.13 are novel proteins and have not been previously described. 193P1E1B v.9 shows 99% identity to a hypothetical protein, MGC4832. 193P1E1B v.10 shows 100% identity to a novel unnamed protein BAC03484.1.
Materials and Methods
RNA Isolation:
Tumor tissues were homogenized in Trizol reagent (Life Technologies, Gibco BRL) using 10 ml/g tissue or 10 ml/108 cells to isolate total RNA. Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA Mini and Midi kits. Total and mRNA were quantified by spectrophotometric analysis (O.D. 260/280 nm) and analyzed by gel electrophoresis.
Oligonucleotides:
The following HPLC purified oligonucleotides were used.









DPNCDN (cDNA synthesis primer):




5′TTTTGATCAAGCTT303′
(SEQ ID NO: 52)


 


Adaptor 1:


5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′
(SEQ ID NO: 53)


3′GGCCCGTCCTAG5′
(SEQ ID NO: 54)


 


Adaptor 2:


5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′
(SEQ ID NO: 55)


3′CGGCTCCTAG5′
(SEQ ID NO: 56)


 


PCR primer 1:


5′CTAATACGACTCACTATAGGGC3′
(SEQ ID NO: 57)


 


Nested primer (NP)1:


5′TCGAGCGGCCGCCCGGGCAGGA3′
(SEQ ID NO: 58)


 


Nested primer (NP)2:


5′AGCGTGGTCGCGGCCGAGGA3′
(SEQ ID NO: 59)






Suppression Subtractive Hybridization (SSH) was used to identify cDNAs corresponding to genes that may be differentially expressed in prostate cancer. The SSH reaction utilized cDNA from prostate cancer xenograft LAPC-9AD2. The gene 193P1E1B was derived from a prostate cancer xenograft LAPC-9AD2 minus prostate cancer xenograft LAPC-9AD tissues. The SSH DNA sequence (FIG. 1) was identified.
The cDNA derived from prostate cancer xenograft LAPC-9AD tissue was used as the source of the “driver” cDNA, while the cDNA from prostate cancer xenograft LAPC-9AD2 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 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.
Tester cDNA was generated by diluting 1 p 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 x reaction buffer (CLONTECH) and 0.5 μl 50 x 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 ml 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: 60) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 61) to amplify β-acon. 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 193P1E1B 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.
A typical RT-PCR expression analysis is shown in FIG. 14. RT-PCR expression analysis was performed on first strand cDNA generated using pools of tissues from multiple samples. The cDNA samples were shown to be normalized using beta-actin PCR. Strong expression of 193P1E1B was observed in prostate cancer xenograft pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, and metastasis pool. Low expression was observed in prostate cancer pool, but no expression was detected in VP1 and VP2.
Example 2
Isolation of Full Length 193P1E1B Encoding cDNA
To isolate genes that are involved in prostate cancer, an experiment was conducted using the prostate cancer xenograft LAPC-9AD2. The gene 193P1E1B was derived from a subtraction consisting of a prostate cancer xenograft LAPC-9AD2 minus prostate cancer xenograft LAPC-9AD. The SSH DNA sequence (FIG. 1) was designated 193P1E1B. Thirteen variants of 193P1E1l were identified (FIGS. 2 and 3). cDNA clone 193P1E1B v.1 and 193P1E1B v.5 were cloned from bladder cancer pool cDNA. 193P1E1B v.9 was cloned from LAPC-4AD cDNA library. All other variants were identified by bioinformatic analysis.
193P1E1B v.1 through v.8 differ from each other by one nucleic acid substitution. 193P1E1B v.1, v.2, v.4 v.7 and v.8 code for the same protein, whereas 193P1E1B v.5 and v.6 contain one amino acid substitution as shown in FIG. 12.
Absence of a 62-nucleotide sequence was identified in 193P1E1B v.9, nucleic acid positions 907-969 of 193P1E1B v.1. This resulted in an 82-amino acid truncation at the amino terminus of 193P1E1B v.9. Other splice variants were identified and referred to as 193P1E1B v.10, v.11, v.12 and v.13. 193P1E1B v.1, v.2, v.3, v.4, v.5, v.6, v.7, v.8, v.11, v.12 and v.13 are novel proteins and have not been previously described. 193P1E1B v.9 shows 99% identity to a hypothetical protein, MGC4832. 193P1E1B v.10 shows 100% identity to a novel unnamed protein BAC03484.1.
Example 3
Chromosomal Mapping of 193P1E1B
Chromosomal localization can implicate genes in disease pathogenesis. Several chromosome mapping approaches are available including fluorescent in situ hybridization (FISH), human/hamster radiation hybrid (RH) panels (Walter et al., 1994; Nature Genetics 7:22; Research Genetics, Huntsville Ala.), human-rodent somatic cell hybrid panels such as is available from the Coriell Institute (Camden, N.J.), and genomic viewers utilizing BLAST homologies to sequenced and mapped genomic clones (NCBI, Bethesda, Md.).
193P1E1B maps to chromosome 13q11, using 193P1E1B sequence and the NCBI BLAST tool. This 13q11 region has been previously implicated in bladder cancer (Wada T, Louhelainen J, Hemminki K, Adolfsson J, Wijkstrom H, Norming U, Borgstrom E, Hansson J, Sandstedt B, Steineck G. Bladder cancer: allelic deletions at and around the retinoblastoma tumor suppressor gene in relation to stage and grade. Clin Cancer Res. 2000 Feb;6(2):610-5.).
Example 4
Expression Analysis of 193P1E1B
Expression of 193P1E1B was analyzed using 2 sets of primers as illustrated in FIG. 14A. First strand cDNA was prepared from vital pool 1 (VP1: liver, lung and kidney), vital pool 2 (VP2, pancreas, colon and stomach), prostate xenograft pool (LAPC-4AD, LAPC-4AI, LAPC-9AD, LAPC-9AI), normal thymus, prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, metastasis cancer pool, pancreas cancer pool, and from prostate cancer metastasis to lymph node from 2 different patients. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using Primer Set A (B) or Primer Set B (C) to 193P1E1B, was performed at 30 cycles of amplification. A schematic diagram depicting the location of the 2 primer sets A and B is shown in FIG. 14A. Primer Set A detected a PCR product of 190 bp which is identical in all variants of 193P1E1B (FIG. 14B). Expression of 193P1E1B was observed in prostate cancer xenograft pool, prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, metastasis cancer pool, pancreas cancer pool, as well as the 2 prostate metastasis to lymph node, but not in VP1 and VP2 (FIG. 14B). In order to test abundance of expression of 193P1E1B v.1 through v.8 compared to 193P1E1B v.9, an experiment was conducted in which RT-PCR was performed using Primer Set B (FIG. 14C). Primer Set B detected a PCR product of 239 bp from 193P1E1B v.1 through v.8, and of 177 bp from 193P1E1B v.9 (FIG. 14C). FIG. 14C shows that the transcipt encoding encoding 193P1E1B v.1 through v.8, is expressed ate higher levels that the transcript encoding 193P1E1B v.9. But both transcripts are expressed at similar proportion in all tissues tested.
Extensive northern blot analysis of 193P1E1B in 16 human normal tissues confirms the expression observed by RT-PCR (FIG. 15). Two transcripts of approximately 3.5 kb and 2 kb are only detected in testis and thymus, but not in any other normal tissue tested.
FIG. 16 shows expression of 193P1E1B in prostate cancer xenografts. RNA was extracted from normal prostate, and from prostate cancer xenografts, LAPC-4AD, LAPC-4AI, LAPC-9AD, and LAPC-9AI. Northern blot with 10 ug of total RNA/lane was probed with 193P1E1B SSH sequence. Northern blot analysis shows expression of 193P1E1B in all 4 tissues, LAPC4AD, LAPC-4AI, LAPC-9AD, LAPC-9AI, with the lowest expression detected in the LAPC-9AD tissue, but not in normal prostate.
To test expression of 193P1E1B in patient cancer specimens, RNA was extracted from a pool of three patients for each of the following, bladder cancer, colon cancer, ovary cancer and metastasis cancer, as well as from normal prostate (NP), normal bladder (NB), normal kidney (NK), normal colon (NC). Northern blots with 10 ug of total RNA/lane were probed with 193P1E1B SSH sequence (FIG. 17). Results show expression of 193P1E1B in bladder cancer pool, colon cancer pool, ovary cancer pool and metastasis cancer pool, but not in any of the normal tissues tested.
Analysis of individual bladder cancer tissues by northern blot shows expression of 193P1E1B in the 2 bladder cancer cell lines and in the 3 bladder cancer patient specimens tested, but not in normal bladder tissues (FIG. 18).
FIG. 19 shows expression of 193P1E1Bin cancer metastasis patient specimens. RNA was extracted from the following cancer metastasis tissues, colon metastasis to lung, lung metastasis to lymph node, lung metastasis to skin, and breast metastasis to lymph node, as well as from normal bladder (NB), normal lung (NL), normal breast (NBr), and normal ovary (NO). Northern blots with 10 ug of total RNA/lane were probed with 193P1E1B sequence. Size standards in kilobases (kb) are indicated on the side. The results show expression of 193P1E1B in all four different cancer metastasis samples but not in the normal tissues tested.
FIG. 20 shows expression of 193P1E1B in pancreatic, ovarian and testicular cancer patient specimens. RNA was extracted from pancreatic cancer (P1), ovarian cancer (P2, P3), and testicular cancer (P4, P5) isolated from cancer patients, as well as from normal pancreas(NPa). Northern blots with 10 ug of total RNA/lane were probed with 193P1E1B sequence. Size standards in kilobases (kb) are indicated on the side. The results show expression of 193P1E1B in pancreatic, ovarian and testicular cancer specimens but not in normal pancreas.
FIG. 21 shows expression of 193P1E1B in normal compared to patient cancer specimens. First strand cDNA was prepared from a panel of normal tissues (stomach, brain, heart, liver, spleen, skeletal muscle, testis prostate, bladder, kidney, colon, lung and pancreas) and from a panel of patient cancer pools (prostate cancer pool, bladder cancer pool, kidney cancer pool, colon cancer pool, lung cancer pool, pancreas cancer pool, ovary cancer pool, breast cancer pool, metastasis cancer pool, LAPC prostate xenograft pool (XP), and from prostate cancer metastasis to lymph node from 2 different patients (PMLN2). Normalization was performed by PCR using primers to actin. Semi-quantitative PCR, using primer Set A as described in FIG. 14, was performed was performed at 26 and 30 cycles of amplification. Samples were run on an agarose gel, and PCR products were quantitated using the Alphalmager software. Relative expression was calculated by normalizing to signal obtained using actin primers. Results show restricted 193P1E1B expression in normal testis amongst all normal tissues tested. 193P1E1B expression was strongly upregulated in cancers of the bladder, colon, lung, pancreas, ovary, breast, and to a lesser extent in prostate and kidney cancers.
193P1E1B was also shown to be expressed in uterus, melanoma and bone cancer patient specimens. First strand cDNA was prepared from a panel of uterus patient cancer specimens (A), melanoma and bone cancer specimens (B). Semi-quantitative PCR, using primers to 193P1E1B, was performed at 26 and 30 cycles of amplification. Samples were run on an agarose gel, and PCR products were quantitated using the Alphalmager software. Expression was recorded as absent, low, or strong. Results show expression of 193P1E1B in the majority of uterus patient cancer specimens tested, as well as in the 2 melanoma specimens and in the bone tumor tested.
193P1E1B expression is reminiscent of a cancer-testis gene. Its restricted normal tissue expression to normal testis, and the upregulation detected in prostate cancer, bladder cancer, kidney cancer, colon cancer, lung cancer, ovary cancer, breast cancer and pancreatic cancer suggest that 193P1E1B is a potential therapeutic target and a diagnostic marker for human cancers.
Example 5
Transcript Variants of 193P1E1B
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. April 2000; 10(4):516-22); Grail and GenScan. For a general discussion of splice variant identification protocols see., e.g., Southan, C., A genomic perspective on human proteases, FEBS Lett. Jun. 8, 2001; 498(2-3):214-8; de Souza, S. J., et al., Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags, Proc. Natl. Acad. Sci USA. Nov. 7, 2000; 97(23):12690-3.
To further confirm the parameters of a transcript variant, a variety of techniques are available in the art, such as full-length cloning, proteomic validation, PCR-based validation, and 5′ RACE validation, etc. (see e.g., Proteomic Validation: Brennan, S. O., et al., Albumin banks peninsula: a new termination variant characterized by electrospray mass spectrometry, Biochem Biophys Acta. Aug. 17, 1999;1433(1-2):321-6; Ferranti P, et al., Differential splicing of pre-messenger RNA produces multiple forms of mature caprine alpha(s1)-casein, Eur J Biochem. Oct. 1, 1997;249(1):1-7. For PCR-based Validation: Wellmann S, et al., Specific reverse transcription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCycler technology, Clin Chem. April 2001;47(4):654-60; Jia, H. P., et al., Discovery of new human beta-defensins using a genomics-based approach, Gene. Jan. 24, 2001; 263(1-2):211-8. For PCR-based and 5′ RACE Validation: Brigle, K. E., et al., Organization of the murine reduced folate carrier gene and identification of variant splice forms, Biochem Biophys Acta. Aug. 7, 1997; 1353(2): 191-8).
It is known in the art that genomic regions are modulated in cancers. When the genomic region to which a gene maps is modulated in a particular cancer, the alternative transcripts or splice variants of the gene are modulated as well. Disclosed herein is that 193P1E1B has a particular expression profile related to cancer. Alternative transcripts and splice variants of 193P1E1B may also be involved in cancers in the same or different tissues, thus serving as tumor-associated markers/antigens.
Using the full-length gene and EST sequences, three transcript variants were identified, designated as 193P1E1B v.7, v.8 and v.9. Compared with 193P1E1B v.1, transcript variant 193P1E1B v.7 has spliced out exons 10 and 11 from variant 193P1E1B v.1, as shown in FIG. 12. Variant 193P1E1B v.8 inserted 36 bp in between 1931 and 1932 of variant 193P1E1B v.1 and variant 193P1E1B v.9 replaced with 36 bp the segment 1136-1163 of variant 193P1E1B v.1. Theoretically, each different combination of exons in spatial order, e.g. exons 2 and 3, is a potential splice variant. Tables LI through LXX are set forth on a variant-by-variant bases. Tables LI, LV, LIX, LXIII, and LXVII show the nucleotide sequence of the transcript variant. Tables LII, LVI, LX, LXIV, and LXVIII show the alignment of the transcript variant with nucleic acid sequence of 193P1E1B v.1. Tables LIII, LVII, LXI, LXV, and LXIX show the amino acid translation of the transcript variant for the identified reading frame orientation. Tables LIV, LVIII, LXII, LXVI, and LXX display alignments of the amino acid sequence encoded by the splice variant with that of 193P1E1Bv.1.
Example 6
Single Nucleotide Polymorphisms of 193P1E1B
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, CIG, G/C and T/A. Genotype refers to the specific base pair sequence of one or more locations in the genome of an individual. Haplotype refers to the base pair sequence of more than one location on the same DNA molecule (or the same chromosome in higher organisms), often in the context of one gene or in the context of several tightly linked genes. SNPs that occur on a cDNA are called cSNPs. These cSNPs may change amino acids of the protein encoded by the gene and thus change the functions of the protein. Some SNPs cause inherited diseases; others contribute to quantitative variations in phenotype and reactions to environmental factors including diet and drugs among individuals. Therefore, SNPs and/or combinations of alleles (called haplotypes) have many applications, including diagnosis of inherited diseases, determination of drug reactions and dosage, identification of genes responsible for diseases, and analysis of the genetic relationship between individuals (P. Nowotny, J. M. Kwon and A. M. Goate, “SNP analysis to dissect human traits,” Curr. Opin. Neurobiol. October 2001; 11(5):637-641; M. Pirmohamed and B. K. Park, “Genetic susceptibility to adverse drug reactions,” Trends Pharmacol. Sci. June 2001; 22(6):298-305; J. H. Riley, C. J. Allan, E. Lai and A. Roses, “The use of single nucleotide polymorphisms in the isolation of common disease genes,” Pharmacogenomics. February 2000; 1(1):3947; R. Judson, J. C. Stephens and A. Windemuth, “The predictive power of haplotypes in clinical response,” Pharmacogenomics. 2000 feb; 1(1):15-26).
SNPs are identified by a variety of art-accepted methods (P. Bean, “The promising voyage of SNP target discovery,” Am. Clin. Lab. October-November 2001; 20(9):18-20; K. M. Weiss, “In search of human variation,” Genome Res. July 1998; 8(7):691-697; M. M. She, “Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies,” Clin. Chem. 2001 Feb; 47(2):164-172). For example, SNPs are identified by sequencing DNA fragments that show polymorphism by gel-based methods such as restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). They can also be discovered by direct sequencing of DNA samples pooled from different individuals or by comparing sequences from different DNA samples. With the rapid accumulation of sequence data in public and private databases, one can discover SNPs by comparing sequences using computer programs (Z. Gu, L. Hillier and P. Y. Kwok, “Single nucleotide polymorphism hunting in cyberspace,” Hum. Mutat. 1998; 12(4):221-225). SNPs can be verified and genotype or haplotype of an individual can be determined by a variety of methods including direct sequencing and high throughput microarrays (P. Y. Kwok, “Methods for genotyping single nucleotide polymorphisms,” Annu. Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K. Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft, “High-throughput SNP genotyping with the Masscode system,” Mol. Diagn. December 2000; 5(4):329-340). Using the methods described above, seven SNPs were identified in the original transcript, 193P1E1B v.1, at positions 57 (A/G), 792 (C/G), 804 (C/A), 1253 (G/A), 1564 (A/G), 2268 (C/T) and 2387 (C/T). The transcripts or proteins with alternative alleles were designated as variants 193P1E1B v.2, v.3, v.4, v.5 and v.6, respectively. FIG. 10 shows the schematic alignment of the SNP variants. FIG. 11 shows the schematic alignment of protein variants, corresponding to nucleotide variants. Nucleotide variants that code for the same amino acid sequence as variant 1 are not shown in FIG. 11. These alleles of the SNPs, though shown separately here, can occur in different combinations (haplotypes) and in any one of the transcript variants (such as 193P1E1B v.9) that contains the sequence context of the SNPs.
Example 7
Production of Recombinant 193P1E1B in Prokaryotic Systems
To express recombinant 193P1E1B in prokaryotic cells, the full or partial length 193P1E1B cDNA sequences can be cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 193P1E1B are expressed in these constructs, amino acids 1 to 412 of variant 5 or variant 2; or amino acids 1 to 388 of variant 10, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 193P1E1B, variants, or analogs thereof. In certain embodiments a region of 193P1E1B is expressed that encodes an amino acid not shared amongst at least variants.
A. In vitro transcription and translation constructs:
pCRII: To generate 193P1E1B sense and anti-sense RNA probes for RNA in situ investigations, pCRII constructs (Invitrogen, Carlsbad Calif.) are generated encoding either all or fragments of a 193P1E1B cDNA. The pCRII vector has Sp6 and T7 promoters flanking the insert to drive the transcription of 193P1E1B RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze the cell and tissue expression of 193P1E1B at the RNA level. Transcribed 193P1E1B RNA representing the cDNA amino acid coding region of the 193P1E1B gene is used in in vitro translation systems such as the TnTTM Coupled Reticulolysate System (Promega, Corp., Madison, Wis.) to synthesize 193P1E1B protein.
B. Bacterial Constructs:
PGEX Constructs: To generate recombinant 193P1E1B proteins in bacteria that are fused to the Glutathione S-transferase (GST) protein, all or parts of a 193P1E1B cDNA protein coding sequence are fused to the GST gene by cloning into pGEX-6P-1 or any other GST-fusion vector of the pGEX family (Amersham Pharmacia Biotech, Piscataway, N.J.). These constructs allow controlled expression of recombinant 193P1E1B protein sequences with GST fused at the amino-terminus and a six histidine epitope (6×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 193P1E1B-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 193P1E1B proteins that are fused to maltose-binding protein (MBP), all or parts of a 193P1E1B cDNA protein coding sequence are fused to the MBP gene by cloning into the pMAL-c2X and pMAL-p2X vectors (New England Biolabs, Beverly, Mass.). These constructs allow controlled expression of recombinant 193P1E1B protein sequences with MBP fused at the amino-terminus and a 6×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 193P1E1B. 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 193P1E1B in bacterial cells, all or parts of a 193P1E1B cDNA protein coding sequence are cloned into the pET family of vectors (Novagen, Madison, Wis.). These vectors allow tightly controlled expression of recombinant 193P1E1B protein in bacteria with and without fusion to proteins that enhance solubility, such as NusA and thioredoxin (Trx), and epitope tags, such as 6×His and S-Tag Tm that aid purification and detection of the recombinant protein. For example, constructs are made utilizing pET NusA fusion system 43.1 such that regions of a 193P1E1B protein are expressed as amino-terminal fusions to NusA.
C. Yeast Constructs:
pESC Constructs: To express 193P1E1B in the yeast species Saccharomyces cerevisiae for generation of recombinant protein and functional studies, all or parts of a 193P1E1B cDNA protein coding sequence are cloned into the pESC family of vectors each of which contain 1 of 4 selectable markers, HIS3, TRP1, LEU2, and URA3 (Stratagene, La Jolla, Calif.). These vectors allow controlled expression from the same plasmid of up to 2 different genes or cloned sequences containing either FlagTM or Myc epitope tags in the same yeast cell. This system is useful to confirm protein-protein interactions of 193P1E1B. In addition, expression in yeast yields similar post-translational modifications, such as glycosylations and phosphorylations, that are found when expressed in eukaryotic cells.
pESP Constructs: To express 193P1E1B in the yeast species Saccharomyces pombe, all or parts of a 193P1E1B cDNA protein coding sequence are cloned into the pESP family of vectors. These vectors allow controlled high level of expression of a 193P1E1B protein sequence that is fused at either the amino terminus or at the carboxyl terminus to GST which aids purification of the recombinant protein. A FlagTM epitope tag allows detection of the recombinant protein with anti-FlagTM antibody.
Example 8
Production of Recombinant 193P1E1B in Higher Eukarvotic Systems
A. Mammalian Constructs:
To express recombinant 193P1E1B in eukaryotic cells, the full or partial length 193P1E1B cDNA sequences can be cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 193P1E1B are expressed in these constructs, amino acids 1 to 412; or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 193P1E1B, variants, or analogs thereof.
The constructs can be transfected into any one of a wide variety of mammalian cells such as 293T cells. Transfected 293T cell lysates can be probed with the anti-193P1E1B polyclonal serum, described herein.
pcDNA4/HisMax Constructs: To express 193P1E1B in mammalian cells, the 193P1E1B ORF, or portions thereof, of 193P1E1B are cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter and the SP16 translational enhancer. The recombinant protein has 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. 
pcDNA3.11MycHis Constructs: To express 193P1E1B in mammalian cells, the 193P1E1B ORF, or portions thereof, of 193P1E1B with a consensus Kozak translation initiation site are cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the myc epitope and 6×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. 
pcDNA3.1/CT-GFP-TOPO Construct: To express 193P1E1B in mammalian cells and to allow detection of the recombinant proteins using fluorescence, the 193P1E1B ORF, or portions thereof, of 193P1E1B with a consensus Kozak translation initiation site are cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the Green Fluorescent Protein (GFP) fused to the carboxyl-terminus facilitating non-invasive, in vivo detection and cell biology studies. The pcDNA3.1 CT-GFP-TOPO vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli. Additional constructs with an amino-terminal GFP fusion are made in pcDNA3.1/NT-GFP-TOPO spanning the entire length of the 193P1E1B proteins.
PAPtag: The 193P1E1B ORF, or portions thereof, of 193P1E1B are cloned into pAPtag-5 (GenHunter Corp. Nashville, Tenn.). This construct generates an alkaline phosphatase fusion at the carboxyl-terminus of the 193P1E1B proteins while fusing the 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 193P1E1B proteins. The resulting recombinant 193P1E1B proteins are optimized for secretion into the media of transfected mammalian cells and can be used to identify proteins such as ligands or receptors that interact with the 193P1E1B proteins. Protein expression is driven from the CMV promoter and the recombinant proteins also contain myc and 6×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: The 193P1E1B ORF, or portions thereof, of 193P1E1B are cloned into pTag-5. This vector is similar to pAPtag but without the alkaline phosphatase fusion. This construct generates 193P1E1B protein with an amino-terminal IgGκ signal sequence and myc and 6×His epitope tags at the carboxyl-terminus that facilitate detection and affinity purification. The resulting recombinant 193P1E1B protein is optimized for secretion into the media of transfected mammalian cells, and is used as immunogen or ligand to identify proteins such as ligands or receptors that interact with the 193P1E1B proteins. Protein expression is driven from the CMV promoter. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli. 
PsecFc: The 193P1E1B ORF, or portions thereof, of 193P1E1B are also cloned into psecFc. The psecFc vector was assembled by cloning the human immunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen, California). This construct generates an IgG1 Fc fusion at the carboxyl-terminus of the 193P1E1B proteins, while fusing the IgGK signal sequence to N-terminus. 193P1E1B fusions utilizing the murine IgG1 Fc region are also used. The resulting recombinant 193P1E1B proteins are optimized for secretion into the media of transfected mammalian cells, and can be used as immunogens or to identify proteins such as ligands or receptors that interact with the 193P1E1B protein. Protein expression is driven from the CMV promoter. The hygromycin resistance gene present in the vector allows for selection of mammalian cells that express the recombinant protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli. 
pSRα Constructs: To generate mammalian cell lines that express 193P1E1B constitutively, 193P1E1B ORF, or portions thereof, of 193P1E1B are cloned into pSRα constructs. Amphotropic and ecotropic retroviruses are 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, 193P1E1B, into the host cell-lines. Protein expression is driven from a long terminal repeat (LTR). The Neomycin resistance gene present in the vector allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permit selection and maintenance of the plasmid in E. coli. The retroviral vectors can thereafter be used for infection and generation of various cell lines using, for example, PC3, NIH 3T3, TsuPr1, 293 or rat-1 cells.
Additional pSRα constructs are made that fuse an epitope tag such as the FLAGTM tag to the carboxyl-terminus of 193P1E1B 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: 62) 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 193P1E1B proteins.
Additional Viral Vectors: Additional constructs are made for viral-mediated delivery and expression of 193P1E1B. High virus titer leading to high level expression of 193P1E1B is achieved in viral delivery systems such as adenoviral vectors and herpes amplicon vectors. The 193P1E1B coding sequences or fragments thereof are amplified by PCR and subcloned into the AdEasy shuffle vector (Stratagene). Recombination and virus packaging are performed according to the manufacturer's instructions to generate adenoviral vectors. Alternatively, 193P1E1B coding sequences or fragments thereof are cloned into the HSV-1 vector (Imgenex) to generate herpes viral vectors. The viral vectors are thereafter used for infection of various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
Regulated Expression Systems: To control expression of 193P1E1B in mammalian cells, coding sequences of 193P1E1B, or portions thereof, are cloned into regulated mammalian expression systems such as the T-Rex System (Invitrogen), the GeneSwitch System (Invitrogen) and the tightly-regulated Ecdysone System (Sratagene). These systems allow the study of the temporal and concentration dependent effects of recombinant 193P1E1B. These vectors are thereafter used to control expression of 193P1E1B in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
B. Baculovirus Expression Systems
To generate recombinant 193P1E1B proteins in a baculovirus expression system, 193P1E1B ORF, or portions thereof, are cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag at the N-terminus. Specifically, pBlueBac-193P1E1B is co-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda) insect cells to generate recombinant baculovirus (see Invitrogen instruction manual for details). Baculovirus is then collected from cell supernatant and purified by plaque assay.
Recombinant 193P1E1B protein is then generated by infection of HighFive insect cells (Invitrogen) with purified baculovirus. Recombinant 193P1E1B protein can be detected using anti-193P1E1B or anti-His-tag antibody. 193P1E1B protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 193P1E1B.
Example 9
Antigenicity Profiles and Secondary Structure
FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9 depict graphically five amino acid profiles of the 193P1E1B amino acid sequence (variant 1), each assessment is available by accessing the ProtScale website on the ExPasy molecular biology server.
These profiles: FIG. 5, Hydrophilicity, (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 6, Hydropathicity, (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132); FIG. 7, Percentage Accessible Residues (Janin J., 1979 Nature 277:491-492); FIG. 8, Average Flexibility, (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255); FIG. 9, Beta-turn (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294); and optionally others available in the art, such as on the ProtScale website, were used to identify antigenic regions of 193P1E1B protein. Each of the above amino acid profiles of 193P1E1B were generated using the following ProtScale parameters for analysis: 1) A window size of 9; 2) 100% weight of the window edges compared to the window center; and, 3) amino acid profile values normalized to lie between 0 and 1.
Hydrophilicity (FIG. 5), Hydropathicity (FIG. 6) and Percentage Accessible Residues (FIG. 7) profiles were used to determine stretches of hydrophilic amino acids (i.e., values greater than 0.5 on the Hydrophilicity and Percentage Accessible Residues profiles, and values less than 0.5 on the Hydropathicity profile). Such regions are likely to be exposed to the aqueous environment, be present on the surface of the protein, and thus available for immune recognition, such as by antibodies.
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 full length 193P1E1B protein (variant 1) indicated, e.g., by the profiles set forth in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and/or FIG. 9 are used to prepare immunogens, either peptides or nucleic acids that encode them, to generate therapeutic and diagnostic anti-193P1E1B antibodies. The immunogen can be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50 contiguous amino acids, or the corresponding nucleic acids that encode them, from 193P1E1B protein. In particular, peptide immunogens of the invention can comprise, a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 412 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5; a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 412 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of FIG. 6; a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 412 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7; a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 412 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile on FIG. 8; and, a peptide region of at least 5 amino acids of FIG. 2 in any whole number increment up to 412 that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of FIG. 9. Peptide immunogens of the invention can also comprise nucleic acids that encode any of the forgoing.
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 193P1E1B, namely the predicted presence and location of alpha helices, extended strands, and random coils, is predicted from the primary amino acid sequence of 193P1E1B variant 1 using the HNN-Hierarchical Neural Network method, accessed from the ExPasy molecular biology server. The analysis indicates that 193P1E1B is composed 29.13% alpha helix, 9.95% extended strand, and 60.92% random coil (FIG. 13).
Analysis of 193P1E1B using a variety of transmembrane prediction algorithms accessed from the ExPasy molecular biology server did not predict the presence of such domains, confirming that 193P1E1B is a soluble protein.
Example 10
Generation of 193P1E1B Polyclonal Antibodies
Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. In addition to immunizing with the full length 193P1E1B protein, computer algorithms are employed in design of immunogens that, based on amino acid sequence analysis contain characteristics of being antigenic and available for recognition by the immune system of the immunized host (see Example 9 entitled “Antigenicity Profiles and Secondary Structure”). Such regions would be predicted to be hydrophilic, flexible, in beta-turn conformations, and be exposed on the surface of the protein (see, e.g., FIG. 5, FIG. 6, FIG. 7, FIG. 8, or FIG. 9 for amino acid profiles that indicate such regions of 193P1E1B).
For example, 193P1E1B recombinant bacterial fusion proteins or peptides containing hydrophilic, flexible, beta-turn regions of 193P1E1B are used as antigens to generate polyclonal antibodies in New Zealand White rabbits. For example, such regions include, but are not limited to, amino acids 20-43, amino acids 100-164, amino acids 241-261, or amino acids 310-331. It is useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. In one embodiment, a peptide encoding amino acids 241-261 of 193P1E1B is conjugated to KLH and used to immunize the rabbit. Alternatively the immunizing agent may include all or portions of a 193P1E1B protein, analogs or fusion proteins thereof. For example, a 193P1E1B amino acid sequence can be fused using recombinant DNA techniques to any one of a variety of fusion protein partners that are well known in the art, such as glutathione-S-transferase (GST) and HIS tagged fusion proteins. Such fusion proteins are purified from induced bacteria using the appropriate affinity matrix.
In one embodiment, a GST-fusion protein containing an entire 193P1E1B coding sequence is produced and purified and used as immunogen. Other recombinant bacterial fusion proteins that can be employed include maltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see the section entitled “Production of 193P1E1B in Prokaryotic Systems” and Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, P. S., Brady, W., 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 Example 8 entitled “Production of Recombinant 193P1E1B in Eukaryotic Systems”), and retain post-translational modifications such as glycosylations found in native protein. In one embodiment, an entire 193P1E1B coding sequence is cloned into the Tag5 mammalian secretion vector. The recombinant protein is purified by metal chelate chromatography from tissue culture supernatants of 293T cells stably expressing the recombinant vector. The purified Tag5 193P1E1B protein is then used as immunogen.
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 Tag5 193P1E1B protein or KLH-coupled peptide encoding amino acids 241-261, the full-length 193P1E1B cDNA is cloned into pCDNA 3.1 myc-his expression vector (invitrogen, see the Example entitled “Production of Recombinant 193P1E1B in Eukaryotic Systems”). After transfection of the constructs into 293T cells, cell lysates are probed with the anti-193P1E1B serum and with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, Calif.) to determine specific reactivity to denatured 193P1E1B protein using the Western blot technique. Immunoprecipitation and flow cytometric analyses of 293T and other recombinant 193P1E1B-expressing cells determine recognition of native protein by the antiserum. In addition, Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometric techniques using cells that endogenously express 193P1E1B are carded out to test specificity.
The anti-serum from the Tag5 193P1E1B immunized rabbit is affinity purified by passage over a column composed of the Tag5 antigen covalently coupled to Affigel matrix (BioRad, Hercules, Calif.). The serum is then further purified by protein G affinity chromatography to isolate the IgG fraction. Serum from rabbits immunized with fusion proteins, such as GST and MBP fusion proteins, are purified by depletion of antibodies reactive to the fusion partner sequence by passage over an affinity column containing the fusion partner either alone or in the context of an irrelevant fusion protein. Sera from other His-tagged antigens and peptide immunized rabbits as well as fusion partner depleted sera are affinity purified by passage over a column matrix composed of the original protein immunogen or free peptide.
Example 11
Generation of 193P1E1B Monoclonal Antibodies (mAbs)
In one embodiment, therapeutic mAbs to 193P1E1B comprise those that react with epitopes of the protein that would disrupt or modulate the biological function of 193P1E1B, for example those that would disrupt its interaction with ligands, proteins, or substrates that mediate its biological activity. Immunogens for generation of such mAbs include those designed to encode or contain an entire 193P1E1B protein or its variants or regions of a193P1E1B protein predicted to be antigenic from computer analysis of the amino acid sequence (see, e.g., FIG. 5, FIG. 6, FIG. 7, FIG. 8, or FIG. 9, and Example 9 entitled “Antigenicity Profiles and Secondary Structure”). Immunogens include peptides, recombinant bacterial proteins, and mammalian expressed Tag 5 proteins and human and murine IgG FC fusion proteins. In addition, cells expressing high levels of 193P1E1B, such as 293T-193P1E1B or 300.19-193P1E1B murine Pre-B cells, are used to immunize mice.
To generate mAbs to 193P1E1B, mice are first immunized intraperitoneally (IP) with, typically, 10-50 μg of protein immunogen or 107 193P1E1B-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 193P1E1B sequence is used to immunize mice by direct injection of the plasmid DNA. For example, an entire coding sequence of 193P1E1B, e.g., amino acids 1-412 of 193P1E1B variant 1, is cloned into the Tag5 mammalian secretion vector and the recombinant vector is used as immunogen. In another example the amino acids are cloned into an Fc-fusion secretion vector in which a 193P1E1B sequence is fused at the amino-terminus to an IgK leader sequence and at the carboxyl-terminus to the coding sequence of the human or murine IgG Fc region. This recombinant vector is then used as immunogen. The plasmid immunization protocols are used in combination with purified proteins expressed from the same vector and with cells expressing 193P1E1B.
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, monoclonal antibodies are derived that distinguish between, e.g., the various 193P1E1B variants, e.g., the amino terminal truncated splice variant 3, encoding amino acids 83-412 and the full length protein encoding amino acids 1-412. In one method, two different Fc-fusion proteins are derived, one encoding amino acids 1-82, and the other encoding amino acids 83-412. These are expressed and purified from stably transfected 293T cells. Balb C mice are initially immunized intraperitoneally with 25 μg of the Tag5-193P1E1B 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 the full length 193P1E1B protein and to amino terminal truncated variant 3 is monitored by Western blotting, immunoprecipitation and flow cytometry using 293T cells transfected with an expression vector encoding each of the respective 193P1E1B cDNAs (see e.g., the Example entitled “Production of Recombinant 193P1E1B in Eukaryotic Systems”). Other recombinant 193P1E1B-expressing cells or cells endogenously expressing 193P1E1B are also used. Mice showing the strongest reactivity are rested and given a final injection of Tag5 antigen in PBS and then sacrificed four days later. The spleens of the sacrificed mice are harvested and fused to SPO/2 myeloma cells using standard procedures (see, e.g., Harlow and Lane, 1988). Supernatants from HAT selected growth wells are screened by ELISA, Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometry to identify 193P1E1B specific antibody-producing clones.
The binding affinity of a 193P1E1B monoclonal antibody is determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which 193P1E1B monoclonal antibodies preferred, e.g., for diagnostic or therapeutic use, as appreciated by one of skill in the art. The BIAcore system (Uppsala, Sweden) is a preferred method for determining binding affinity. The BIAcore system uses surface plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Morton and Myszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecular interactions in real time. BIAcore analysis conveniently generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants.
Example 12
HLA Class I and Class II Binding Assays
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 193P1E1B. 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 193P1E1B protein sequences are analyzed using a text string search software program to identify potential peptide sequences containing appropriate HLA binding motifs; such programs are readily produced in accordance with information in the art in view of known motif/supermotif disclosures. Furthermore, such calculations can be made mentally.
Identified A2-, A3-, and DR-supermotif sequences are scored using polynomial algorithms to predict their capacity to bind to specific HLA-Class I or Class II molecules. These polynomial algorithms account for the impact of different amino acids at different positions, and are essentially based on the premise that the overall affinity (or AG) of peptide-HLA molecule interactions can be approximated as a linear polynomial function of the type:

“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 j, 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 193P1E1B are scanned utilizing motif identification software, to identify 8-, 9-10- and 11-mer sequences containing the HLA-A2-supermotif main anchor specificity. Typically, these sequences are then scored using the protocol described above and the peptides corresponding to the positive-scoring sequences are synthesized and tested for their capacity to bind purified HLA-A*0201 molecules in vitro (HLA-A*0201 is considered a prototype A2 supertype molecule).
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 193P1E1B protein sequence(s) scanned above is also examined for the presence of peptides with the HLA-A3-supermotif primary anchors. Peptides corresponding to the HLA A3 supermotif-bearing sequences are then synthesized and tested for binding to HLA-A*0301 and HLA-A*1101 molecules, the molecules encoded by the two most prevalent A3-supertype alleles. The peptides that bind at least one of the two alleles with binding affinities of ≦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 193P1E1B protein(s) scanned above is also analyzed for the presence of 8-, 9- 10-, or 11-mer peptides with the HLA-B7-supermotif. Corresponding peptides are synthesized and tested for binding to HLA-B*0702, the molecule encoded by the most common B7-supertype allele (i.e., the prototype B7 supertype allele). Peptides binding B*0702 with 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 193P1E1B 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 .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® M450) 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 20×106cells/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 92-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 μg/ml of IL-7. Recombinant human IL-10 is added the next day at a final concentration of 10 μg/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 92 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 μg/ml and recombinant human IL2 is added the next day and again 2-3 days later at 50 UI/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/mi. 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, 25μM 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 193P1E1B. Briefly, PBMCs are isolated from patients, re-stimulated with peptide-pulsed monocytes and assayed for the ability to recognize peptide-pulsed target cells as well as transfected cells endogenously expressing the antigen.
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 193P1E1B-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 193P1E1B-Derived Sequences with HLA-DR Binding Motifs
Peptide epitopes bearing an HLA class II supermotif or motif are identified and confirmed as outlined below using methodology similar to that described for HLA Class I peptides.
Selection of HLA-DR-Supermotif-Bearing Epitopes.
To identify 193P1E1B-derived, HLA class II HTL epitopes, a 193P1E1B antigen is analyzed for the presence of sequences bearing an HLA-DR-motif or supermotif. Specifically, 15-mer sequences are selected comprising a DR-supermotif, comprising a 9-mer core, add 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 193P1E1B-derived peptides identified above are tested for their binding capacity for various common HLA-DR molecules. All peptides are initially tested for binding to the DR molecules in the primary panel: DR1, DR4w4, and DR7. Peptides binding at least two of these three DR molecules are then tested for binding to DR2w2 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. 193P1E1B-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 193P1E1B antigens are analyzed for sequences carrying one of the two DR3-specific binding motifs reported by Geluk et al. (J. Immunol. 152:5742-5748, 1994). The corresponding peptides are then synthesized and confirmed as having the ability to bind DR3 with an affinity of 1 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 193P1E1B-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 193P1E1B-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.l/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 193P1E1B expression vectors.
The results demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized 193P1E1B antigen. The choice of transgenic mouse model to be used for such an analysis depends upon the epitope(s) that are being evaluated. In addition to HLA-A*0201/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 193P1E1B-derived CTL and HTL peptide vaccine compositions. The vaccine composition used herein comprise peptides to be administered to a patient with a 193P1E1B-expressing tumor. The peptide composition can comprise multiple CTL and/or HTL epitopes. The epitopes are identified using methodology as described herein. This example also illustrates that enhanced immunogenicity can be achieved by inclusion of one or more HTL epitopes in a CTL vaccine composition; such a peptide composition can comprise an HTL epitope conjugated to a CTL epitope. The CTL epitope can be one that binds to multiple HLA family members at an affinity of 500 nM or less, or analogs of that epitope. The peptides may be lipidated, if desired.
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 at, 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 193P1E1B-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 193P1E1B clearance. The number of epitopes used depends on observations of patients who spontaneously clear 193P1E1B. For example, if it has been observed that patients who spontaneously clear 193P1E1B-expressing cells generate an immune response to at least three (3) epitopes from 193P1E1B antigen, then at least three epitopes should be included for HLA class I. A similar rationale is used to determine HLA class II epitopes.
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.
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 193P1E1B, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing nucleic acid vaccine compositions. Related to this embodiment, computer programs can be derived in accordance with principles in the art, which identify in a target sequence, the greatest number of epitopes per sequence length.
A vaccine composition comprised of selected, peptides, when administered, is safe, efficacious, and elicits an immune response similar in magnitude to an immune response that controls or clears cells that bear or overexpress 193P1E1B.
Example 22
Construction of “Minigene” Multi-Epitope DNA Plasmids
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 supenmotif-bearing epitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearing peptide epitopes derived 193P1E1B, are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from 193P1E1B to provide broad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct. The selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.
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 (1×=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., Bamelt et al., Aids Res. and Human Retrovinises 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 107pfu/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-A11 or HLA-B7 transgenic mouse models to assess CTL induction by HLA-A3 or HLA-B7 motif or supermotif epitopes. The use of prime boost protocols in humans is described below in the Example entitled “Induction of CTL Responses Using a Prime Boost Protocol.”
Example 24
Peptide Compositions for Prophylactic Uses
Vaccine compositions of the present invention can be used to prevent 193P1E1B expression in persons who are at risk for tumors that bear this antigen. For example, a polyepitopic peptide epitope composition (or a nucleic acid comprising the same) containing multiple CTL and HTL epitopes such as those selected in the above Examples, which are also selected to target greater than 80% of the population, is administered to individuals at risk for a 193P1E1B-associated tumor.
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 193P1E1B-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 193P1E1B Sequences
A native 193P1E1B polyprotein sequence is analyzed, preferably using computer algorithms defined for each class I and/or class II supermotif or motif, to identify “relatively short” regions of the polyprotein that comprise multiple epitopes. The “relatively short” regions are preferably less in length than an entire native antigen. This relatively short sequence that contains multiple distinct or overlapping, “nested” epitopes can be used to generate a minigene construct. The construct is engineered to express the peptide, which corresponds to the native protein sequence. The “relatively short” peptide is generally less than 250 amino acids in length, often less than 100 amino acids in length, preferably less than 75 amino acids in length, and more preferably less than 50 amino acids in length. The protein sequence of the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, i.e., it has a high concentration of epitopes. As noted herein, epitope motifs may be nested or overlapping (i.e., frame shifted relative to one another). For example, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes.
The vaccine composition will include, for example, multiple CTL epitopes from 193P1E1B antigen and at least one HTL epitope. This polyepitopic native sequence is administered either as a peptide or as a nucleic acid sequence which encodes the peptide. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide.
The embodiment of this example provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally, such an embodiment provides for the possibility of motif-bearing epitopes for an HLA makeup(s) that is presently unknown. Furthermore, this embodiment (excluding an analoged embodiment) directs the immune response to multiple peptide sequences that are actually present in native 193P1E1B, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing peptide or nucleic acid vaccine compositions.
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 193P1E1B peptide epitopes of the present invention are used in conjunction with epitopes from other target tumor-associated antigens, to create a vaccine composition that is useful for the prevention or treatment of cancer that expresses 193P1E1B and such other antigens. For example, a vaccine composition can be provided as a single polypeptide that incorporates multiple epitopes from 193P1E1B as well as tumor-associated antigens that are often expressed with a target cancer associated with 193P1E1B expression, or can be administered as a composition comprising a cocktail of one or more discrete epitopes. Alternatively, the vaccine can be administered as a minigene construct or as dendritic cells which have been loaded with the peptide epitopes in vitro.
Example 27
Use of Peptides to Evaluate an Immune Response
Peptides of the invention may be used to analyze an immune response for the presence of specific antibodies, CTL or HTL directed to 193P1E1B. Such an analysis can be performed in a manner described by Ogg et al., Science 279:2103-2106, 1998. In this Example, peptides in accordance with the invention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen.
In this example highly sensitive human leukocyte antigen tetrameric complexes (“tetramers”) are used for a cross-sectional analysis of, for example, 193P1E1B HLA-A*0201 -specific CTL frequencies from HLA A*0201 -positive individuals at different stages of disease or following immunization comprising a 193P1E1B peptide containing an A*0201 motif. Tetrameric complexes are synthesized as described (Musey et al., N. Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201 in this example) and P2-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 193P1E1B epitope, and thus the status of exposure to 193P1E1B, or exposure to a vaccine that elicits a protective or therapeutic response.
Example 28
Use of Peptide Epitopes to Evaluate Recall Responses
The peptide epitopes of the invention are used as reagents to evaluate T cell responses, such as acute or recall responses, in patients. Such an analysis may be performed on patients who have recovered from 193P1E1B-associated disease or who have been vaccinated with a 193P1E1B vaccine.
For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any 193P1E1B vaccine. PBMC are collected from vaccinated individuals and HLA typed. Appropriate peptide epitopes of the invention that, optimally, bear supermotifs to provide cross-reactivity with multiple HLA supertype family members, are then used for analysis of samples derived from individuals who bear that HLA type.
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 (2mM), penicillin (50U/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 II/well of complete RPMI. On days 3 and 10, 100 μl of complete RPMI and 20 U/ml final concentration of rIL-2 are added to each well. On day 7 the cultures are transferred into a 96-well flat-bottom plate and restimulated with peptide, rIL-2 and 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 (ASHI, 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 193P1E1B or a 193P1E1B 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 193P1E1B antigen, or PHA. Cells are routinely plated in replicates of 4-6 wells for each condition. After seven days of culture, the medium is removed and replaced with fresh medium containing 10 U/ml IL-2. Two days later, 1 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 193P1E1B
Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients having cancer that expresses 193P1E1B. The main objectives of the trial are to determine an effective dose and regimen for inducing CTLs in cancer patients that express 193P1E1B, to establish the safety of inducing a CTL and HTL response in these patients, and to see to what extent activation of CTLs improves the clinical picture of these patients, as manifested, e.g., by the reduction and/or shrinking of lesions. Such a study is designed, for example, as follows:
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 193P1E1B.
Clinical manifestations or antigen-specific T-cell responses are monitored to assess the effects of administering the peptide compositions. The vaccine composition is found to be both safe and efficacious in the treatment of 193P1E1B-associated disease.
Example 31
Induction of CTL Responses Using a Prime Boost Protocol
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 193P1E1B 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 193P1E1B 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 193P1E1B antigens can be induced by incubating, in tissue culture, the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of APC, such as DC, and immunogenic peptides. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cells, i.e., tumor cells.
Example 33
An Alternative Method of Identifying and Confirming Motif-Bearing Peptides
Another method of identifying and confirming motif-bearing peptides is to elute them from cells bearing defined MHC molecules. For example, EBV transformed B cell lines used for tissue typing have been extensively characterized to determine which HLA molecules they express. In certain cases these cells express only a single type of HLA molecule. These cells can be transfected with nucleic acids that express the antigen of interest, e.g. 193P1E1B. Peptides produced by endogenous antigen processing of peptides produced as a result of transfection will then bind to HLA molecules within the cell and be transported and displayed on the cell's surface. Peptides are then eluted from the HLA molecules by exposure to mild acid conditions and their amino acid sequence determined, e.g., by mass spectral analysis (e.g., Kubo et at., 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 193P1E1B to isolate peptides corresponding to 193P1E1B that have been presented on the cell surface. Peptides obtained from such an analysis will bear motif(s) that correspond to binding to the single HLA allele that is expressed in the cell.
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 193P1E1B-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring 193P1E1B. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06 software (National Biosciences) and the coding sequence of 193P1E1B. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5′ sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to a 193P1E1B-encoding transcript.
Example 35
Purification of Naturally-Occurring or Recombinant 193P1E1B Using 193P1E1B-Specific Antibodies
Naturally occurring or recombinant 193P1E1B is substantially purified by immunoaffinity chromatography using antibodies specific for 193P1E1B. An immunoaffinity column is constructed by covalently coupling anti-193P1E1B antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing 193P1E1B are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of 193P1E1B (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/193P1E1B binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and GCR.P is collected.
Example 36
Identification of Molecules Which Interact with 193P1E1B
193P1E1B, or biologically active fragments thereof, are labeled with 121 1 Bolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J. 133:529.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled 193P1E1B, washed, and any wells with labeled 193P1E1B complex are assayed. Data obtained using different concentrations of 193P1E1B are used to calculate values for the number, affinity, and association of 193P1E1B with the candidate molecules.
Example 37
In Vivo Assay for 193P1E1B Tumor Growth Promotion
The effect of a 193P1E1B protein on tumor cell growth can be confirmed in vivo by gene overexpression in a variety of cancer cells such as those in Table I. For example, SCID mice can be injected SQ on each flank with 1×106 prostate, kidney, colon or bladder cancer cells (such as PC3, LNCaP, SCaBER, UM-UC-3, HT1376, SK-CO, Caco, RT4, T24, Caki, A-498 and SW839 cells) containing tkNeo empty vector or 193P1E1B.
At least two strategies can be used:
(1) Constitutive 193P1E1B expression under regulation of a promoter such as a constitutive promoter obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2, 211, 504 published Jul. 5, 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, provided such promoters are compatible with the host cell systems.
(2) Regulated expression under control of an inducible vector system, such as ecdysone, tet, etc., can be used provided such promoters are compatible with the host cell systems. Tumor volume is then monitored at the appearance of palpable tumors or by following serum markers such as PSA. Tumor development is followed over time to validate that 193P1E1B-expressing cells grow at a faster rate and/or that tumors produced by 193P1E1B-expressing cells demonstrate characteristics of altered aggressiveness (e.g., enhanced metastasis, vascularization, reduced responsiveness to chemotherapeutic drugs). Tumor volume is evaluated by caliper measurements. Additionally, mice can be implanted with the same cells orthotopically in the prostate, bladder, colon or kidney to determine if 193P1E1B has an effect on local growth, e.g., in the prostate, bladder, colon or kidney or on the ability of the cells to metastasize, specifically to lungs or lymph nodes (Saffran et al., Proc Natl Acad Sci USA. 2001, 98: 2658; Fu, X., et al., Int. J. Cancer, 1991. 49: 938-939; Chang, S., et al., Anticancer Res., 1997, 17: 3239-3242; Peralta, E. A., et al., J. Urol., 1999. 162:1806-1811). For instance, the orthotopic growth of PC3 and PC3-193P1E1B can be compared in the prostate of SCID mice. Such experiments reveal the effect of 193P1E1B on orthotopic tumor growth, metastasis and/or angiogenic potential.
Furthermore, this assay is useful to confirm the inhibitory effect of candidate therapeutic compositions, such as 193P1E1B antibodies or intrabodies, and 193P1E1B antisense molecules or ribozymes, or 193P1E1B directed small molecules, on cells that express a 193P1E1B protein.
Example 38
193P1E1B Monoclonal Antibody-mediated Inhibition of Prostate Tumors In Vivo.
The significant expression of 193P1E1B, in cancer tissues, together with its restricted expression in normal tissues makes 193P1E1B an excellent target for antibody therapy. Similarly, 193P1E1B is a target for T cell-based immunotherapy. Thus, the therapeutic efficacy of anti-193P1E1B mAbs is evaluated, e.g., in human prostate cancer xenograft mouse models using androgen-independent LAPC-4 and LAPC-9 xenografts (Craft, N., et al. Cancer Res, 1999. 59(19): p. 5030-5036), kidney cancer xenografts (AGS-K3, AGS-K6), kidney cancer metastases to lymph node (AGS-K6 met) xenografts, and kidney cancer cell lines transfected with 193P1E1B, such as 769P-193P E1B, A498-193P1E1B.
Antibody efficacy on tumor growth and metastasis formation is studied, e.g., in mouse orthotopic prostate cancer xenograft models and mouse kidney xenograft models. The antibodies can be unconjugated, as discussed in this example, or can be conjugated to a therapeutic modality, as appreciated in the art. Anti-193P1E1B mAbs inhibit formation of both the androgen-dependent LAPC-9 and androgen-independent PC3-193P1E1B tumor xenografts. Anti-193P1E1B mAbs also retard the growth of established orthotopic tumors and prolonged survival of tumor-bearing mice. These results indicate the utility of anti-193P1E1B mAbs in the treatment of local and advanced stages of, e.g., prostate cancer. (See, e.g., Saffran, D., et al., PNAS 10:1073-1078. Similarly, anti-193P1E1B mAbs inhibit formation of AGS-K3 and AGS-K6 tumors in SCID mice, and prevent or retard the growth A498-193P1E1B tumor xenografts. These results indicate the use of anti-193P1E1B mAbs in the treatment of prostate and/or kidney cancer.
Administration of the anti-193P1E1B mAbs leads to retardation of established orthotopic tumor growth and inhibition of metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies indicate that 193P1E1B is an attractive target for immunotherapy and demonstrate the therapeutic use of anti-193P1E1B mAbs for the treatment of local and metastatic cancer. This example demonstrates that unconjugated 193P1E1B monoclonal antibodies are effective to inhibit the growth of human prostate tumor xenografts and human kidney xenografts grown in SCID mice.
Tumor Inhibition Using Multiple Unconjugated 193P1E1B mAbs
Materials and Methods
193P1E1B Monoclonal Antibodies:
Monoclonal antibodies are obtained against 193P1E1B, as described in Example 11 entitled: Generation of 193P1E1B Monoclonal Antibodies (mAbs), or may be obtained commercially. The antibodies are characterized by ELISA, Western blot, FACS, and immunoprecipitation for their capacity to bind 193P1E1B. Epitope mapping data for the anti-193P1E1B mAbs, as determined by ELISA and Western analysis, recognize epitopeson a 193P1E1B protein. Immunohistochemical analysis of cancer tissues and cells is performed with these antibodies.
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, e.g., LAPC-9 prostate tumor xenografts.
Cancer Xenografts and Cell Lines
The LAPC-9 xenograft, which expresses a wild-type androgen receptor and produces prostate-specific antigen (PSA), is passaged in 6- to 8-week-old male ICR-severe combined immunodeficient (SCID) mice (Taconic Farms) by subcutaneous (s.c.) trocar implant (Craft, N., et al., 1999, Cancer Res. 59:5030-5036). The AGS-K3 and AGS-K6 kidney xenografts are also passaged by subcutaneous implants in 6- to 8-week old SCID mice. Single-cell suspensions of tumor cells are prepared as described in Craft, et al. The prostate carcinoma cell line PC3 (American Type Culture Collection) is maintained in RPMI supplemented with L-glutamine and 10% FBS, and the kidney carcinoma line A498 (American Type Culture Collection) is maintained in DMEM supplemented with L-glutamine and 10% FBS.
PC3-193P1E1B and A498-193P1E1B cell populations are generated by retroviral gene transfer as described in Hubert, R. S., et al., STEAP: A Prostate-specific Cell-surface Antigen Highly Expressed in Human Prostate Tumors, Proc Nat. Acad. Sci. USA, 1999. 96(25): p. 14523-14528. Anti-193P1E1B staining is detected by using, e.g., an FITC-conjugated goat anti-mouse antibody (Southern Biotechnology Associates) followed by analysis on a Coulter Epics-XL flow cytometer.
Xenograft Mouse Models.
Subcutaneous (s.c.) tumors are generated by injection of 1×106 LAPC-9, AGS-K3, AGS-K6, PC3, PC3-193P1E1B, A498 or A498-193P1E1B cells mixed at a 1:1 dilution with Matrigel (Collaborative Research) in the right flank of male SCID mice. To test antibody efficacy on tumor formation, i.p. antibody injections are started on the same day as tumor-cell injections. As a control, mice are injected with either purified mouse IgG (ICN) or PBS; or a purified monoclonal antibody that recognizes an irrelevant antigen not expressed in human cells. In preliminary studies, no difference is found between mouse IgG or PBS on tumor growth. Tumor sizes are determined by vemier caliper measurements, and the tumor volume is calculated as length×width×height. Mice with s.c. tumors greater than 1.5 cm in diameter are sacrificed. PSA levels are determined by using a PSA ELISA kit (Anogen, Mississauga, Ontario). Circulating levels of anti-193P1E1B mAbs are determined by a capture ELISA kit (Bethyl Laboratories, Montgomery, Tex.). (See, e.g., (Saffran, D., et al., PNAS 10:1073-1078 .
Orthotopic prostate injections are performed under anesthesia by using ketamine/xylazine. For prostate orthotopic studies, an incision is made through the abdominal muscles to expose the bladder and seminal vesicles, which then are delivered through the incision to expose the dorsal prostate. LAPC-9 cells (5×105) mixed with Matrigel are injected into each dorsal lobe in a 10 μl volume. To monitor tumor growth, mice are bled on a weekly basis for determination of PSA levels. For kidney orthotopic models, an incision is made through the abdominal muscles to expose the kidney. AGS-K3 or AGS-K6 cells mixed with Matrigel are injected under the kidney capsule. The mice are segregated into groups for appropriate treatments, with anti-193P1E1B or control mAbs being injected i.p.
Anti-193P1E1B mAbs Inhibit Growth of 193P1E1B-Expressing Xenograft-Cancer Tumors
The effect of anti-193P1E1B mAbs on tumor formation is tested by using, e.g., LAPC-9 and/or AGS-K3 orthotopic models. As compared with the s.c. tumor model, the orthotopic model, which requires injection of tumor cells directly in the mouse prostate or kidney, respectively, results in a local tumor growth, development of metastasis in distal sites, deterioration of mouse health, and subsequent death (Saffran, D., et al., PNAS supra; Fu, X., et al., Int J Cancer, 1992. 52(6): p. 987-90; Kubota, T., J Cell Biochem, 1994. 56(1): p. 4-8). The features make the orthotopic model more representative of human disease progression and allow for tracking of the therapeutic effect of mAbs on clinically relevant end points.
Accordingly, tumor cells are injected into the mouse prostate or kidney, and the mice are segregated into two groups and treated with either: a) 200-500 μg of anti-193P1E1B Ab, or b) PBS for two to five weeks.
As noted, a major advantage of the orthotopic prostate-cancer model is the ability to study the development of metastases. Formation of metastasis in mice bearing established orthotopic tumors is studied by IHC analysis on lung sections using an antibody against a prostate-specific cell-surface protein STEAP expressed at high levels in LAPC-9 xenografts (Hubert, R. S., et al., Proc Natl. Acad. Sci. USA, 1999. 96(25): p. 14523-14528) or anti-G250 antibody for kidney cancer models. G250 is a clinically relevant marker for renal clear cell carcinoma, which is selectively expressed on tumor but not normal kidney cells (Grabmaier K et al., Int J Cancer. 2000, 85: 865).
Mice bearing established orthotopic LAPC-9 tumors are administered 500-1000 μg injections of either anti-193P1E1B mAb or PBS over a 4-week period. Mice in both groups are allowed to establish a high tumor burden (PSA levels greater than 300 μg/ml), to ensure a high frequency of metastasis formation in mouse lungs. Mice then are killed and their prostate/kidney and lungs are analyzed for the presence of tumor cells by IHC analysis. These studies demonstrate a broad anti-tumor efficacy of anti-193P1E1B antibodies on initiation and/or progression of prostate and kidney cancer in xenograft mouse models. Anti-193P1E1B antibodies inhibit tumor formation of both androgen-dependent and androgen-independent prostate tumors as well as retarding the growth of already established tumors and prolong the survival of treated mice. Moreover, anti-193P1E1B mAbs demonstrate a dramatic inhibitory effect on the spread of local prostate tumor to distal sites, even in the presence of a large tumor burden. Similar therapeutic effects are seen in the kidney cancer model. Thus, anti-193P1E1B mAbs are efficacious on major clinically relevant end points (tumor growth), prolongation of survival, and health.
Example 39
Therapeutic and Diagnostic Use of Anti-193P1E1B Antibodies in Humans.
Anti-193P1E1B monoclonal antibodies are safely and effectively used for diagnostic, prophylactic, prognostic and/or therapeutic purposes in humans. Western blot and immunohistochemical analysis of cancer tissues and cancer xenografts with anti-193P1E1B mAb show strong extensive staining in carcinoma but significantly lower or undetectable levels in normal tissues. Detection of 193P1E1B in carcinoma and in metastatic disease demonstrates the usefulness of the mAb as a diagnostic and/or prognostic indicator. Anti-193P1E1B antibodies are therefore used in diagnostic applications such as immunohistochemistry of kidney biopsy specimens to detect cancer from suspect patients.
As determined by flow cytometry, anti-193P1E1B mAb specifically binds to carcinoma cells. Thus, anti-193P1E1B antibodies are used in diagnostic whole body imaging applications, such as radioimmunoscintigraphy and radioimmunotherapy, (see, e.g., Potamianos S., et. al. Anticancer Res 20(2A):925-948 (2000)) for the detection of localized and metastatic cancers that exhibit expression of 193P1E1B. Shedding or release of an extracellular domain of 193P1E1B into the extracellular milieu, such as that seen for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology 27:563-568 (1998)), allows diagnostic detection of 193P1E1B by anti-193P1E1B antibodies in serum and/or urine samples from suspect patients.
Anti-193P1E1B antibodies that specifically bind 193P1E1B are used in therapeutic applications for the treatment of cancers that express 193P1E1B. Anti-193P1E1B antibodies are used as an unconjugated modality and as conjugated form in which the antibodies are attached to one of various therapeutic or imaging modalities well known in the art, such as a prodrugs, enzymes or radioisotopes. In preclinical studies, unconjugated and conjugated anti-193P1E1B antibodies are tested for efficacy of tumor prevention and growth inhibition in the SCID mouse cancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6, (see, e.g., the Example entitled “193P1E1B Monoclonal Antibody-mediated Inhibition of Bladder and Lung Tumors In Vivo”). Either conjugated and unconjugated anti-193P1E1B antibodies are used as a therapeutic modality in human clinical trials either alone or in combination with other treatments as described in following Examples.
Example 40
Human Clinical Trials for the Treatment and Diagnosis of Human Carcinomas Through use of Human Anti-193P1E1B Antibodies In Vivo
Antibodies are used in accordance with the present invention which recognize an epitope on 193P1E1B, and are used in the treatment of certain tumors such as those listed in Table I. Based upon a number of factors, including 193P1E1B expression levels, tumors such as those listed in Table I are presently preferred indications. In connection with each of these indications, three clinical approaches are successfully pursued.
I.) Adjunctive therapy: In adjunctive therapy, patients are treated with anti-193P1E1B antibodies in combination with a chemotherapeutic or antineoplastic agent and/or radiation therapy. Primary cancer targets, such as those listed in Table I, are treated under standard protocols by the addition anti-193P1E1B antibodies to standard first and second line therapy. Protocol designs address effectiveness as assessed by reduction in tumor mass as well as the ability to reduce usual doses of standard chemotherapy. These dosage reductions allow additional and/or prolonged therapy by reducing dose-related toxicity of the chemotherapeutic agent. Anti-193P1E1B antibodies are utilized in several adjunctive clinical trials in combination with the chemotherapeutic or antineoplastic agents adriamycin (advanced prostrate carcinoma), cisplatin (advanced head and neck and lung carcinomas), taxol (breast cancer), and doxorubicin (preclinical).
II.) Monotherapy: In connection with the use of the anti-193P1E1B antibodies in monotherapy of tumors, the antibodies are administered to patients without a chemotherapeutic or antineoplastic agent. In one embodiment, monotherapy is conducted clinically in end stage cancer patients with extensive metastatic disease. Patients show some disease stabilization. Trials demonstrate an effect in refractory patients with cancerous tumors.
III.) Imaging Agent: Through binding a radionuclide (e.g., iodine or yttrium (I131, Y90) to anti-193P1E1B antibodies, the radiolabeled antibodies are utilized as a diagnostic and/or imaging agent. In such a role, the labeled antibodies localize to both solid tumors, as well as, metastatic lesions of cells expressing 193P1E1B. In connection with the use of the anti-193P1E1B antibodies as imaging agents, the antibodies are used as an adjunct to surgical treatment of solid tumors, as both a pre-surgical screen as well as a post-operative follow-up to determine what tumor remains and/or returns. In one embodiment, a (111In)-193P1E1B antibody is used as an imaging agent in a Phase I human clinical trial in patients having a carcinoma that expresses 193P1E1B (by analogy see, e.g., Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991)). Patients are followed with standard anterior and posterior gamma camera. The results indicate that primary lesions and metastatic lesions are identified
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-193P1E1B 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-193P1E1B antibodies relative to the affinity of a known antibody for its target is one parameter used by those of skill in the art for determining analogous dose regimens. Further, anti-193P1E1B antibodies that are fully human antibodies, as compared to the chimeric antibody, have slower clearance; accordingly, dosing in patients with such fully human anti-193P1E1B antibodies can be lower, perhaps in the range of 50 to 300 mg/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-193P1E1B antibodies. Conventional intravenous delivery is one standard delivery technique for many tumors. However, in connection with tumors in the peritoneal cavity, such as tumors of the ovaries, biliary duct, other ducts, and the like, intraperitoneal administration may prove favorable for obtaining high dose of antibody at the tumor and to also minimize antibody clearance. In a similar manner, certain solid tumors possess vasculature that is appropriate for regional perfusion. Regional perfusion allows for a high dose of antibody at the site of a tumor and minimizes short term clearance of the antibody.
Clinical Development Plan (CDP)
Overview: The CDP follows and develops treatments of anti-193P1E1B antibodies in connection with adjunctive therapy, monotherapy, and as an imaging agent. Trials initially demonstrate safety and thereafter confirm efficacy in repeat doses. Trails are open label comparing standard chemotherapy with standard therapy plus anti-193P1E1B antibodies. As will be appreciated, one criteria that can be utilized in connection with enrollment of patients is 193P1E1B expression levels in their tumors as determined by biopsy.
As with any protein or antibody infusion-based therapeutic, safety concerns are related primarily to (i) cytokine release syndrome, i.e., hypotension, fever, shaking, chills; (ii) the development of an immunogenic response to the material (i.e., development of human antibodies by the patient to the antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that express 193P1E1B. Standard tests and follow-up are utilized to monitor each of these safety concerns. Anti-193P1E1B antibodies are found to be safe upon human administration.
Example 41
Human Clinical Trial Adjunctive Therapy with Human Anti-193P1E1B Antibody and Chemotherapeutic Agent
A phase I human clinical trial is initiated to assess the safety of six intravenous doses of a human anti-193P1E1B antibody in connection with the treatment of a solid tumor, e.g., a cancer of a tissue listed in Table I. In the study, the safety of single doses of anti-193P1E1B antibodies when utilized as an adjunctive therapy to an antineoplastic or chemotherapeutic agent as defined herein, such as, without limitation: cisplatin, topotecan, doxorubicin, adriamycin, taxol, or the like, is assessed. The trial design includes delivery of six single doses of an anti-193P1E1B antibody with dosage of antibody escalating from approximately about 25 mg/m2to about 275 mg/m2over 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
75
125
175
225
275



mg/m2
mg/m2
mg/m2
mg/m2
mg/m2
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 193P1E1B. Standard tests and follow-up are utilized to monitor each of these safety concerns. Patients are also assessed for clinical outcome, and particularly reduction in tumor mass as evidenced by MRI or other imaging.
The anti-193P1E1B antibodies are demonstrated to be safe and efficacious, Phase II trials confirm the efficacy and refine optimum dosing.
Example 42
Human Clinical Trial: Monotherapy with Human Anti-193P1E1B Antibody
Anti-193P1E1B antibodies are safe in connection with the above-discussed adjunctive trial, a Phase II human clinical trial confirms the efficacy and optimum dosing for monotherapy. Such trial is accomplished, and entails the same safety and outcome analyses, to the above-described adjunctive trial with the exception being that patients do not receive chemotherapy concurrently with the receipt of doses of anti-193P1E1B antibodies.
Example 43
Human Clinical Trial: Diagnostic Imaging with Anti-193P1E1B Antibody
Once again, as the adjunctive therapy discussed above is safe within the safety criteria discussed above, a human clinical trial is conducted concerning the use of anti-193P1E1B antibodies as a diagnostic imaging agent. The protocol is designed in a substantially similar manner to those described in the art, such as in Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991). The antibodies are found to be both safe and efficacious when used as a diagnostic modality.
Example 44
Homology Comparison of 193P1E1B to Known Sequences
The 193P1E1B protein has several forms, including 3 SNPs and 5 splice variants (FIG. 4G). Three variants, namely 193P1E1B v. 1, v.5 and v.6, consist of 412 amino acids each, with calculated molecular weight of 46.25 kDa, and pl of 5.18, and differ from each other by one amino acid. 193P1E1B v.10, v.9 and v.12 are progressively smaller proteins, with 388, 330, and 73 amino acids respectively. These variants differ with regards to their molecular weights and isoelectic points, as shown in Table L. All variants of 193P1E1B are predicted to be nuclear proteins, with possible localization to the mitochondria (193P1E3B v.1, v.5, v.6, v.9, v.10, v.11 and v.13) or cytoplasm (193P1E3B v.12). Motif analysis revealed no known motifs.
All protein variants of 193P1E1B show best homology to a human un-named protein (gi 21748775) of unknown function, with 193P1E1B v.5 showing 100% identity with gi 21748775 over the entire length of the protein, and 193P1E1B sharing 99% identity with the same protein. Similarly, the other variants show highest homology to the human un-named protein (gi 21748775). The variant with the lowest homology to gi 21748775 is 193P1E1B v.12, with 89% identity and 89% homology over the first 39 amino acids of the protein (FIGS. 4A-D).
The 193P1E1B protein shows homology to a protein of known function, namely the arginine repressor (gi14349114) of E. coli, also known as carbamate kinase. Variant 193P1E1B v.1 shows 30% identity and 57% homology with that protein (FIG. 4E). This homology indicates that 193P1E1B may regulate ATP synthesis and metabolism (Marina A et al., Eur J Biochem 1998, 253:280; Alcantara C et al., FEBS Left. 2000, 484:261), a key factor in cell growth and biological function.
In addition, 193P1E1B also exhibit some homology to human double-stranded RNA-specific adenosine deaminase (ADAR-c isoform) (gi 7669475). 193P1E1Bv.1 shares 26% identity and 40% homology with ADAR-c (FIG. 4F). Similar results were obtained with 193P1E1Bv.5, v.6, v.9, v.10 and v.13. This suggests that 193P1E1B has the ability to bind specifically to double stranded RNA or DNA (Schwartz T., et al., Nature Struc. Biol. 2001, 8:761). Adenosine deaminases acting on RNA have been shown to be involved in RNA editing (Raitskin, O., et al., Proc. Natl. Acad. Sci 2001, 98:6571). Recent studies have associated adenosine deaminase with cancer and cellular proliferation (Eroglu A, et al., Med Oncol. 2000, 17:319-24; Barry C. P., and, Lind, S. E., Cancer Res. 2000, 60:1887-94). In addition, adenosine deaminase is highly expressed in tumor tissue relative to normal tissues in such cancers as colon, leukemia and other lymphoid cancers (Blatt, J., et al., N Engl J Med. 1980; 303:918; Eroglu, A., et al., Med Oncol. 2000, 17:319). Adenosine deaminase has been considered a potential marker for lymphoid malignancies (Blatt J et al., N Engl J Med. 1980;303: 918). In addition, inhibition of adenosine deaminase was found to result in cell death of epithelial cells (Barry, C. P., and, Lind, S. E., Cancer Res. 2000, 60:1887).
This information indicates that 193P1E1B plays a role in the transformation of mammalian cells, supports cell survival and proliferation, and regulates gene transcription by regulating events in the nucleus. Accordingly, when 193P1E1B functions as a regulator of cell transformation, tumor formation, or as a modulator of transcription involved in activating genes associated with inflammation, tumorigenesis or proliferation, 193P1E1B is used for therapeutic, diagnostic, prognostic and/or preventative purposes.
Example 45
Identification and Confirmation of Potential Signal Transduction Pathways
Many mammalian proteins have been reported to interact with signaling molecules and to participate in regulating signaling pathways. (J Neurochem. 2001; 76:217-223). In particular, adenosine deaminase has been found to associate with G-proteins, thereby regulating several signaling pathways (Ciruela F et al., FEBS Lett. 1996, 380:219). Using immunoprecipitation and Western blotting techniques, proteins are identified that associate with 193P1E1B and mediate signaling events. Several pathways known to play a role in cancer biology can be regulated by 193P1E1B, including phospholipid pathways such as P13K, AKT, etc, adhesion and migration pathways, including FAK, Rho, Rac-1, etc, as well as mitogenic/survival cascades such as ERK, p38, etc .(Cell Growth Differ. 2000, 11:279; J Biol Chem. 1999, 274:801; Oncogene. 2000, 19:3003, J. Cell Biol. 1997, 138:913).
To confirm that 193P1E1B directly or indirectly activates known signal transduction pathways in cells, luciferase (luc) based transcriptional reporter assays are carried out in cells expressing individual genes. These transcriptional reporters contain consensus-binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways. The reporters and examples of these associated transcription factors, signal transduction pathways, and activation stimuli are listed below.

    
    
        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
    
    


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 193P1E1B are mapped and used for the identification and validation of therapeutic targets. When 193P1E1B is involved in cell signaling, it is used as target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 46
Regulation of Transcription
The nuclear localization of 193P1E1B and its ability to regulate adenosine deaminase indicate that it is effectively used as a modulator of the transcriptional regulation of eukaryotic genes. Regulation of gene expression is confirmed, e.g., by studying gene expression in cells expressing or lacking 193P1E1B. For this purpose, two types of experiments are performed.
In the first set of experiments, RNA from parental and 193P1E1B-expressing cells are extracted and hybridized to commercially available gene arrays (Clontech) (Smid-Koopman E et al. Br J Cancer. 2000. 83:246). Resting cells as well as cells treated with FBS, pheromones, or growth factors are compared. Differentally 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, 193P1E1B plays a role in gene regulation, and it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 47
Involvement in Tumor Progression
The 193P1E1B gene can contribute to the growth of cancer cells. The role of 193P1E1B in tumor growth is confirmed in a variety of primary and transfected cell lines including prostate, colon, bladder and kidney cell lines, as well as NIH 3T3 cells engineered to stably express 193P1E1B. Parental cells lacking 193P1E1B and cells expressing 193P1E1B are evaluated for cell growth using a well-documented proliferation assay (Fraser S P, et al., Prostate 2000;44:61, Johnson DE, Ochieng J, Evans SL. Anticancer Drugs. 1996, 7:288). The effect of 193P1E1B can also be observed on cell cycle progression. Control and 193P1E1B-expressing cells are grown in low serum overnight, and treated with 10% FBS for 48 and 72 hrs. Cells are analyzed for BrdU and propidium iodide incorporation by FACS analysis.
To confirm the role of 193P1E1B in the transformation process, its effect in colony forming assays is investigated. Parental NIH-3T3 cells lacking 193P1E1B are compared to NIH-3T3 cells expressing 193P1E1B, using a soft agar assay under stringent and more permissive conditions (Song Z. et al. Cancer Res. 2000;60:6730).
To confirm the role of 193P1E1B in invasion and metastasis of cancer cells, a well-established assay is used. A non-limiting example is the use of an assay which provides a basement membrane or an analog thereof used to detect whether cells are invasive (e.g., a Transwell Insert System assay (Becton Dickinson) (Cancer Res. 1999; 59:6010)). Control cells, including prostate, and bladder cell lines lacking 193P1E1B are compared to cells expressing 193P1E1B. Cells are loaded with the fluorescent dye, calcein, and plated in the top well of a support structure coated with a basement membrane analog (e.g. the Transwell insert) and used in the assay. Invasion is determined by fluorescence of cells in the lower chamber relative to the fluorescence of the entire cell population.
193P1E1B can also play a role in cell cycle and apoptosis. Parental cells and cells expressing 193P1E1B are compared for differences in cell cycle regulation using a well-established BrdU assay (Abdel-Malek ZA. J Cell Physiol. 1988, 136:247). In short, cells are grown under both optimal (full serum) and limiting (low serum) conditions are labeled with BrdU and stained with anti-BrdU Ab and propidium iodide. Cells are analyzed for entry into the G1, S, and G2M phases of the cell cycle. Alternatively, the effect of stress on apoptosis is evaluated in control parental cells and cells expressing 193P1E1B, including normal and tumor prostate, and kidney cells. Engineered and parental cells are treated with various chemotherapeutic agents, such as etoposide, flutamide, etc, and protein synthesis inhibitors, such as cycloheximide. Cells are stained with annexin V-FITC and cell death is measured by FACS analysis. The modulation of cell death by 193P1E1B can play a critical role in regulating tumor progression and tumor load.
When 193P1E1B plays a role in cell growth, transformation, invasion or apoptosis, it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 48
Involvement in Angiogenesis
Angiogenesis or new capillary blood vessel formation is necessary for tumor growth (Hanahan D, Folkman J. Cell. 1996, 86:353; Folkman J. Endocrinology. 1998 139:441). Based on the effect of phsophodieseterase inhibitors on endothelial cells, 193P1E1B plays a role in angiogenesis (DeFouw L et al., Microvasc Res 2001, 62:263). Several assays have been developed to measure angiogenesis in vitro and in vivo, such as the tissue culture assays endothelial cell tube formation and endothelial cell proliferation. Using these assays as well as in vitro neo-vascularization, the role of 193P1E1B in angiogenesis, enhancement or inhibition, is confirmed. For example, endothelial cells engineered to express 193P1E1B are evaluated using tube formation and proliferation assays. The effect of 193P1E1B is also confirmed in animal models in vivo. For example, cells either expressing or lacking 193P1E1B are implanted subcutaneously in immunocompromised mice. Endothelial cell migration and angiogenesis are evaluated 5-15 days later using immunohistochemistry techniques. 193P1E1B affects angiogenesis, and it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 49
Involvement in Cell Adhesion
Cell adhesion plays a critical role in tissue colonization and metastasis. 193P1E1B can participate in cellular organization, and as a consequence cell adhesion and motility. To confirm that 193P1E1B regulates cell adhesion, control cells lacking 193P1E1B are compared to cells expressing 193P1E1B, using techniques previously described (see, e.g., Haier et al., Br. J. Cancer. 1999, 80:1867; Lehr and Pienta, J. Natl. Cancer Inst. 1998, 90:118). Briefly, in one embodiment, cells labeled with a fluorescent indicator, such as calcein, are incubated on tissue culture wells coated with media alone or with matrix proteins. Adherent cells are detected by fluorimetric analysis and percent adhesion is calculated. In another embodiment, cells lacking or expressing 193P1E1B are analyzed for their ability to mediate cell-cell adhesion using similar experimental techniques as described above. Both of these experimental systems are used to identify proteins, antibodies and/or small molecules that modulate cell adhesion to extracellular matrix and cell-cell interaction. Cell adhesion plays a critical role in tumor growth, progression, and, colonization, and 193P1E1B is involved in these processes. Thus, it serves as a diagnostic, prognostic, preventative and/or therapeutic modality.
Example 50
Protein-Protein Association
Several adenosine deaminasess have been shown to interact with other proteins, thereby regulating gene transcription, protein function, as well as cell growth (Raitskin et al above; Morimoto C, and Schlossman SF, Immunol Rev. 1998, 161:55.). Using immunoprecipitation techniques as well as two yeast hybrid systems, proteins are identified that associate with 193P1E1B. Immunoprecipitates from cells expressing 193P1E1B and cells lacking 193P1E1B are compared for specific protein-protein associations.
Studies are performed to confirm the extent of association of 193P1E1B with effector molecules, such as nuclear proteins, transcription factors, kinases, phosphates, etc. Studies comparing 193P1E1B positive and 193P1E1B negative cells as well as studies comparing unstimulated/resting cells and cells treated with epithelial cell activators, such as cytokines, growth factors, androgen and anti-integrin Ab reveal unique interactions.
In addition, protein-protein interactions are confirmed using two yeast hybrid methodology (Curr. Opin. Chem. Biol. 1999, 3:64). A vector carrying a library of proteins fused to the activation domain of a transcription factor is introduced into yeast expressing a 193P1E1B-DNA-binding domain fusion protein and a reporter construct. Protein-protein interaction is detected by colorimetric reporter activity. Specific association with effector molecules and transcription factors directs one of skill to the mode of action of 193P1E1B, and thus identifies therapeutic, prognostic, preventative and/or diagnostic targets for cancer. This and similar assays are also used to identify and screen for small molecules that interact with 193P1E1B. Thus it is found that 193P1E1B associates with proteins and small molecules. Accordingly, 193P1E1Band these proteins and small molecules are used 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 URL, addresses 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 193P1E1B:







a. Malignant Tissues









Prostate



Bladder



Kidney



Colon



Lung



Ovary



Breast



Pancreas



Testis



Uterus



Skin



Bone

















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



















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


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


in related, natural proteins.



































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


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



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




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





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






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







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








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









4
−3
2
1
−3
−3
−3
−3
−2
−1
3
−3
−1
I










5
−2
−1
0
−1
1
2
0
−1
−2
−3
−2
K











4
2
−3
−3
−2
−2
−2
−1
1
−2
−1
L












5
−2
−2
0
−1
−1
−1
1
−1
−1
M













6
−2
0
0
1
0
−3
−4
−2
N














7
−1
−2
−1
−1
−2
−4
−3
P















5
1
0
−1
−2
−2
−1
Q
















5
−1
−1
−3
−3
−2
R

















4
1
−2
−3
−2
S


















5
0
−2
−2
T



















4
−3
−1
V




















11
2
W





















7
Y
















TABLE IV







(A): HLA Class I Supermotifs/Motifs












POSITION
POSITION



POSITION
3 (Primary
C Terminus 



2 (Primary Anchor)
Anchor)
(Primary Anchor)














SUPERMOTIF





A1
TILVMS

FWY



(SEQ ID NO:124)


A2
LIVMATQ

IVMATL



(SEQ ID NO:125)

(SEQ ID NO:126)


A3
VSMATLI

RK



(SEQ ID NO:127)


A24
YFWIVLMT

FIYWLM



(SEQ ID NO:128)

(SEQ ID NO:129)


B7
P

VILFMWYA





(SEQ ID NO:130)


B27
RHK

FYLWMIVA





(SEQ ID NO:131)


B44
ED

FWYLIMVA





(SEQ ID NO:132)


B58
ATS

FWYLIVMA





(SEQ ID NO:133)


B62
QLIVMP

FWYMIVLA



(SEQ ID NO:134)

(SEQ ID NO:135)


MOTIFS


A1
TSM

Y


A1

DEAS
Y




(SEQ ID




NO:247)



A2.1
LMVQIAT

VLIMAT



(SEQ ID NO:136)

(SEQ ID NO:137)


A3
LMVISATFCGD

KYRHFA



(SEQ ID NO:138)

(SEQ ID NO:139)


A11
VTMLISAGNCDF

KRYH



(SEQ ID NO:140)

(SEQ ID NO:141)


A24
YFWM

FLIW



(SEQ ID NO:142)

(SEQ ID NO:143)


A*3101
MVTALIS

RK



(SEQ ID NO:144)


A*3301
MVALFIST

RK



(SEQ ID NO:145)


A*6801
AVTMSLI

RK



(SEQ ID NO:146)


B*0702
P

LMFWYAIV





(SEQ ID NO:147)


B*3501
P

LMFWYIVA





(SEQ ID NO:148)


B51
P

LIVFWYAM





(SEQ ID NO:149)


B*5301
P

IMFWYALV





(SEQ ID NO:150)


B*5401
P

ATIVLMFWY





(SEQ ID NO:151)





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













TABLE 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
(SEQ ID NO:152)


W

(SEQ ID NO:153)
R

WDE


DR1
preferred
MFLIVWY


PAMQ

VMATSPLIC
M

AVM



deleterious
(SEQ ID NO:154)

C
(SEQ ID NO:155)
CWD
(SEQ ID NO:156)
GDE
D






CH
FD


DR7
preferred
MFLIVWY
M
W
A

IVMSACTPL
M

IV



deleterious
(SEQ ID NO:157)
C

G

(SEQ ID NO:158)
GRD
N
G


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


Motif a preferred

LIVMFY


D




(SEQ ID NO:159)


Motif b preferred

LIVMFAY


DNQEST

KRH




(SEQ ID NO:160)


(SEQ ID NO:161)


DR Supermotif

MFLIVWY




VMSTACPLI



(SEQ ID NO:162)




(SEQ ID NO:163)





Italicized residues indicate less preferred or “tolerated” residues













TABLE IV







(D): HLA Class I Supermotifs

















SUPER-












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





A1


1° Anchor






1° Anchor





TILVMS






FWY





(SEQ ID NO:164)









A2


1° Anchor






1° Anchor





LIVMATQ






LIVMAT





(SEQ ID NO:165)






(SEQ ID NO:166)


A3
Preferred

1° Anchor
YFW


YFW
YFW
P
1° Anchor





VSMATLI
(4/5)


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





(SEQ ID NO:167)










deleterious
DE (3/5);

DE










P (5/5)

(4/5)








A24


1° Anchor






1° Anchor





YFWIVLMT






FIYWLM





(SEQ ID NO:168)






(SEQ ID NO:169)


B7
Preferred
FWY (5/5)
1° Anchor
FWY




FWY
1° Anchor




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




(3/5)
VILFMWYA




(SEQ ID NO:170)







(SEQ ID NO:171)



deleterious
DE (3/5); P(5/5);



DE
G
QN
DE





G(4/5); A(3/5); QN(3/5)



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



B27


1° Anchor






1° Anchor





RHK






FYLWMIVA












(SEQ ID NO:172)


B44


1° Anchor






1° Anchor





ED






FWYLIMVA












(SEQ ID NO:173)


B58


1° Anchor






1° Anchor





ATS






FWYLIVMA












(SEQ ID NO:174)


B62


1° Anchor






1° Anchor





QLIVMP






FWYMIVLA





(SEQ ID NO:175)






(SEQ ID NO:176)





Italicized residues indicate less preferred or “tolerated” residues













TABLE IV





(E): HLA Class I Motifs



























PO-













SI-








9 or C-
C-



TION
1
2
3
4
5
6
7
8
terminus
terminus





A1
pre-
GFYW
1° Anchor
DEA
YFW

P
DEQN
YFW
1° Anchor



9-mer
ferred
(SEQ ID
STM




(SEQ ID

Y




NO:177)





NO:178)



del-
DE

RHKLIVMP
A
G
A



ete-


(SEQ ID



rious


NO:179)


A1
pre-
GRHK
ASTC-
1° Anchor
GSTC

ASTC (SEQ
LIVM (SEQ
DE
1° Anchor


9-mer
ferred
(SEQ ID
LIVM *
DEAS (SEQ
(SEQ ID

ID NO:184)
ID NO:185)

Y




NO:180)
(SEQ ID
ID NO:182)
NO:183)





NO:181)



del-
A
RHKD-

DE
PQN
RHK
PG
GP



ete-

EPYFW



rious

(SEQ ID





NO:186)


A1
pre-
YFW
1° Anchor
DEAQN
A
YFWQN

PASTC
GDE
P
1° Anchor


10-
ferred

STM
(SEQ ID

(SEQ ID

(SEQ ID


Y


mer



NO:187)

NO:188)

NO:189)



del-
GP

RHKGLIVM
DE
RHK
QNA *
RHKYFW
RHK
A



ete-


(SEQ ID



(SEQ ID



rious


NO:190)



NO:191)


A1
pre-
YFW
STCLIVM
1° Anchor
A
YFW

PG
G
YFW
1° Anchor


10-
ferred

(SEQ ID
DEAS (SEQ






Y


mer


NO:192)
ID NO:193)



del-
RHK
RHKD-


P
G

PRHK (SEQ
QN



ete-

EPYFW





ID NO:195)



rious

(SEQ ID





NO:194)


A2.1
pre-
YFW
1° Anchor
YFW
STC
YFW

A
P
1° Anchor


9-mer
ferred

LMIV-






VLIMAT





QAT






(SEQ ID





(SEQ ID






NO:197)





NO:196)



del-
DEP

DERKH


RKH
DERKH



ete-


(SEQ ID



(SEQ ID



rious


NO:198)



NO:199)






PO-



SI-









C-



TION:
1
2
3
4
5
6
7
8
9
Terminus





A2.1
pre-
AYFW
1° Anchor
LVIM (SEQ
G

G

FYWLVIM

1° Anchor


10-
ferred

LMIV-
ID NO:201)




(SEQ ID

VLIMAT


mer


QAT





NO:202)

(SEQ ID





(SEQ ID







NO:203)





NO:200)



del-
DEP

DE
RKHA
P

RKH
DERKH
RKH



ete-



(SEQ ID



(SEQ ID



rious



NO:204)



NO:205)


A3
pre-
RHK
1° Anchor
YFW
PRHKYFW
A
YFW

P
1° Anchor



ferred

LMVIS-

(SEQ ID




KYRHFA





ATFCGD

NO:207)




(SEQ ID





(SEQ ID






NO:208)





NO:206)



del-
DEP

DE



ete-



rious


A11
pre-
A
1° Anchor
YFW
YFW
A
YFW
YFW
P
1° Anchor



ferred

VTLMIS-






KRYH





AGNCDF






(SEQ ID





(SEQ ID






NO:210)





NO:209)



del-
DEP





A
G



ete-



rious


A24
pre-
YFWRHK
1° Anchor

STC


YFW
YFW
1° Anchor


9-mer
ferred
(SEQ ID
YFWM






FLIW




NO:211)
(SEQ ID






(SEQ ID





NO:212)






NO:213)



del-
DEG

DE
G
QNP
DERHK
G
AQN



ete-





(SEQ ID



rious





NO:214)


A24
Pre-

1° Anchor

P
YFWP

P


1° Anchor


10-
ferred

YFWM


(SEQ ID




FLIW


mer


(SEQ ID


NO:216)




(SEQ ID





NO:215)







NO:217)



Del-


GDE
QN
RHK
DE
A
QN
DEA



ete-



rious


A31-
Pre-
RHK
1° Anchor
YFW
P

YFW
YFW
AP
1° Anchor


01
ferred

MVTA-






RK





LIS





(SEQ ID





NO:218)



Del-
DEP

DE

ADE
DE
DE
DE



ete-



rious


A33-
Pre-

1° Anchor
YFW



AYFW

1° Anchor


01
ferred

MVAL-




(SEQ ID

RK





FIST




NO:220)





(SEQ ID





NO:219)



Del-
GP

DE



ete-



rious


A68-
Pre-
YFWSTC
1° Anchor


YFWLIVM

YFW
P
1° Anchor


01
ferred
(SEQ ID
AVTM-


(SEQ ID



RK




NO:221)
SLI


NO:223)





(SEQ ID





NO:222)



del-
GP

DEG

RHK


A



ete-



rious


B07-
Pre-
RHKFWY
1° Anchor
RHK

RHK
RHK
RHK
PA
1° Anchor


02
ferred
(SEQ ID
P






LMFW-




NO:224)







YAIV












(SEQ ID












NO:225)



del-
DEQNP

DEP
DE
DE
GDE
QN
DE



ete-
(SEQ ID



rious
NO:226)


B35-
Pre-
FWYL-
1° Anchor
FWY



FWY

1° Anchor


01
ferred
IVM
P






LMFW-




(SEQ ID







YIVA




NO:227)







(SEQ ID












NO:228)



del-
AGP



G
G



ete-



rious


B51
Pre-
LIVM-
1° Anchor
FWY
STC
FWY

G
FWY
1° Anchor



ferred
FWY
P






LIVF-




(SEQ ID







WYAM




NO:229)







(SEQ ID












NO:230)



del-
AGPD-



DE
G
DEQN
GDE



ete-
ERHK





(SEQ ID



rious
STC





NO:232)




(SEQ ID




NO:231)


B53-
pre-
LIVM-
1° Anchor
FWY
STC
FWY

LIVMFWY
FWY
1° Anchor


01
ferred
FWY
P




(SEQ ID

IMFW-




(SEQ ID





NO:234)

YALV




NO:233)







(SEQ ID












NO:235)



del-
AGPQN




G
RHKQN
DE



ete-
(SEQ ID





(SEQ ID



rious
NO:236)





NO:237)


B54-
pre-
FWY
1° Anchor
FWYLIVM

LIVM

ALIVM
EWYAP
1° Anchor


01
ferred

P
(SEQ ID

(SEQ ID

(SEQ ID
(SEQ ID
ATIVL-






NO:238)

NO:239)

NO:240)
NO:241)
MFWY












(SEQ ID












NO:242)



del-
GPQNDE

GDESTC

RHKDE
DE
QNDGE
DE



ete-
(SEQ ID

(SEQ ID

(SEQ ID

(SEQ ID



rious
NO:243)

NO:244)

NO:245)

NO:246)
















TABLE IV







(F):


Summary of HLA-supertypes


Overall phenotypic frequencies of HLA-supertypes in different ethnic populations









Phenotypic frequency













Specificity





Ave-

















Supertype
Position 2

C-Terminus

Caucasian
N.A. Black
Japanese
Chinese
Hispanic
rage




















B7
P

AILMVFWY
(SEQ ID NO:248)
43.2
55.1
57.1
43.0
49.3
49.5


A3
AILMVST
(SEQ ID NO:249)
RK

37.5
42.1
45.8
52.7
43.1
44.2


A2
AILMVT
(SEQ ID NO:250)
AILMVT
(SEQ ID NO:251)
45.8
39.0
42.4
45.9
43.0
42.2


A24
YF (WIVLMT)
(SEQ ID NO:252)
FI (YWLM)
(SEQ ID NO:253)
23.9
38.9
58.6
40.1
38.3
40.0


B44
E (D)

FWYLIMVA
(SEQ ID NO:254)
43.0
21.2
42.9
39.1
39.0
37.0


A1
TI (LVMS)
(SEQ ID NO:255)
FWY

47.1
16.1
21.8
14.7
26.3
25.2


B27
RHK

FYL (WMI)
(SEQ ID NO:256)
28.4
26.1
13.3
13.9
35.3
23.4


B62
QL (IVMP)
(SEQ ID NO:257)
FWY (MIV)
(SEQ ID NO:258)
12.6
4.8
36.5
25.4
11.1
18.1


B58
ATS

FWY (LIV)
(SEQ ID NO:259)
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,
99.5
98.1
100.0
99.5
99.4
99.3


B44 and A1
99.9
99.6
100.0
99.8
99.9
99.8


A2, A3, B7, A24,


B44, A1, B27,


B62, and 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




terminal)/b6/petB
superoxide


lg
19%
Immunoglobulin domain
domains are one hundred amino acids





long and include a conserved





intradomain disulfide bond.


WD40
18%
WD domain, G-beta repeat
tandem repeats of about 40 residues,





each containing a Trp-Asp motif.





Function in signal transduction and





protein interaction


PDZ
23%
PDZ domain
may function in targeting signaling





molecules to sub-membranous sites


LRR
28%
Leucine Rich Repeat
short sequence motifs involved in





protein-protein interactions


Pkinase
23%
Protein kinase domain
conserved catalytic core common to





both serine/threonine and tyrosine





protein kinases containing an ATP





binding site and a catalytic site


PH
16%
PH domain
pleckstrin homology involved in





intracellular signaling or as constituents





of the cytoskeleton


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





extracellular domain of membrane-





bound proteins or in secreted proteins


Rvt
49%
Reverse transcriptase




(RNA-dependent DNA




polymerase)


Ank
25%
Ank repeat
Cytoplasmic protein, associates integral





membrane proteins to the cytoskeleton


Oxidored_q1
32%
NADH-
membrane associated. Involved in




Ubiquinone/plastoquinone
proton translocation across the




(complex I), various chains
membrane


Efhand
24%
EF hand
calcium-binding domain, consists of a12





residue loop flanked on both sides by a





12 residue alpha-helical domain


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




protease
a catalytic aspartyl residue


Collagen
42%
Collagen triple helix repeat
extracellular structural proteins involved




(20 copies)
in formation of connective tissue. The





sequence consists of the G-X-Y and the





polypeptide chains forms a triple helix.


Fn3
20%
Fibronectin type III domain
Located in the extracellular ligand-





binding region of receptors and is about





200 amino acid residues long with two





pairs of cysteines involved in disulfide





bonds


7tm_1
19%
7 transmembrane receptor
seven hydrophobic transmembrane




(rhodopsin family)
regions, with the N-terminus located





extracellularly while the C-terminus is





cytoplasmic. Signal through G proteins
















TABLE VI





Motifs and Post-translational Modifications of 193P1E1B

















N-glycosylation site



Number of matches: 3










1
246-249 NKSE (SEQ ID NO:63)



2
316-319 NSSS (SEQ ID NO:64)



3
340-343 NLTD (SEQ ID NO:65)









cAMP- and cGMP-dependent protein kinase



phosphorylation site











107-110 KKNS (SEQ ID NO:66)









Protein kinase C phosphorylation site



Number of matches: 10










1
22-24 TAR



2
53-55 TLK



3
103-105 SPR



4
152-154 SPR



5
149-151 SEK



6
103-105 SPR



7
152-154 SPR



8
203-205 TPK



9
217-219 TPK



10
203-205 TPK









Casein kinase II phosphorylation site



Number of matches: 12










1
16-19 STLD (SEQ ID NO:67)



2
34-37 SDFE (SEQ ID NO:68)



3
53-56 TLKD (SEQ ID NO:69)



4
110-113 SVHE (SEQ ID NO:70)



5
119-122 SDPE (SEQ ID NO:71)



6
124-127 SNCE (SEQ ID NO:72)



7
276-279 SDAE (SEQ ID NO:73)



8
318-321 SSND (SEQ ID NO:74)



9
336-339 TCFE (SEQ ID NO:75)



10
350-353 SSYE (SEQ ID NO:76)



11
360-363 TPPE (SEQ ID NO:77)



12
408-411 SNKE (SEQ ID NO:78)









N-myristoylation site











239-244 GLKNAR (SEQ ID NO:79)

















TABLE VII





Search Peptides
















variant 1:



9-mers, 10-mers and 15-mers








(SEQ ID NO: 80)









MDPIRSFCGKLRSLASTLDCETARLQRALDGEESDFEDYPMRILYDLHSE



 


VQTLKDDVNIPELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSD


 


FGLERYIVSQVLPNPPQAVNLLDKARLENQEGIDFIKATKVLMEKNSMDI


 


MKIREYFQKYGYSPRVKKNSVHEQEAINSDNYKEEPVIVTPPTKQSLVKV


 


LKTPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGLKNARNNKSEE


 


AIDTESRLNDNVFATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTK


 


NSIALVSTNYPLSKTNSSSNDLEVEDRTSLVLNSDTCFENLTDPSSPTIS


 


SYENLLRTPTPPEVTKIPEDILQLLSKYNSNLATPIAIKAVPPSKRFLKH


 


GQNIRDVSNKEN


 









variant 5:




9-mers


PVASSCISEKSPRSPQL
(SEQ ID NO: 81)


 


10-mers


PPVASSCISEKSPRSPQLS
(SEQ ID NO: 82)


 


15-mers


DDLSDPPVASSCISEKSPRSPQLSDFGLE
(SEQ ID NO: 83)


 


Variant 6:


9-mers


NKSEEAIDAESRLND NV
(SEQ ID NO: 84)


 


10-mers


NNKSEEAIDAESRLND NVF
(SEQ ID NO: 85)


 


15-mers


LKNARNNKSEEAIDAESRLND NVFATPSP
(SEQ ID NO: 86)


 


Variant 10:


9-mers


KIPEDILQKFQWIYPTQKLNKMR
(SEQ ID NO: 87)


 


10-mers


TKIPEDILQKFQWIYPTQKLNKMR
(SEQ ID NO: 88)


 


15-mers


TPPEVTKIPEDILQKFQWIYPTQKLNKMR
(SEQ ID NO: 89)


 


Variant 12:


9-mers


RALDGEESLLSKYNSN
(SEQ ID NO: 90)


 


10-mers


QRALDGEESLLSKYNSNL
(SEQ ID NO: 91)


 


15-mers


ETARLQRALDGEESLLSKYNSNLATPIA
(SEQ ID NO: 92)









Tables VIII-XXI:










TABLE VIII





Start
Subsequence
Score















V1-HLA-A1-9mers-193P1EIB


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


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


position for each peptide is the start position plus eight.









19
DCETARLQR
45.000


98
LSDFGLERY
37.500


341
LTDPSSPTI
25.000


132
GIDFIKATK
20.000


78
LSDPPVASS
15.000


129
NQEGIDFIK
13.500


272
QLEKSDAEY
9.000


28
ALDGEESDF
5.000


253
DTESRLNDN
2.250


31
GEESDFEDY
2.250


33
ESDFEDYPM
1.500


306
VSTNYPLSK
1.500


63
LSNCENFQK
1.500


225
ISEYTMCLN
1.350


37
EDYPMRILY
1.250


228
YTMCLNEDY
1.250


319
SNDLEVEDR
1.250


71
KTDVKDDLS
1.250


232
LNEDYTMGL
1.125


358
TPTPPEVTK
1.000


215
CVTPKLEHF
1.000


389
KAVPPSKRF
1.000


17
TLDCETARL
1.000


277
DAEYTNSPL
0.900


321
DLEVEDRTS
0.900


323
EVEDRTSLV
0.900


344
PSSPTISSY
0.750


349
ISSYENLLR
0.750


333
NSDTCFENL
0.750


275
KSDAEYTNS
0.750


48
HSEVQTLKD
0.675


233
NEDYTMGLK
0.500


382
LATPIAIKA
0.500


281
TNSPLVPTF
0.500


251
AIDTESRLN
0.500


370
DILQLLSKY
0.500


263
FATPSPIIQ
0.500


302
SIALVSTNY
0.500


97
QLSDFGLER
0.500


219
KLEHFGISE
0.450


381
NLATPIAIK
0.400


236
YTMGLKNAR
0.250


16
STLDCETAR
0.250


391
VPPSKRFLK
0.250


267
SPIIQQLEK
0.250


209
KMDDFECVT
0.250


121
LLDKARLEN
0.250


189
VTPPTKQSL
0.250


60
IPELSNCEN
0.225


367
IPEDILQLL
0.225


171
VHEQEAINS
0.225


35
DFEDYPMRI
0.225


142
LMEKNSMDI
0.225


182
YKEEPVIVT
0.225


175
EAINSDNYK
0.200


201
LKTPKCALK
0.200


110
QVLPNPPQA
0.200


83
VASSCISGK
0.200


102
GLERYIVSQ
0.180


146
NSMDIMKIR
0.150


173
EQEAINSDN
0.135


247
KSEEAIDTE
0.135


290
CTPGLKIPS
0.125


147
SMDIMKIRE
0.125


264
ATPSPIIQQ
0.125


30
DGEESDFED
0.113


86
SCISGKSPR
0.100


330
LVLNSDTCF
0.100


188
IVTPPTKQS
0.100


118
AVNLLDKAR
0.100


205
KCALKMDDF
0.100


160
YGYSPRVKK
0.100


137
KATKVLMEK
0.100


390
AVPPSKRFL
0.100


126
RLENQEGID
0.090


183
KEEPVIVTP
0.090


65
NCENFQKTD
0.090


212
DFECVTPKL
0.090


12
RSLASTLDC
0.075


178
NSDNYKEEP
0.075


5
RSFCGKLRS
0.075


316
NSSSNDLEV
0.075


350
SSYENLLRT
0.075


195
QSLVKVLKT
0.075


194
KQSLVKVLK
0.060


287
PTFCTPGLK
0.050


57
DVNIPELSN
0.050


112
LPNPPQAVN
0.050


280
YTNSPLVPT
0.050


106
YIVSQVLPN
0.050


224
GISEYTMCL
0.050


154
REYFQKYGY
0.050


257
RLNDNVFAT
0.050


369
EDILQLLSK
0.050


55
KDDVNIPEL
0.050


152
KIREYFQKY
0.050


366
KIPEDILQL
0.050


67
ENFQKTDVK
0.050


75
KDDLSDPPV
0.050


214
ECVTPKLEH
0.050







V5-HLA-A1-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









2
VASSCISEK
0.200


5
SCISEKSPR
0.100


6
CISEKSPRS
0.020


3
ASSCISEKS
0.015


7
ISEKSPRSP
0.014


9
EKSPRSPQL
0.010


4
SSCISEKSP
0.002


1
PVASSCISE
0.001


8
SEKSPRSPQ
0.000







V6-HLA-A1-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









8
DAESRLNDN
0.900


6
AIDAESRLN
0.500


2
KSEEAIDAE
0.135


3
SEEAIDAES
0.090


5
EAIDAESRL
0.010


4
EEAIDAESR
0.050


1
NKSEEAIDA
0.003


7
IDAESRLND
0.000


9
AESRLNDNV
0.000







V10-HLA-A1-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus eight.









2
IPEDILQKF
2.250


1
KIPEDILQK
1.000


12
WIYPTQKLN
0.100


13
IYPTQKLNK
0.050


6
ILQKFQWIY
0.050


5
DILQKFQWI
0.010


10
FQWIYPTQK
0.003


3
PEDILQKFQ
0.003


15
PTQKLNKMR
0.003


4
EDILQKFQW
0.003


14
YPTQKLNKM
0.003


8
QKFQWIYPT
0.001


9
KFQWIYPTQ
0.001


11
QWIYPTQKL
0.001


7
LQKFQWIYP
0.000







V12-HLA-A1-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus eight.









4
DGEESLLSK
22.500


5
GEESLLSKY
2.250


2
ALDGEESLL
0.500


7
ESLLSKYNS
0.030


8
SLLSKYNSN
0.010


1
RALDGEESL
0.010


3
LDGEESLLS
0.003


6
EESLLSKYN
0.001


















TABLE IX





Start
Subsequence
Score















V1-HLA-A1-10mers-193P1EIB


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


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


position for each peptide is the start position plus nine.









219
KLEHFGISEY
45.000


126
RLENQEGIDF
45.000


33
ESDFEDYPMR
15.000


341
LTDPSSPTIS
12.500


147
SMDIMKIREY
12.500


30
DGEESDFEDY
11.250


390
AVPPSKRFLK
10.000


78
LSDPPVASSC
7.500


173
EQEAINSDNY
6.750


36
FEDYPMRILY
6.250


323
EVEDRTSLVL
4.500


153
IREYFQKYGY
4.500


232
LNEDYTMGLK
4.500


60
IPELSNCENF
2.250


253
DTESRLNDNV
2.250


277
DAEYTNSPLV
1.800


247
KSEEAIDTES
1.350


367
IPEDILQLLS
1.125


357
RTPTPPEVTK
1.000


305
LVSTNYPLSK
1.000


62
ELSNCENFQK
1.000


19
DCETARLQRA
0.900


321
DLEVEDRTSL
0.900


65
NCENFQKTDV
0.900


301
NSIALVSTNY
0.750


333
NSDTCFENLT
0.750


178
NSDNYKEEPV
0.750


98
LSDFGLERYI
0.750


225
ISEYTMCLNE
0.675


129
NQEGIDFIKA
0.675


71
KTDVKDDLSD
0.625


17
TLDCETARLQ
0.500


263
FATPSPIIQQ
0.500


210
MDDFECVTPK
0.500


132
GIDFIKATKV
0.500


348
TISSYENLLR
0.500


289
FCTPGLKIPS
0.500


280
YTNSPLVPTF
0.500


97
QLSDFGLERY
0.500


248
SEEAIDTESR
0.450


368
PEDILQLLSK
0.250


190
TPPTKQSLVK
0.250


189
VTPPTKQSLV
0.250


128
ENQEGIDFIK
0.250


251
AIDTESRLND
0.250


46
DLHSEVQTLK
0.200


266
PSPIIQQLEK
0.150


85
SSCISGKSPR
0.150


15
ASTLDCETAR
0.150


318
SSNDLEVEDR
0.150


271
QQLEKSDAEY
0.150


48
HSEVQTLKDD
0.135


343
DPSSPTISSY
0.125


258
LNDNVFATPS
0.125


233
NEDYTMGLKN
0.125


319
SNDLEVEDRT
0.125


380
SNLATPIAIK
0.100


188
IVTPPTKQSL
0.100


185
EPVIVTPPTK
0.100


110
QVLPNPPQAV
0.100


214
ECVTPKLEHF
0.100


27
RALDGEESDF
0.100


131
EGIDFIKATK
0.100


382
LATPIAIKAV
0.100


329
SLVLNSDTCF
0.100


215
CVTPKLEHFG
0.100


117
QAVNLLDKAR
0.100


389
KAVPPSKRFL
0.100


102
GLERYIVSQV
0.090


272
QLEKSDAEYT
0.090


337
CFENLTDPSS
0.090


379
NSNLATPIAI
0.075


275
KSDAEYTNSP
0.075


94
RSPQLSDFGL
0.075


349
ISSYENLLRT
0.075


282
NSPLVPTFCT
0.075


291
TPGLKIPSTK
0.050


331
VLNSDTCFEN
0.050


54
LKDDVNIPEL
0.050


381
NLATPIAIKA
0.050


16
STLDCETARL
0.050


44
LYDLHSEVQT
0.050


236
YTMGLKNARN
0.050


28
ALDGEESDFE
0.050


112
LPNPPQAVNL
0.050


75
KDDLSDPPVA
0.050


286
VPTFCTPGLK
0.050


121
LLDKARLENQ
0.050


170
SVHEQEAINS
0.050


231
CLNEDYTMGL
0.050


141
VLMEKNSMDI
0.050


74
VKDDLSDPPV
0.050


150
IMKIREYFQK
0.050


120
NLLDKARLEN
0.050


209
KMDDFECVTP
0.050


290
CTPGLKIPST
0.050


136
IKATKVLMEK
0.050


183
KEEPVIVTPP
0.045


351
SYENLLRTPT
0.045


35
DFEDYPMRIL
0.045







V5-HLA-A1-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









5
SSCISEKSPR
0.150


8
ISEKSPRSPQ
0.135


6
SCISEKSPRS
0.020


2
PVASSCISEK
0.020


3
VASSCISEKS
0.010


10
EKSPRSPQLS
0.005


4
ASSCISEKSP
0.002


7
CISEKSPRSP
0.001


1
PPVASSCISE
0.000


9
SEKSPRSPQL
0.000







V6-HLA-A1-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









3
KSEEAIDAES
2.700


9
DAESRLNDNV
0.900


4
SEEAIDAESR
0.450


7
AIDAESRLND
0.250


6
EAIDAESRLN
0.010


1
NNKSEEAIDA
0.001


10
AESRLNDNVF
0.001


8
IDAESRLNDN
0.001


5
EEAIDAESRL
0.001


2
NKSEEAIDAE
0.000







V10-HLA-A1-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus nine.









13
WIYPTQKLNK
10.000


1
TKIPEDILQK
0.500


6
DILQKFQWIY
0.500


3
IPEDILQKFQ
0.225


2
KIPEDILQKF
0.100


15
YPTQKLNKMR
0.025


4
PEDILQKFQW
0.013


10
KFQWIYPTQK
0.010


9
QKFQWIYPTQ
0.001


7
ILQKFQWIYP
0.001


14
IYPTQKLNKM
0.001


12
QWIYPTQKLN
0.001


5
EDILQKFQWI
0.001


8
LQKFQWIYPT
0.000


11
FQWIYPTQKL
0.000







V12-HLA-A1-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus nine.









5
DGEESLLSKY
11.250


3
ALDGEESLLS
2.500


4
LDGEESLLSK
0.050


6
GEESLLSKYN
0.045


8
ESLLSKYNSN
0.015


9
SLLSKYNSNL
0.010


2
RALDGEESLL
0.010


7
EESLLSKYNS
0.001


1
QRALDGEESL
0.001


















TABLE X





Start
Subsequence
Score















V1-HLA-A0201-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









43
ILYDLHSEV
1551.288


257
RLNDNVFAT
407.580


111
VLPNPPQAV
118.238


366
KIPEDILQL
96.947


209
KMDDFECVT
48.131


374
LLSKYNSNL
36.316


340
NLTDPSSPT
30.553


304
ALVSTNYPL
21.362


229
TMCLNEDYT
14.504


224
GISEYTMCL
12.043


17
TLDCETARL
8.545


10
KLRSLASTL
5.682


140
KVLMEKNSM
5.629


199
KVLKTPKCA
5.629


39
YPMRILYDL
5.459


295
KIPSTKNSI
5.021


278
AEYTNSPLV
4.328


46
DLHSEVQTL
3.685


261
NVFATPSPI
3.378


348
TISSYENLL
2.937


322
LEVEDRTSL
2.895


329
SLVLNSDTC
2.434


208
LKMDDFECV
2.319


383
ATPIAIKAV
2.222


390
AVPPSKRFL
2.056


110
QVLPNPPQA
1.608


350
SSYENLLRT
1.468


66
CENFQKTDV
1.352


200
VLKTPKCAL
1.271


280
YTNSPLVPT
1.095


283
SPLVPTFCT
1.044


314
KTNSSSNDL
1.038


207
ALKMDDFEC
1.009


119
VNLLDKARL
0.877


270
IQQLEKSDA
0.856


142
LMEKNSMDI
0.820


95
SPQLSDFGL
0.809


371
ILQLLSKYN
0.697


352
YENLLRTPT
0.667


254
TESRLNDNV
0.663


133
IDFIKATKV
0.608


145
KNSMDIMKI
0.548


52
QTLKDDVNI
0.536


189
VTPPTKQSL
0.504


231
CLNEDYTMG
0.458


316
NSSSNDLEV
0.454


190
TPPTKQSLV
0.454


195
QSLVKVLKT
0.414


373
QLLSKYNSN
0.414


103
LERYIVSQV
0.402


300
KNSIALVST
0.392


141
VLMEKNSMD
0.384


64
SNCENFQKT
0.379


114
NPPQAVNLL
0.321


265
TPSPIIQQL
0.321


378
YNSNLATPI
0.313


158
QKYGYSPRV
0.309


380
SNLATPIAI
0.252


202
KTPKCALKM
0.242


307
STNYPLSKT
0.238


286
VPTFCTPGL
0.237


97
QLSDFGLER
0.232


223
FGISEYTMC
0.224


50
EVQTLKDDV
0.224


20
CETARLQRA
0.222


230
MCLNEDYTM
0.204


130
QEGIDFIKA
0.184


328
TSLVLNSDT
0.180


282
NSPLVPTFC
0.178


45
YDLHSEVQT
0.176


14
LASTLDCET
0.176


7
FCGKLRSLA
0.149


36
FEDYPMRIL
0.144


367
IPEDILQLL
0.143


331
VLNSDTCFE
0.139


75
KDDLSDPPV
0.135


131
EGIDEIKAT
0.131


128
ENQEGIDFI
0.130


58
VNIPELSNC
0.127


12
RSLASTLDC
0.120


323
EVEDRTSLV
0.120


382
LATPIAIKA
0.117


324
VEDRTSLVL
0.116


168
KNSVHEQEA
0.114


291
TPGLKIPST
0.112


135
FIKATKVLM
0.110


106
YIVSQVLPN
0.108


341
LTDPSSPTI
0.099


55
KDDVNIPEL
0.096


250
EAIDTESRL
0.091


9
GKLRSLAST
0.088


399
KHGQNIRDV
0.078


117
QAVNLLDKA
0.078


298
STKNSIALV
0.078


320
NDLEVEDRT
0.077


232
LNEDYTMGL
0.062


70
QKTDVKDDL
0.060


99
SDFGLERYI
0.059


354
NLLRTPTPP
0.055


237
TMGLKNARN
0.054







X-V5-HLA-A0201-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









6
CISEKSPRS
0.042


9
EKSPRSPQL
0.002


2
VASSCISEK
0.001


3
ASSCISEKS
0.000


5
SCISEKSPR
0.000


4
SSCISEKSP
0.000


1
PVASSCISE
0.000


8
SEKSPRSPQ
0.000


7
ISEKSPRSP
0.000







V6-HLA-A0201-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









9
AESRLNDNV
0.663


5
EAIDAESRL
0.091


1
NKSEEAIDA
0.028


6
AIDAESRLN
0.001


7
IDAESRLND
0.000


2
KSEEAIDAE
0.000


3
SEEAIDAES
0.000


8
DAESRLNDN
0.000


4
EEAIDAESR
0.000







V10-HLA-A0201-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus eight.









5
DILQKFQWI
4.160


6
ILQKFQWIY
1.480


14
YPTQKLNKM
0.343


12
WIYPTQKLN
0.151


8
QKFQWIYPT
0.088


1
KIPEDILQK
0.068


10
FQWIYPTQK
0.058


11
QWIYPTQKL
0.003


7
LQKFQWIYP
0.001


2
IPEDILQKF
0.000


9
KFQWIYPTQ
0.000


4
EDILQKFQW
0.000


3
PEDILQKFQ
0.000


15
PTQKLNKMR
0.000


13
IYPTQKLNK
0.000







V12-HLA-A0201-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus eight.









2
ALDGEESLL
8.545


1
RALDGEESL
2.205


8
SLLSKYNSN
0.414


3
LDGEESLLS
0.001


6
EESLLSKYN
0.001


5
GEESLLSKY
0.000


7
ESLLSKYNS
0.000


4
DGEESLLSK
0.000


















TABLE XI





Start
Subsequence
Score















V1-HLA-A0201-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









141
VLMEKNSMDI
269.051


366
KIPEDILQLL
96.947


231
CLNEDYTMGL
87.586


373
QLLSKYNSNL
79.041


340
NLTDPSSPTI
42.774


42
RILYDLHSEV
35.385


199
KVLKTPKCAL
24.206


110
QVLPNPPQAV
22.517


102
GLERYIVSQV
10.238


322
LEVEDRTSLV
9.426


355
LLRTPTPPEV
8.986


374
LLSKYNSNLA
8.446


13
SLASTLDCET
7.452


194
KQSLVKVLKT
6.082


381
NLATPIAIKA
4.968


228
YTMCLNEDYT
4.747


16
STLDCETARL
4.501


132
GIDFIKATKV
3.825


382
LATPIAIKAV
3.777


229
TMCLNEDYTM
3.588


188
IVTPPTKQSL
3.178


285
LVPTFCTPGL
3.178


207
ALKMDDFECV
2.266


127
LENQEGIDFI
2.138


162
YSPRVKKNSV
2.088


118
AVNLLDKARL
1.869


303
IALVSTNYPL
1.866


51
VQTLKDDVNI
1.798


189
VTPPIKQSLV
1.642


206
CALKMDDFEC
1.481


216
VTPKLEHFGI
1.429


261
NVFATPSPII
1.385


272
QLEKSDAEYT
1.285


130
QEGIDFIKAT
1.266


45
YDLHSEVQTL
1.161


269
IIQQLEKSDA
1.161


389
KAVPPSKRFL
1.142


157
FQKYGYSPRV
1.135


120
NLLDKARLEN
1.130


295
KIPSTKNSIA
0.980


332
LNSDTCFENL
0.905


94
RSPQLSDFGL
0.809


331
VLNSDTCFEN
0.735


264
ATPSPIIQQL
0.682


220
LEHFGISEYT
0.664


49
SEVQTLKDDV
0.663


223
FGISEYTMCL
0.641


109
SQVLPNPPQA
0.504


315
TNSSSNDLEV
0.454


208
LKMDDFECVT
0.416


97
QLSDFGLERY 
0.344


282
NSPLVPTFCT
0.282


74
VKDDLSDPPV
0.269


290
CTPGLKIPST
0.238


5
RSFCGKLRSL
0.237


112
LPNPPQAVNL
0.237


296
IPSTKNSIAL
0.237


152
KIREYFQKYG
0.234


34
SDFEDYPMRI
0.220


54
LKDDVNIPEL
0.190


306
VSTNYPLSKT
0.190


349
ISSYENLLRT
0.190


142
LMEKNSMDIM
0.180


281
TNSPLVPTFC
0.178


69
FQKTDVKDDL
0.171


63
LSNCENFQKT
0.157


358
TPTPPEVTKI
0.157


99
SDFGLERYIV
0.147


371
ILQLLSKYNS
0.127


276
SDAEYTNSPL
0.122


168
KNSVHEQEAI
0.117


257
RLNDNVFATP
0.116


271
QQLEKSDAEY
0.115


304
ALVSTNYPLS
0.112


9
GKLRSLASTL
0.110


327
RTSLVLNSDT
0.104


321
DLEVEDRTSL
0.103


346
SPTISSYENL
0.102


224
GISEYTMCLN
0.097


178
NSDNYKEEPV
0.089


20
CETARLQRAL
0.083


133
IDFIKATKVL
0.077


398
LKHGQNIRDV
0.076


57
DVNIPELSNC
0.075


329
SLVLNSDTCF
0.075


333
NSDTCFENLT
0.074


372
LQLLSKYNSN
0.071


365
TKIPEDILQL
0.068


379
NSNLATPIAI
0.068


43
ILYDLHSEVQ
0.067


209
KMDDFECVTP
0.062


129
NQEGIDFIKA
0.061


378
YNSNLATPIA
0.061


328
TSLVLNSDTC
0.059


14
LASTLDCETA
0.057


354
NLLRTPTPPE
0.055


77
DLSDPPVASS
0.053


111
VLPNPPQAVN
0.052


256
SRLNDNVFAT
0.051


98
LSDFGLERYI
0.051







V5-HLA-A0201-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









9
SEKSPRSPQL
0.015


7
CISEKSPRSP
0.002


3
VASSCISEKS
0.001


6
SCISEKSPRS
0.000


5
SSCISEKSPR
0.000


4
ASSCISEKSP
0.000


2
PVASSCISEK
0.000


8
ISEKSPRSPQ
0.000


10
EKSPRSPQLS
0.000


1
PPVASSCISE
0.000







V6-HLA-A0201-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









5
EEAIDAESRL
0.031


9
DAESRLNDNV
0.002


8
IDAESRLNDN
0.002


1
NNKSEEAIDA
0.001


7
AIDAESRLND
0.001


10
AESRLNDNVF
0.001


3
KSEEAIDAES
0.000


2
NKSEEAIDAE
0.000


6
EAIDAESRLN
0.000


4
SEEAIDAESR
0.000







V10-HLA-A0201-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus nine.









11
FQWIYPTQKL
82.694


2
KIPEDILQKF
0.338


7
ILQKFQWIYP
0.237


8
LQKFQWIYPT
0.103


6
DILQKFQWIY
0.033


13
WIYPTQKLNK
0.030


5
EDILQKFQWI
0.011


14
IYPTQKLNKM
0.003


15
YPTQKLNKMR
0.000


3
IPEDILQKFQ
0.000


9
QKFQWIYPTQ
0.000


4
PEDILQKFQW
0.000


1
TKIPEDILQK
0.000


10
KFQWIYPTQK
0.000


12
QWIYPTQKLN
0.000







V12-HLA-A0201-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus nine.









9
SLLSKYNSNL
79.041


2
RALDGEESLL
4.501


3
ALDGEESLLS
0.030


1
QRALDGEESL
0.001


6
GEESLLSKYN
0.001


4
LDGEESLLSK
0.000


8
ESLLSKYNSN
0.000


7
EESLLSKYNS
0.000


5
DGEESLLSKY
0.000


















TABLE XII





Start
Subsequence
Score















V1-HLA-A3-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









239
GLKNARNNK
60.000


381
NLATPIAIK
45.000


97
QLSDFGLER
24.000


152
KIREYFQKY
16.200


132
GIDFIKATK
9.000


129
NQEGIDFIK
4.050


397
FLKHGQNIR
4.000


272
QLEKSDAEY
4.000


387
AIKAVPPSK
3.000


28
ALDGEESDF
3.000


194
KQSLVKVLK
2.700


137
KATKVLMEK
2.700


304
ALVSTNYPL
2.700


197
LVKVLKTPK
2.000


374
LLSKYNSNL
1.800


10
KLRSLASTL
1.800


224
GISEYTMCL
1.620


142
LMEKNSMDI
1.200


43
ILYDLHSEV
1.000


209
KMDDFECVT
0.900


200
VLKTPKCAL
0.900


257
RLNDNVFAT
0.900


366
KIPEDILQL
0.810


267
SPIIQQLEK
0.600


207
ALKMDDFEC
0.600


306
VSTNYPLSK
0.600


391
VPPSKRFLK
0.600


302
SIALVSTNY
0.600


17
TLDCETARL
0.600


46
DLHSEVQTL
0.540


358
TPTPPEVTK
0.450


236
YTMGLKNAR
0.450


215
CVTPKLEHF
0.450


219
KLEHFGISE
0.360


186
PVIVTPPTK
0.300


83
VASSCISGK
0.300


111
VLPNPPQAV
0.300


63
LSNCENFQK
0.300


330
LVLNSDTCF
0.300


16
STLDCETAR
0.300


261
NVFATPSPI
0.300


228
YTMCLNEDY
0.300


329
SLVLNSDTC
3.000


2
DPIRSFCGK
0.270


102
GLERYIVSQ
0.270


370
DILQLLSKY
0.270


118
AVNLLDKAR
0.200


116
PQAVNLLDK
0.180


402
QNIRDVSNK
0.180


295
KIPSTKNSI
0.180


348
TISSYENLL
0.180


154
REYFQKYGY
0.180


160
YGYSPRVKK
0.150


287
PTFCTPGLK
0.150


340
NLTDPSSPT
0.150


149
DIMKIREYF
0.135


211
DDFECVTPK
0.135


157
FQKYGYSPR
0.120


31
GEESDFEDY
0.108


389
KAVPPSKRF
0.101


229
TMCLNEDYT
0.100


151
MKIREYFQK
0.090


140
KVLMEKNSM
0.090


314
KTNSSSNDL
0.090


205
KCALKMDDF
0.090


293
GLKIPSTKN
0.090


175
EAINSDNYK
0.090


39
YPMRILYDL
0.061


67
ENFQKTDVK
0.060


202
KTPKCALKM
0.060


150
IMKIREYFQ
0.060


86
SCISGKSPR
0.060


53
TLKDDVNIP
0.060


47
LHSEVQTLK
0.045


199
KVLKTPKCA
0.045


110
QVLPNPPQA
0.045


364
VTKIPEDIL
0.045


141
VLME KNSMD
0.045


52
QTLKDDVNI
0.045


189
VTPPTKQSL
0.045


341
LTDPSSPTI
0.045


349
ISSYENLLR
0.040


121
LLDKARLEN
0.040


147
SMDIMKIRE
0.040


191
PPTKQSLVK
0.040


196
SLVKVLKTP
0.034


146
NSMDIMKIR
0.034


201
LKTPKCALK
0.030


231
CLNEDYTMG
0.030


373
QLLSKYNSN
0.030


34
SDFEDYPMR
0.030


24
RLQRALDGE
0.030


98
LSDFGLERY
0.030


354
NLLRTPTPP
0.030


355
LLRTPTPPE
0.030


13
SLASTLDCE
0.030


363
EVTKIPEDI
0.027


369
EDILQLLSK
0.027


233
NEDYTMGLK
0.027


319
SNDLEVEDR
0.024







V5-HLA-A3-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









2
VASSCISEK
0.300


5
SCISEKSPR
0.060


6
CISEKSPRS
0.006


1
PVASSCISE
0.000


3
ASSCISEKS
0.000


9
EKSPRSPQL
0.000


8
SEKSPRSPQ
0.000


4
SSCISEKSP
0.000


7
ISEKSPRSP
0.000







V6-HLA-A3-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









4
EEAIDAESR
0.004


5
EAIDAESRL
0.003


2
KSEEAIDAE
0.001


1
NKSEEAIDA
0.001


9
AESRLNDNV
0.001


6
AIDAESRLN
0.000


3
SEEAIDAES
0.000


8
DAESRLNDN
0.000


7
IDAESRLND
0.000







V10-HLA-A3-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus eight.









6
ILQKFQWIY
36.000


1
KIPEDILQK
27.000


10
FQWIYPTQK
9.000


5
DILQKFQWI
0.081


2
IPEDILQKF
0.045


13
IYPTQKLNK
0.040


15
PTQKLNKMR
0.010


12
WIYPTQKLN
0.007


8
QKFQWIYPT
0.007


14
YPTQKLNKM
0.003


11
QWIYPTQKL
0.001


7
LQKFQWIYP
0.001


4
EDILQKFQW
0.000


9
KFQWIYPTQ
0.000


3
PEDILQKFQ
0.000







V12-HLA-A3-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus eight.









2
ALDGEESLL
0.900


5
GEESLLSKY
0.054


8
SLLSKYNSN
0.030


4
DGEESLLSK
0.027


1
RALDGEESL
0.009


7
ESLLSKYNS
0.000


3
LDGEESLLS
0.000


6
EESLLSKYN
0.000


















TABLE XIII





Start
Subsequence
Score















V1-HLA-A3-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









150
IMKIREYFQK
60.000


196
SLVKVLKTPK
30.000


200
VLKTPKCALK
20.000


219
KLEHFGISEY
18.000


62
ELSNCENFQK
18.000


305
LVSTNYPLSK
12.000


46
DLHSEVQTLK
9.000


390
AVPPSKRFLK
9.000


97
QLSDFGLERY
6.000


231
CLNEDYTMGL
5.400


401
GQNIRDVSNK
5.400


126
RLENQEGIDF
4.000


329
SLVLNSDTCF
3.000


102
GLERYIVSQV
2.700


373
QLLSKYNSNL
2.700


141
VLMEKNSMDI
2.700


357
RTPTPPEVTK
1.500


348
TISSYENLLR
0.800


366
KIPEDILQLL
0.608


207
ALKMDDFECV
0.600


147
SMDIMKI REY
0.600


381
NLATPIAIKA
0.600


387
AK AVPPSKR
0.600


340
NLTDPSSPTI
0.600


229
TMCLNEDYTM
0.600


386
IAIKAVPPSK
0.450


261
NVFATPSPII
0.450


199
KVLKTPKCAL
0.405


190
TPPTKQSLVK
0.400


82
PVASSCISGK
0.300


291
TPGLKIPSTK
0.300


142
LMEKNSMDIM
0.300


280
YTNSPLVPTF
0.300


271
QQLEKSDAEY
0.270


355
LLRTPTPPEV
0.200


374
LLSKYNSNLA
0.200


321
DLEVEDRTSL
0.180


380
SNLATPIAIK
0.135


143
MEKNSMDIMK
0.120


371
ILQLLSKYNS
0.120


239
GLKNARNNKS
0.120


96
PQLSDFGLER
0.108


13
SLASTLDCET
0.100


43
ILYDLHSEVQ
0.100


272
QLEKSDAEYT
0.100


159
KYGYSPRVKK
0.090


136
IKATKVLMEK
0.090


216
VTPKLEHFGI
0.090


188
IVTPPTKQSL
0.090


257
RLNDNVFATP
0.900


185
EPVIVTPPTK
0.090


128
ENQEGIDFIK
0.081


264
ATPSPIIQQL
0.068


110
QVLPNPPQAV
0.068


1
MDPIRSFCGK
0.060


210
MDDFECVTPK
0.060


115
PPQAVNLLDK
0.060


304
ALVSTNYPLS
0.060


295
KIPSTKNSIA
0.060


293
GLKIPSTKNS
0.060


53
TLKDDVNIPE
0.060


118
AVNLLDKARL
0.600


132
GIDFIKATKV
0.060


331
VLNSDTCFEN
0.060


174
QEAINSDNYK
0.060


66
CENFQKTDVK
0.060


10
KLRSLASTLD
0.060


286
VPTFCTPGLK
0.060


285
LVPTFCTPGL
0.060


318
SSNDLEVEDR
0.060


209
KMDDFECVTP
0.060


120
NLLDKARLEN
0.060


77
DLSDPPVASS
0.054


194
KQSLVKVLKT
0.054


129
NQEGIDFIKA
0.054


151
MKIREYFQKY
0.054


301
NSIALVSTNY
0.045


354
NLLRTPTPPE
0.045


16
STLDCETARL
0.045


287
PTFCTPGLKI
0.045


397
FLKHGQNIRD
0.040


323
EVEDRTSLVL
0.036


173
EQEAINSDNY
0.036


193
TKQSLVKVLK
0.030


238
MGLKNARNNK
0.030


28
ALDGEESDFE
0.030


42
RILYDLHSEV
0.030


117
QAVNLLDKAR
0.030


27
RALDGEESDF
0.030


111
VLPNPPQAVN
0.030


92
SPRSPQLSDF
0.030


121
LLDKARLENQ
0.030


358
TPTPPEVTKI
0.027


303
IALVSTNYPL
0.027


34
SDFEDYPMRI
0.027


363
EVTKIPEDIL
0.027


145
KNSMDIMKIR
0.027


69
FQKTDVKDDL
0.027


36
FEDYPMRILY
0.024


40
PMRILYDLHS
0.024







V5-HLA-A3-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









2
PVASSCISEK
0.300


5
SSCISEKSPR
0.020


9
SEKSPRSPQL
0.002


6
SCISEKSPRS
0.001


3
VASSCISEKS
0.001


7
CISEKSPRSP
0.000


8
ISEKSPRSPQ
0.000


1
PPVASSCISE
0.000


4
ASSCISEKSP
0.000


10
EKSPRSPQLS
0.000







V6-HLA-A3-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









4
SEEAIDAESR
0.012


10
AESRLNDNVF
0.006


7
AIDAESRLND
0.004


3
KSEEAIDAES
0.001


1
NNKSEEAIDA
0.001


9
DAESRLNDNV
0.001


5
EEAIDAESRL
0.001


8
IDAESRLNDN
0.000


2
NKSEEAIDAE
0.000


6
EAIDAESRLN
0.000







V10-HLA-A3-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus nine.









13
WIYPTQKLNK
30.000


2
KIPEDILQKF
2.025


6
DILQKFQWIY
1.620


10
KFQWIYPTQK
0.180


1
TKIPEDILQK
0.135


11
FQWIYPTQKL
0.135


8
LQKFQWIYPT
0.041


7
ILQKFQWIYP
0.040


15
YPTQKLNKMR
0.020


5
EDILQKFQWI
0.001


14
IYPTQKLNKM
0.000


4
PEDILQKFQW
0.000


9
QKFQWIYPTQ
0.000


3
IPEDILQKFQ
0.000


12
QWIYPTQKLN
0.000







V12-HLA-A3-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus nine.









9
SLLSKYNSNL
2.700


3
ALDGEESLLS
0.120


4
LDGEESLLSK
0.090


2
RALDGEESLL
0.009


5
DGEESLLSKY
0.003


1
QRALDGEESL
0.001


7
EESLLSKYNS
0.000


6
GEESLLSKYN
0.000


8
ESLLSKYNSN
0.000


















TABLE XIV





Start
Subsequence
Score















V1-HLA-A1101-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









197
LVKVLKTPK
2.000


194
KQSLVKVLK
1.800


129
NQEGIDFIK
1.800


239
GLKNARNNK
1.200


137
KATKVLMEK
1.200


132
GIDFIKATK
1.200


391
VPPSKRFLK
0.600


267
SPIIQQLEK
0.600


387
AIKAVPPSK
0.400


236
YTMGLKNAR
0.400


381
NLATPIAIK
0.400


186
PVIVTPPTK
0.300


16
STLDCETAR
0.300


118
AVNLLDKAR
0.200


287
PTFCTPGLK
0.200


358
TPTPPEVTK
0.200


83
VASSCISGK
0.200


97
QLSDFGLER
0.160


116
PQAVNLLDK
0.120


159
KYGYSPRVK
0.120


157
FQKYGYSPR
0.120


151
MKIREYFQK
0.090


140
KVLMEKNSM
0.090


175
EAINSDNYK
0.090


2
DPIRSFCGK
0.090


397
FLKHGQNIR
0.080


402
QNIRDVSNK
0.060


63
LSNCENFQK
0.060


202
KTPKCALKM
0.060


233
NEDYTMGLK
0.060


86
SCISGKSPR
0.060


199
KVLKTPKCA
0.045


160
YGYSPRVKK
0.040


191
PPTKQSLVK
0.040


306
VSTNYPLSK
0.040


261
NVFATPSPI
0.040


330
LVLNSDTCF
0.030


110
QVLPNPPQA
0.030


314
KTNSSSNDL
0.030


67
ENFQKTDVK
0.024


224
GISEYTMCL
0.024


19
DCETARLQR
0.024


366
KIPEDILQL
0.024


47
LHSEVQTLK
0.020


228
YTMCLNEDY
0.020


215
CVTPKLEHF
0.020


201
LKTPKCALK
0.020


369
EDILQLLSK
0.018


52
QTLKDDVNI
0.015


295
KIPSTKNSI
0.012


144
EKNSMDIMK
0.012


211
DDFECVTPK
0.012


304
ALVSTNYPL
0.012


10
KLRSLASTL
0.012


152
KIREYFQKY
0.012


298
STKNSIALV
0.010


189
VTPPTKQSL
0.010


341
LTDPSSPTI
0.010


364
VTKIPEDIL
0.010


396
RFLKHGQNI
0.009


142
LMEKNSMDI
0.008


349
ISSYENLLR
0.008


34
SDFEDYPMR
0.008


319
SNDLEVEDR
0.008


43
ILYDLHSEV
0.008


39
YPMRILYDL
0.008


154
REYFQKYGY
0.007


323
EVEDRTSLV
0.006


165
RVKKNSVHE
0.006


50
EVQTLKDDV
0.006


363
EVTKIPEDI
0.006


205
KCALKMDDF
0.006


217
TPKLEHFGI
0.006


95
SPQLSDFGL
0.006


270
IQQLEKSDA
0.006


230
MCLNEDYTM
0.006


383
ATPIAIKAV
0.005


389
KAVPPSKRF
0.005


200
VLKTPKCAL
0.004


146
NSMDIMKIR
0.004


382
LATPIAIKA
0.004


222
HFGISEYTM
0.004


272
QLEKSDAEY
0.004


28
ALDGEESDF
0.004


111
VLPNPPQAV
0.004


288
TFCTPGLKI
0.004


135
FIKATKVLM
0.004


388
IKAVPPSKR
0.004


17
TLDCETARL
0.004


374
LLSKYNSNL
0.004


348
TISSYENLL
0.004


181
NYKEEPVIV
0.004


302
SIALVSTNY
0.004


257
RLNDNVFAT
0.004


249
EEAIDTESR
0.004


292
PGLKIPSTK
0.003


71
KTDVKDDLS
0.003


357
RTPTPPEVT
0.003


117
QAVNLLDKA
0.003


327
RTSLVLNSD
0.003







V5-HLA-A1101-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









2
VASSCISEK
0.200


5
SCISEKSPR
0.060


1
PVASSCISE
0.000


6
CISEKSPRS
0.000


9
EKSPRSPQL
0.000


8
SEKSPRSPQ
0.000


3
ASSCISEKS
0.000


4
SSCISEKSP
0.000


7
ISEKSPRSP
0.000







V6-HLA-A1101-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









4
EEAIDAESR
0.004


5
EAIDAESRL
0.001


9
AESRLNDNV
0.001


1
NKSEEAIDA
0.000


2
KSEEAIDAE
0.000


8
DAESRLNDN
0.000


3
SEEAIDAES
0.000


7
IDAESRLND
0.000


6
AIDAESRLN
0.000







V10-HLA-A1101-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus eight.









1
KIPEDILQK
2.400


10
FQWIYPTQK
1.200


13
IYPTQKLNK
0.800


15
PTQKLNKMR
0.010


6
ILQKFQWIY
0.008


14
YPTQKLNKM
0.002


2
IPEDILQKF
0.002


5
DILQKFQWI
0.002


7
LQKFQWIYP
0.001


9
KFQWIYPTQ
0.001


12
WIYPTQKLN
0.000


11
QWIYPTQKL
0.000


4
EDILQKFQW
0.000


8
QKFQWIYPT
0.000


3
PEDILQKFQ
0.000







V12-HLA-A1101-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus eight.









4
DGEESLLSK
0.012


1
RALDGEESL
0.009


2
ALDGEESLL
0.004


5
GEESLLSKY
0.002


8
SLLSKYNSN
0.001


3
LDGEESLLS
0.000


7
ESLLSKYNS
0.000


6
EESLLSKYN
0.000


















TABLE XV





Start
Subsequence
Score















V1-HLA-A11-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









390
AV PPSKRFLK
6.000


305
LVSTNYPLSK
4.000


357
TPTP PEVTK
3.000


401
GQNIRDVSNK
1.800


159
KYGYSPRVKK
1.200


150
IMKIREYFQK
1.200


196
SLVKVLKTPK
0.600


200
VLKTPKCALK
0.400


190
TPPTKQ SLVK
0.400


62
ELSNCENFQK
0.360


386
IAIKAVPPSK
0.300


291
TPGLKIPSTK
0.200


286
VPTFCTPGL K
0.200


82
PVASSCISGK
0.200


396
RFLKHGQ NIR
0.180


348
TISSYENLLR
0.160


46
DLHSEVQTLK
0.120


143
MEKNSMDIMK
0.120


199
KVLKTPKCAL
0.090


185
EPVIVTPPTK
0.090


387
AIKAVPPSKR
0.080


380
SNLATPIAIK
0.060


174
QEAI NSDNYK
0.060


66
CENFQKTDVK
0.060


136
IKATKVLMEK
0.040


115
PPQAVNLLDK
0.040


156
YFQKYGYSPR
0.040


261
NVFATPSPII
0.040


232
LNEDYTMGLK
0.040


96
PQLSDFGLER
0.036


128
ENQEGIDFIK
0.036


238
MGLKNARNNK
0.030


110
QVLPNPPQAV
0.030


117
QAVNLLDKAR
0.030


216
VTPKLEHFGI
0.030


235
DYTMGLKNAR
0.024


126
RLENQEGIDF
0.024


188
IVTPPTKQSL
0.020


193
TKQSLVKVLK
0.020


1
MDPIRSFCGK
0.020


210
MDDFECVTPK
0.020


118
AVNLLDKARL
0.020


285
LVPTFCTPGL
0.020


42
RILYDLHSEV
0.018


141
VLMEKNSMDI
0.016


16
STLDCETARL
0.015


145
KNSMDIMKIR
0.012


129
NQEGIDFIKA
0.012


219
KLEHFGISEY
0.012


132
GIDFIKATKV
0.012


368
PEDILQLLSK
0.012


366
KIPEDILQLL
0.012


295
KIPSTKNSIA
0.012


323
EVEDRTSLVL
0.012


248
SEEAIDTESR
0.012


102
GLERYIVSQV
0.012


377
KYNSNLATPI
0.012


264
ATPSPIIQQL
0.010


280
YTNSPLVPTF
0.010


189
VTPPT KQSLV
0.010


27
RALDGEESDF
0.009


131
EGIDFIKATK
0.009


140
KVLMEKNSMD
0.009


109
SQVLPNPPQA
0.009


271
QQLEKSDAEY
0.009


231
CLNEDYTMGL
0.008


18
LDCETARLQR
0.008


381
NLATPIAIKA
0.008


229
TMCLNEDYTM
0.008


51
VQTLKDDVNI
0.006


373
QLLSK YNSNL
0.006


69
FQKTDVKDDL
0.006


303
IALVSTNYPL
0.006


329
SLVLNSDTCF
0.006


124
KARLENQEGI
0.006


363
EVTKIPEDIL
0.006


71
KTDVKDDLSD
0.006


157
FQKYGYSPRV
0.006


165
RVKKNSVHEQ
0.006


158
QKYGYSPRVK
0.004


266
PSPIIQQLEK
0.004


269
IIQQLEKSDA
0.004


287
PTFCTPGLKI
0.004


85
SSCISGKSPR
0.004


39
YPMRILYDLH
0.004


374
LLSKYNSNLA
0.004


355
LLRTPTPPEV
0.004


3
PIRSFCGKLR
0.004


296
IPSTKNSIAL
0.004


15
ASTLDCETAR
0.004


97
QLSDFGLERY
0.004


207
ALKMDDFECV
0.004


170
SVHEQEAINS
0.004


318
SSNDLEVEDR
0.004


142
LMEKNSMDIM
0.004


391
VPPSKRFLKH
0.004


340
NLTDPSSPTI
0.004


194
KQSLVKVLKT
0.004


105
RYIVSQVLPN
0.004


314
KTNSSSNDLE
0.003







V5-HLA-A1101-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









2
PVASSCISEK
0.200


5
SSCISEKSPR
0.004


9
SEKSPRSPQL
0.001


6
SCISEKSPRS
0.000


3
VASSCISEKS
0.000


1
PPVASSCISE
0.000


7
CISEKSPRSP
0.000


8
ISEKSPRSPQ
0.000


4
ASSCISEKSP
0.000


10
EKSPRSPQLS
0.000







V6-HLA-A1101-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









4
SEEAIDAESR
0.012


1
NNKSEEAIDA
0.001


7
AIDAESRLND
0.001


9
DAESRLNDNV
0.001


10
AESRLNDNVF
0.001


5
EEAIDAESRL
0.000


3
KSEEAIDAES
0.000


8
IDAESRLNDN
0.000


2
NKSEEAIDAE
0.000


6
EAIDAESRLN
0.000







V10-HLA-A1101-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus nine.









13
WIYPTQKLNK
1.600


10
KFQWIYPTQK
0.600


1
TKIPEDILQK
0.060


15
YPTQKLNKMR
0.020


2
KIPEDILQKF
0.012


11
FQWIYPTQKL
0.012


14
IYPTQKLNKM
0.004


6
DILQKFQWIY
0.004


8
LQKFQWIYPT
0.001


7
ILQKFQWIYP
0.001


4
PEDILQKFQW
0.000


3
IPEDILQKFQ
0.000


5
EDILQKFQWI
0.000


9
QKFQWIYPTQ
0.000


12
QWIYPTQKLN
0.000







V12-HLA-A1101-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus nine.









4
LDGEESLLSK
0.040


2
RALDGEESLL
0.009


9
SLLSKYNSNL
0.006


3
ALDGEESLLS
0.001


1
QRALDGEESL
0.000


6
GEESLLSKYN
0.000


5
DGEESLLSKY
0.000


7
EESLLSKYNS
0.000


8
ESLLSKYNSN
0.000


















TABLE XVI





Start
Subsequence
Score















V1-HLA-A24-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









212
DFECVTPKL
46.200


134
DFIKATKVL
30.000


6
SFCGKLRSL
20.000


396
RFLKHGQNI
18.000


366
KIPEDILQL
14.400


314
KTNSSSNDL
14.400


367
IPEDILQLL
12.096


10
KLRSLASTL
9.600


35
DFEDYPMRI
9.000


189
VTPPTKQSL
8.640


39
YPMRILYDL
8.400


265
TPSPIIQQL
8.064


309
NYPLSKTNS
7.500


114
NPPQAVNLL
7.200


250
EAIDTESRL
7.200


389
KAVPPSKRF
7.200


390
AVPPSKRFL
7.200


232
LNEDYTMGL
7.200


161
GYSPRVKKN
6.600


95
SPQLSDFGL
6.000


119
VNLLDKARL
6.000


304
ALVSTNYPL
6.000


277
DAEYTNSPL
6.000


181
NYKEEPVIV
6.000


288
TFCTPGLKI
5.500


235
DYTMGLKNA
5.000


262
VFATPSPII
5.000


155
EYFQKYGYS
5.000


224
GISEYTMCL
4.800


46
DLHSEVQTL
4.800


21
ETARLQRAL
4.800


333
NSDTCFENL
4.800


348
TISSYENLL
4.800


149
DIMKIREYF
4.200


200
VLKTPKCAL
4.000


286
VPTFCTPGL
4.000


364
VTKIPEDIL
4.000


17
TLDCETARL
4.000


205
KCALKMDDF
4.000


374
LLSKYNSNL
4.000


295
KIPSTKNSI
3.600


330
LVLNSDTCF
3.000


244
RNNKSEEAI
3.000


281
TNSPLVPTF
2.880


222
HFGISEYTM
2.500


215
CVTPKLEHF
2.400


255
ESRLNDNVF
2.400


145
KNSMDIMKI
2.200


28
ALDGEESDF
2.000


128
ENQEGIDFI
1.800


140
KVLMEKNSM
1.800


202
KTPKCALKM
1.650


380
SNLATPIAI
1.500


105
RYIVSQVLP
1.500


142
LMEKNSMDI
1.500


169
NSVHEQEAI
1.500


80
DPPVASSCI
1.500


52
QTLKDDVNI
1.500


377
KYNSNLATP
1.500


363
EVTKIPEDI
1.400


341
LTDPSSPTI
1.200


378
YNSNLATPI
1.200


180
DNYKEEPVI
1.000


217
TPKLEHFGI
1.000


159
KYGYSPRVK
1.000


261
NVFATPSPI
1.000


337
CFENLTDPS
0.900


351
SYENLLRTP
0.900


55
KDDVNIPEL
0.880


230
MCLNEDYTM
0.750


38
DYPMRILYD
0.750


322
LEVEDRTSL
0.720


193
TKQSLVKVL
0.720


113
PNPPQAVNL
0.720


104
ERYIVSQVL
0.672


70
QKTDVKDDL
0.672


227
EYTMCLNED
0.660


347
PTISSYENL
0.600


33
ESDFEDYPM
0.500


44
LYDLHSEVQ
0.500


135
FIKATKVLM
0.500


279
EYTNSPLVP
0.500


100
DFGLERYIV
0.500


90
GKSPRSPQL
0.480


3
PIRSFCGKL
0.440


324
VEDRTSLVL
0.400


297
PSTKNSIAL
0.400


36
FEDYPMRIL
0.400


152
KIREYFQKY
0.380


91
KSPRSPQLS
0.360


257
RLNDNVFAT
0.360


199
KVLKTPKCA
0.300


357
RTPTPPEVT
0.300


12
RSLASTLDC
0.300


127
LENQEGIDF
0.300


168
KNSVHEQEA
0.264


209
KMDDFECVT
0.240


185
EPVIVTPPT
0.210


282
NSPLVPTFC
0.210


400
HGQNIRDVS
0.210







V5-HLA-A24-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









9
EKSPRSPQL
0.480


3
ASSCISEKS
0.154


6
CISEKSPRS
0.120


7
ISEKSPRSP
0.015


5
SCISEKSPR
0.015


2
VASSCISEK
0.011


4
SSCISEKSP
0.010


8
SEKSPRSPQ
0.001


1
PVASSCISE
0.001







V6-HLA-A24-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









5
EAIDAESRL
7.200


8
DAESRLNDN
0.180


6
AIDAESRLN
0.100


2
KSEEAIDAE
0.036


3
SEEAIDAES
0.023


9
AESRLNDNV
0.012


1
NKSEEAIDA
0.012


7
IDAESRLND
0.001


4
EEAIDAESR
0.001







V10-HLA-A24-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus eight.









11
QWIYPTQKL
7.920


2
IPEDILQKF
6.653


5
DILQKFQWI
2.160


13
IYPTQKLNK
0.750


14
YPTQKLNKM
0.660


9
KFQWIYPTQ
0.210


6
ILQKFQWIY
0.150


12
WIYPTQKLN
0.120


1
KIPEDILQK
0.036


4
EDILQKFQW
0.015


7
LQKFQWIYP
0.010


8
QKFQWIYPT
0.010


10
FQWIYPTQK
0.010


15
PTQKLNKMR
0.002


3
PEDILQKFQ
0.000







V12-HLA-A24-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus eight.









1
RALDGEESL
14.400


2
ALDGEESLL
4.000


8
SLLSKYNSN
0.180


7
ESLLSKYNS
0.150


5
GEESLLSKY
0.020


4
DGEESLLSK
0.018


6
EESLLSKYN
0.012


3
LDGEESLLS
0.012


















TABLE XVII





Start
Subsequence
Score















V1-HLA-A24-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









38
DYPMRILYDL
420.000


377
KYNSNLATPI
180.000


35
DFEDYPMRIL
36.000


366
KIPEDILQLL
24.192


105
RYIVSQVLPN
15.000


389
KAVPPSKRFL
14.400


94
RSPQLSDFGL
12.000


199
KVLKTPKCAL
12.000


264
ATPSPIIQQL
10.080


351
SYENLLRTPT
9.000


309
NYPLSKTNSS
9.000


161
GYSPRVKKNS
8.400


5
RSFCGKLRSL
8.000


231
CLNEDYTMGL
7.200


16
STLDCETARL
7.200


112
LPNPPQAVNL
7.200


323
EV EDRTSLVL
7.200


27
RALDGEESDF
7.200


2
DPIRSFCGKL
6.600


227
EYTMCLNEDY
6.000


181
NYKEEPVIVT
6.000


321
DLEVEDRTSL
6.000


118
AVNLL DKARL
6.000


373
QLLSKYNSNL
6.000


126
RLENQ EGIDF
6.000


285
LVPTFCTPGL
6.000


223
FGISEYTMCL
6.000


303
IALVSTNYPL
6.000


188
IVTPPTKQSL
5.760


332
LNSDTCFENL
5.760


69
FQKTDVKDDL
5.600


44
LYDLHSEVQT
5.000


279
EYTNSPLVPT
5.000


296
IPSTKNSIAL
4.000


363
EVTKIPEDIL
4.000


89
SGKSPRSPQL
4.000


346
SPTISSYENL
4.000


134
DFIKATKVLM
3.750


280
YTNSPLVPTF
3.600


60
IPELSNCENF
3.000


214
ECVTPKLEHF
3.000


329
SLVLN SDTCF
3.000


124
KARL ENQEGI
2.000


92
SPRSPQLSDF
2.000


168
KNS VHEQEAI
2.000


141
VLMEKNSMDI
1.800


260
DNVFATPSPI
1.500


379
NSNLATPIAI
1.500


216
VTPKLEHFGI
1.500


358
TPTPPEVTKI
1.320


340
NLTDPSSPTI
1.200


98
LSDFGLERYI
1.200


159
KYGYSPRVKK
1.100


51
VQTLKDDVNI
1.000


261
NVFATPSPII
1.000


113
PNPPQAVNLL
0.864


337
CFENLTDPSS
0.750


142
LMEKNSMDIM
0.750


211
DDFECVTPKL
0.739


9
GKLRSLASTL
0.720


365
TKIPEDILQL
0.720


347
PTISSYENLL
0.720


45
YDLHSEVQTL
0.720


235
DYTMGLKNAR
0.720


103
LERYIVSQVL
0.672


6
SFCGKLRSLA
0.600


247
KSEEAIDTES
0.554


54
LKDDVNIPEL
0.528


155
EYFQKYGYSP
0.500


222
HFGISEYTMC
0.500


100
DFGLERYIVS
0.500


229
TMCLNEDYTM
0.500


276
SDAEYTNSPL
0.480


20
CETARLQRAL
0.480


192
PTKQ SLVKVL
0.480


313
SKTNSSSNDL
0.480


148
MDIMKIREYF
0.420


133
IDFIKATKVL
0.400


249
EEAIDTESRL
0.400


42
RILYDLHSEV
0.396


219
KLEHFGISEY
0.330


295
KIPSTKNSIA
0.300


137
KATKVLMEKN
0.264


300
KNSIALVSTN
0.240


254
TESRLNDNVF
0.240


395
KRFLK HGQNI
0.240


327
RTSLVL NSDT
0.240


63
LSNCENFQKT
0.238


194
KQSLVKVLKT
0.220


294
LKIPSTKNSI
0.216


110
QVLPNPPQAV
0.216


367
IPEDILQLLS
0.216


30
DGEESDFEDY
0.216


102
GLERYIVSQV
0.210


301
NSIALVSTNY
0.210


388
IKAVPPSKRF
0.200


271
QQLEKSDAEY
0.198


59
NIPELSNCEN
0.198


129
NQEGIDFIKA
0.198


120
NLLDKARLEN
0.198







V5-HLA-A24-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









9
SEKSPRSPQL
0.400


3
VASSCISEKS
0.154


6
SCISEKSPRS
0.150


8
ISEKSPRSPQ
0.015


10
EKSPRSPQLS
0.014


7
CISEKSPRSP
0.012


4
ASSCISEKSP
0.010


5
SSCISEKSPR
0.010


1
PPVASSCISE
0.002


2
PVASSCISEK
0.001







V6-HLA-A24-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









3
KSEEAIDAES
0.554


5
EEAIDAESRL
0.400


10
AESRLNDNVF
0.240


6
EAIDAESRLN
0.180


9
DAESRLNDNV
0.180


1
NNKSEEAIDA
0.100


8
IDAESRLNDN
0.014


7
AIDAESRLND
0.010


4
SEEAIDAESR
0.002


2
NKSEEAIDAE
0.001







V10-HLA-A24-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus nine.









14
IYPTQKLNKM
49.500


2
KIPEDILQKF
13.306


11
FQWIYPTQKL
5.280


5
EDILQKFQWI
0.216


10
KFQWIYPTQK
0.150


6
DILQKFQWIY
0.150


12
QWIYPTQKLN
0.150


8
LQKFQWIYPT
0.100


3
IPEDILQKFQ
0.022


7
ILQKFQWIYP
0.015


15
YPTQKLNKMR
0.012


13
WIYPTQKLNK
0.012


1
TKIPEDILQK
0.002


9
QKFQWIYPTQ
0.001


4
PEDILQKFQW
0.001







V12-HLA-A24-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus nine.









2
RALDGEESLL
14.400


9
SLLSKYNSNL
6.000


1
QRALDGEESL
0.400


5
DGEESLLSKY
0.238


8
ESLLSKYNSN
0.180


3
ALDGEESLLS
0.100


6
GEESLLSKYN
0.018


7
EESLLSKYNS
0.010


4
LDGEESLLSK
0.001


















TABLE XVIII





Start
Subsequence
Score















V1-HLA-B7-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









39
YPMRILYDL
240.000


265
TPSPIIQQL
80.000


114
NPPQAVNLL
80.000


95
SPQLSDFGL
80.000


286
VPTFCTPGL
80.000


390
AVPPSKRFL
60.000


10
KLRSLASTL
40.000


163
SPRVKKNSV
40.000


367
IPEDILQLL
24.000


304
ALVSTNYPL
12.000


250
EAIDTESRL
12.000


80
DPPVASSCI
8.000


217
TPKLEHFGI
8.000


200
VLKTPKCAL
6.000


364
VTKIPEDIL
6.000


140
KVLMEKNSM
5.000


189
VTPPTKQSL
4.000


3
PIRSFCGKL
4.000


314
KTNSSSNDL
4.000


374
LLSKYNSNL
4.000


224
GISEYTMCL
4.000


190
TPPTKQSLV
4.000


119
VNLLDKARL
4.000


366
KIPEDILQL
4.000


46
DLHSEVQTL
4.000


348
TISSYENLL
4.000


21
ETARLQRAL
4.000


277
DAEYTNSPL
3.600


92
SPRSPQLSD
3.000


283
SPLVPTFCT
3.000


185
EPVIVTPPT
2.000


261
NVFATPSPI
2.000


296
IPSTKNSIA
2.000


363
EVTKIPEDI
2.000


291
TPGLKIPST
2.000


232
LNEDYTMGL
1.200


17
TLDCETARL
1.200


333
NSDTCFENL
1.200


50
EVQTLKDDV
1.000


135
FIKATKVLM
1.000


230
MCLNEDYTM
1.000


202
KTPKCALKM
1.000


383
ATPIAIKAV
0.600


343
DPSSPTISS
0.600


112
LPNPPQAVN
0.600


322
LEVEDRTSL
0.600


110
QVLPNPPQA
0.500


199
KVLKTPKCA
0.500


22
TARLQRALD
0.450


113
PNPPQAVNL
0.400


70
QKTDVKDDL
0.400


297
PSTKNSIAL
0.400


145
KNSMDIMKI
0.400


169
NSVHEQEAI
0.400


347
PTISSYENL
0.400


90
GKSPRSPQL
0.400


134
DFIKATKVL
0.400


310
YPLSKTNSS
0.400


346
SPTISSYEN
0.400


378
YNSNLATPI
0.400


6
SFCGKLRSL
0.400


193
TKQSLVKVL
0.400


180
DNYKEEPVI
0.400


295
KIPSTKNSI
0.400


244
RNNKSEEAI
0.400


104
ERYIVSQVL
0.400


128
ENQEGIDFI
0.400


380
SNLATPIAI
0.400


52
QTLKDDVNI
0.400


323
EVEDRTSLV
0.300


117
QAVNLLDKA
0.300


15
ASTLDCETA
0.300


33
ESDFEDYPM
0.300


382
LATPIAIKA
0.300


242
NARNNKSEE
0.300


207
ALKMDDFEC
0.300


111
VLPNPPQAV
0.300


391
VPPSKRFLK
0.300


124
KARLENQEG
0.300


358
TPTPPEVTK
0.300


14
LASTLDCET
0.300


384
TPIAIKAVP
0.200


360
TPPEVTKIP
0.200


2
DPIRSFCGK
0.200


298
STKNSIALV
0.200


152
KIREYFQKY
0.200


43
ILYDLHSEV
0.200


203
TPKCALKMD
0.200


316
NSSSNDLEV
0.200


267
SPIIQQLEK
0.200


103
LERYIVSQV
0.200


255
ESRLNDNVF
0.200


36
FEDYPMRIL
0.180


355
LLRTPTPPE
0.150


280
YTNSPLVPT
0.150


57
DVNIPELSN
0.150


340
NLTDPSSPT
0.150


7
FCGKLRSLA
0.150


118
AVNLLDKAR
0.150


307
STNYPLSKT
0.150







V5-HLA-B7-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









9
EKSPRSPQL
0.400


3
ASSCISEKS
0.060


2
VASSCISEK
0.030


6
CISEKSPRS
0.020


5
SCISEKSPR
0.010


4
SSCISEKSP
0.010


1
PVASSCISE
0.005


7
ISEKSPRSP
0.003


8
SEKSPRSPQ
0.002







V6-HLA-B7-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









5
EAIDAESRL
12.000


9
AESRLNDNV
0.060


6
AIDAESRLN
0.018


8
DAESRLNDN
0.018


1
NKSEEAIDA
0.010


2
KSEEAIDAE
0.003


7
IDAESRLND
0.002


4
EEAIDAESR
0.001


3
SEEAIDAES
0.001







V10-HLA-B7-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus eight.









14
YPTQKLNKM
20.000


11
QWIYPTQKL
0.600


5
DILQKFQWI
0.400


2
IPEDILQKF
0.120


12
WIYPTQKLN
0.020


6
ILQKFQWIY
0.020


8
QKFQWIYPT
0.010


7
LQKFQWIYP
0.010


10
FQWIYPTQK
0.010


1
KIPEDILQK
0.010


4
EDILQKFQW
0.002


13
IYPTQKLNK
0.001


15
PTQKLNKMR
0.001


9
KFQWIYPTQ
0.001


3
PEDILQKFQ
0.000







V12-HLA-B7-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus eight.









1
RALDGEESL
12.000


2
ALDGEESLL
3.600


7
ESLLSKYNS
0.020


8
SLLSKYNSN
0.020


4
DGEESLLSK
0.003


3
LDGEESLLS
0.002


6
EESLLSKYN
0.002


5
GEESLLSKY
0.001


















TABLE XIX





Start
Subsequence
Score















V1-HLA-B7-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









296
IPSTKNSIAL
80.000


2
DPIRSFCGKL
80.000


112
LPNPPQAVNL
80.000


346
SPTISSYENL
80.000


118
AVNLLDKARL
60.000


363
EVTKIPEDIL
30.000


199
KVLKTPKCAL
30.000


188
IVTPPTKQSL
20.000


285
LVPTFCTPGL
20.000


303
IALVSTNYPL
12.000


389
KAVPPSKRFL
12.000


264
ATPSPIIQQL
12.000


124
KARLENQEGI
12.000


358
TPTPPEVTKI
8.000


323
EVEDRTSLVL
6.000


231
CLNEDYTMGL
4.000


366
KIPEDILQLL
4.000


103
LERYIVSQVL
4.000


69
FQKTDVKDDL
4.000


92
SPRSPQLSDF
4.000


94
RSPQLSDFGL
4.000


5
RSFCGKLRSL
4.000


223
FGISEYTMCL
4.000


332
LNSDTCFENL
4.000


16
STLDCETARL
4.000


89
SGKSPRSPQL
4.000


373
QLLSKYNSNL
4.000


261
NVFATPSPII
3.000


242
NARNNKSEEA
3.000


355
LLRTPTPPEV
2.000


163
SPRVKKNSVH
2.000


321
DLEVEDRTSL
1.800


110
QVLPNPPQAV
1.500


141
VLMEKNSMDI
1.200


255
ESRLNDNVFA
1.000


229
TMCLNEDYTM
1.000


207
ALKMDDFECV
0.600


382
LATPIAIKAV
0.600


39
YPMRILYDLH
0.600


57
DVNIPELSNC
0.500


197
LVKVLKTPKC
0.500


133
IDFIKATKVL
0.400


379
NSNLATPIAI
0.400


217
TPKLEHFGIS
0.400


191
PPTKQSLVKV
0.400


365
TKIPEDILQL
0.400


343
DPSSPTISSY
0.400


9
GKLRSLASTL
0.400


168
KNSVHEQEAI
0.400


276
SDAEYTNSPL
0.400


38
DYPMRILYDL
0.400


216
VTPKLEHFGI
0.400


211
DDFECVTPKL
0.400


45
YDLHSEVQTL
0.400


51
VQTLKDDVNI
0.400


20
CETARLQRAL
0.400


260
DNVFATPSPI
0.400


192
PTKQSLVKVL
0.400


313
SKTNSSSNDL
0.400


340
NLTDPSSPTI
0.400


267
SPIIQQLEKS
0.400


113
PNPPQAVNLL
0.400


249
EEAIDTESRL
0.400


80
DPPVASSCIS
0.400


347
PTISSYENLL
0.400


310
YPLSKTNSSS
0.400


228
YTMCLNEDYT
0.300


14
LASTLDCETA
0.300


22
TARLQRALDG
0.300


142
LMEKNSMDIM
0.300


206
CALKMDDFEC
0.300


390
AVPPSKRFLK
0.225


283
SPLVPTFCTP
0.200


25
LQRALDGEES
0.200


291
TPGLKIPSTK
0.200


265
TPSPIIQQLE
0.200


185
EPVIVTPPTK
0.200


180
DNYKEEPVIV
0.200


203
TPKCALKMDD
0.200


286
VPTFCTPGLK
0.200


403
NIRDVSNKEN
0.200


391
VPPSKRFLKH
0.200


189
VTPPTKQSLV
0.200


114
NPPQAVNLLD
0.200


384
TPIAIKAVPP
0.200


42
RILYDLHSEV
0.200


95
SPQLSDFGLE
0.200


315
TNSSSNDLEV
0.200


190
TPPTKQSLVK
0.200


157
FQKYGYSPRV
0.200


360
TPPEVTKIPE
0.200


162
YSPRVKKNSV
0.200


35
DFEDYPMRIL
0.180


277
DAEYTNSPLV
0.180


306
VSTNYPLSKT
0.150


339
ENLTDPSSPT
0.150


282
NSPLVPTFCT
0.150


60
IPELSNCENF
0.120


243
ARNNKSEEAI
0.120


367
IPEDILQLLS
0.120







V5-HLA-B7-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









9
SEKSPRSPQL
0.400


3
VASSCISEKS
0.060


4
ASSCISEKSP
0.030


6
SCISEKSPRS
0.020


1
PPVASSCISE
0.020


5
SSCISEKSPR
0.010


7
CISEKSPRSP
0.010


8
ISEKSPRSPQ
0.007


2
PVASSCISEK
0.005


10
EKSPRSPQLS
0.002







V6-HLA-B7-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









5
EEAIDAESRL
0.400


9
DAESRLNDNV
0.180


1
NNKSEEAIDA
0.100


6
EAIDAESRLN
0.060


7
AIDAESRLND
0.013


10
AESRLNDNVF
0.006


3
KSEEAIDAES
0.006


8
IDAESRLNDN
0.002


2
NKSEEAIDAE
0.001


4
SEEAIDAESR
0.000







V10-HLA-B7-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus nine.









11
FQWIYPTQKL
6.000


15
YPTQKLNKMR
0.200


14
IYPTQKLNKM
0.100


8
LQKFQWIYPT
0.100


3
IPEDILQKFQ
0.060


5
EDILQKFQWI
0.040


6
DILQKFQWIY
0.020


2
KIPEDILQKF
0.020


13
WIYPTQKLNK
0.010


7
ILQKFQWIYP
0.010


12
QWIYPTQKLN
0.002


9
QKFQWIYPTQ
0.001


10
KFQWIYPTQK
0.001


1
TKIPEDILQK
0.001


4
PEDILQKFQW
0.000







V12-HLA-B7-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus nine.









2
RALDGEESLL
12.000


9
SLLSKYNSNL
4.000


1
QRALDGEESL
0.400


8
ESLLSKYNSN
0.020


3
ALDGEESLLS
0.018


5
DGEESLLSKY
0.006


7
EESLLSKYNS
0.002


4
LDGEESLLSK
0.001


6
GEESLLSKYN
0.001


















TABLE XX





Start
Subsequence
Score















V1-HLA-B3501-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









217
TPKLEHFGI
36.000


152
KIREYFQKY
24.000


39
YPMRILYDL
20.000


95
SPQLSDFGL
20.000


114
NPPQAVNLL
20.000


286
VPTFCTPGL
20.000


265
TPSPIIQQL
20.000


255
ESRLNDNVF
15.000


163
SPRVKKNSV
12.000


367
IPEDILQLL
12.000


80
DPPVASSCI
8.000


250
EAIDTESRL
6.000


366
KIPEDILQL
6.000


140
KVLMEKNSM
6.000


135
FIKATKVLM
6.000


389
KAVPPSKRF
6.000


10
KLRSLASTL
6.000


33
ESDFEDYPM
4.500


202
KTPKCALKM
4.000


190
TPPTKQSLV
4.000


230
MCLNED YTM
3.000


364
VTKIPEDIL
3.000


98
LSDFGLERY
3.000


200
VLKTPKCAL
3.000


169
NSVHEQEAI
3.000


302
SIALVSTNY
2.000


283
SPLVPTFCT
2.000


296
IPSTKNSIA
2.000


112
LPNPPQAVN
2.000


310
YPLSKTNSS
2.000


370
DILQLLSKY
2.000


224
GISEYTMCL
2.000


185
EPVIVTPPT
2.000


228
YTMCLNEDY
2.000


314
KTNSSSNDL
2.000


346
SPTISSYEN
2.000


205
KCALKMDDF
2.000


291
TPGLKIPST
2.000


343
DPSSPTISS
2.000


312
LSKTNSSSN
1.500


46
DLHSEVQTL
1.500


119
VNLLDKARL
1.500


333
NSDTCFENL
1.500


375
LSKYNSNLA
1.500


145
KNSMDIMKI
1.200


5
RSFCGKLRS
1.000


215
CVTPKLEHF
1.000


390
AVPPSKRFL
1.000


304
ALVSTNYPL
1.000


189
VTPPTKQSL
1.000


350
SSYENLLRT
1.000


149
DIMKIREYF
1.000


374
LLSKYNSNL
1.000


316
NSSSNDLEV
1.000


330
LVLNSDTCF
1.000


344
PSSPTISSY
1.000


348
TISSYENLL
1.000


91
KSPRSPQLS
1.000


12
RSLASTLDC
1.000


281
TNSPLVPTF
1.000


21
ETARLQRAL
1.000


277
DAEYTNSPL
0.900


244
RNNKSEEAI
0.800


128
ENQEGIDFI
0.800


295
KIPSTKNSI
0.800


15
ASTLDCETA
0.750


272
QLEKSDAEY
0.600


60
IPELSNCEN
0.600


143
MEKNSMDIM
0.600


180
DNYKEEPVI
0.600


52
QTLKDDVNI
0.600


298
STKNSIALV
0.600


92
SPRSPQLSD
0.600


232
LNEDYTMGL
0.600


203
TPKCALKMD
0.600


379
NSNLATPIA
0.500


162
YSPRVKKNS
0.500


328
TSLVLNSDT
0.500


297
PSTKNSIAL
0.500


301
NSIALVSTN
0.500


282
NSPLVPTFC
0.500


195
QSLVKVLKT
0.500


84
ASSCISGKS
0.500


17
TLDCETARL
0.450


28
ALDGEESDF
0.450


207
ALKMDDFEC
0.450


275
KSDAEYTNS
0.450


380
SNLATPIAI
0.400


261
NVFATPSPI
0.400


154
REYFQKYGY
0.400


363
EVTKIPEDI
0.400


360
TPPEVTKIP
0.400


43
ILYDLHSEV
0.400


378
YNSNLATPI
0.400


257
RLNDNVFAT
0.400


386
IAIKAVPPS
0.300


8
CGKLRSLAS
0.300


117
QAVNLLDKA
0.300


14
LASTLDCET
0.300


3
PIRSFCGKL
0.300







V5-HLA-B3501-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









3
ASSCISEKS
0.500


6
CISEKSPRS
0.200


9
EKSPRSPQL
0.100


4
SSCISEKSP
0.500


2
VASSCISEK
0.030


7
ISEKSPRSP
0.015


5
SCISEKSPR
0.015


8
SEKSPRSPQ
0.003


1
PVASSCISE
0.001







V6-HLA-B3501-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









5
EAIDAESRL
6.000


8
DAESRLNDN
0.090


2
KSEEAIDAE
0.060


6
AIDAESRLN
0.045


1
NKSEEAIDA
0.030


9
AESRLNDNV
0.020


3
SEEAIDAES
0.003


7
IDAESRLND
0.002


4
EEAIDAESR
0.002







V10-HLA-B3501-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus eight.









14
YPTQKLNKM
40.000


2
IPEDILQKF
12.000


6
ILQKFQWIY
2.000


5
DILQKFQWI
0.400


11
QWIYPTQKL
0.100


12
WIYPTQKLN
0.100


1
KIPEDILQK
0.060


4
EDILQKFQW
0.050


7
LQKFQWIYP
0.030


10
FQWIYPTQK
0.010


8
QKFQWIYPT
0.010


9
KFQWIYPTQ
0.002


13
IYPTQKLNK
0.001


15
PTQKLNKMR
0.001


3
PEDILQKFQ
0.000







V12-HLA-B3501-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus eight.









1
RALDGEESL
12.000


7
ESLLSKYNS
0.500


2
ALDGEESLL
0.450


8
SLLSKYNSN
0.100


5
GEESLLSKY
0.060


3
LDGEESLLS
0.030


6
EESLLSKYN
0.010


4
DGEESLLSK
0.006


















TABLE XXI





Start
Subsequence
Score















V1-HLA-B3501-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









92
SPRSPQLSDF
60.000


343
DPSSPTISSY
40.000


346
SPTISSYENL
20.000


2
DPIRSFCGKL
20.000


296
IPSTKNSIAL
20.000


112
LPNPPQAVNL
20.000


27
RALDGEESDF
18.000


358
TPTPPEVTKI
12.000


301
NSIALVSTNY
10.000


94
RSPQLSDFGL
10.000


5
RSFCGKLRSL
10.000


124
KARLENQEGI
7.200


389
KAVPPSKRFL
6.000


217
TPKLEHFGIS
6.000


60
IPELSNCENF
6.000


271
QQLEKSDAEY
4.000


366
KIPEDILQLL
4.000


97
QLSDFGLERY
4.000


303
IALVSTNYPL
3.000


89
SGKSPRSPQL
3.000


69
FQKTDVKDDL
3.000


229
TMCLNEDYTM
3.000


16
STLDCETARL
3.000


255
ESRLNDNVFA
2.250


310
YPLSKTNSSS
2.000


199
KVLKTPKCAL
2.000


231
CLNEDYTMGL
2.000


332
LNSDTCFENL
2.000


379
NSNLATPIAI
2.000


80
DPPVASSCIS
2.000


267
SPIIQQLEKS
2.000


30
DGEESDFEDY
1.800


375
LSKYNSNLAT
1.500


118
AVNLLDKARL
1.500


168
KNSVHEQEAI
1.200


367
IPEDILQLLS
1.200


219
KLEHFGISEY
1.200


188
IVTPPTKQSL
1.000


280
YTNSPLVPTF
1.000


162
YSPRVKKNSV
1.000


329
SLVLNSDTCF
1.000


363
EVTKIPEDIL
1.000


264
ATPSPIIQQL
1.000


223
FGISEYTMCL
1.000


373
QLLSKYNSNL
1.000


285
LVPTFCTPGL
1.000


214
ECVTPKLEHF
1.000


126
RLENQEGIDF
0.900


207
ALKMDDFECV
0.900


242
NARNNKSEEA
0.900


250
EAIDTESRLN
0.900


340
NLTDPSSPTI
0.800


141
VLMEKNSMDI
0.800


216
VTPKLEHFGI
0.600


323
EVEDRTSLVL
0.600


147
SMDIMKIREY
0.600


382
LATPIAIKAV
0.600


32
EESDFEDYPM
0.600


157
FQKYGYSPRV
0.600


203
TPKCALKMDD
0.600


51
VQTLKDDVNI
0.600


142
LMEKNSMDIM
0.600


137
KATKVLMEKN
0.600


403
NIRDVSNKEN
0.600


247
KSEEAIDTES
0.600


98
LSDFGLERYI
0.600


173
EQEAINSDNY
0.600


355
LLRTPTPPEV
0.600


163
SPRVKKNSVH
0.600


306
VSTNYPLSKT
0.500


282
NSPLVPTFCT
0.500


345
SSPTISSYEN
0.500


169
NSVHEQEAIN
0.500


328
TSLVLNSDTC
0.500


349
ISSYENLLRT
0.500


63
LSNCENFQKT
0.500


25
LQRALDGEES
0.450


245
NNKSEEAIDT
0.450


14
LASTLDCETA
0.450


206
CALKMDDFEC
0.450


321
DLEVEDRTSL
0.450


260
DNVFATPSPI
0.400


261
NVFATPSPII
0.400


42
RILYDLHSEV
0.400


191
PPTKQSLVKV
0.400


360
TPPEVTKIPE
0.400


138
ATKVLMEKNS
0.300


298
STKNSIALVS
0.300


180
DNYKEEPVIV
0.300


192
PTKQSLVKVL
0.300


170
SVHEQEAINS
0.300


197
LVKVLKTPKC
0.300


239
GLKNARNNKS
0.300


8
CGKLRSLAST
0.300


139
TKVLMEKNSM
0.300


103
LERYIVSQVL
0.300


178
NSDNYKEEPV
0.300


83
VASSCISGKS
0.300


95
SPQLSDFGLE
0.300


293
GLKIPSTKNS
0.300







V5-HLA-B3501-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









9
SEKSPRSPQL
0.300


3
VASSCISEKS
0.300


6
SCISEKSPRS
0.100


5
SSCISEKSPR
0.075


4
ASSCISEKSP
0.050


7
CISEKSPRSP
0.020


1
PPVASSCISE
0.020


8
ISEKSPRSPQ
0.015


10
EKSPRSPQLS
0.010


2
PVASSCISEK
0.001







V6-HLA-B3501-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









6
EAIDAESRLN
0.900


3
KSEEAIDAES
0.600


1
NNKSEEAIDA
0.450


9
DAESRLNDNV
0.180


5
EEAIDAESRL
0.100


10
AESRLNDNVF
0.100


8
IDAESRLNDN
0.020


7
AIDAESRLND
0.003


2
NKSEEAIDAE
0.002


4
SEEAIDAESR
0.000







V10-HLA-B3501-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus nine.









2
KIPEDILQKF
4.000


6
DILQKFQWIY
2.000


11
FQWIYPTQKL
1.000


8
LQKFQWIYPT
0.300


15
YPTQKLNKMR
0.200


14
IYPTQKLNKM
0.200


3
IPEDILQKFQ
0.120


5
EDILQKFQWI
0.040


7
ILQKFQWIYP
0.010


12
QWIYPTQKLN
0.010


13
WIYPTQKLNK
0.010


10
KFQWIYPTQK
0.002


4
PEDILQKFQW
0.002


1
TKIPEDILQK
0.002


9
QKFQWIYPTQ
0.001







V12-HLA-B3501-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus nine.









2
RALDGEESLL
18.000


5
DGEESLLSKY
1.200


9
SLLSKYNSNL
1.000


8
ESLLSKYNSN
0.500


1
QRALDGEESL
0.100


3
ALDGEESLLS
0.045


7
EESLLSKYNS
0.010


6
GEESLLSKYN
0.003


4
LDGEESLLSK
0.002
















TABLE XII







V1-HLA-A1-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









Pos
123456789
score





 98
LSDFGLERY
31


 31
GEESDFEDY
28


 37
EDYPMRILY
26


272
QLEKSDAEY
26


 48
HSEVQTLKD
24


228
YTMCLNEDY
24


344
PSSPTISSY
23


152
KIREYFQKY
21


 78
LSDPPVASS
20


341
LTDPSSPTI
20


182
YKEEPVIVT
19


302
SIALVSTNY
19


 71
KTDVKDDLS
18


121
LLDKARLEN
18


324
VEDRTSLVL
18


368
PEDILQLLS
18


 19
DCETARLQR
17


 54
LKDDVNIPE
17


154
REYFQKYGY
17


220
LEHFGISEY
17


333
NSDTCFENL
17


370
DILQLLSKY
17


147
SMDIMKIRE
16


183
KEEPVIVTP
16


219
KLEHFGISE
16


225
ISEYTMCLN
16


253
DTESRLNDN
16


148
MDIMKIREY
15


171
VHEQEAINS
15


174
QEAINSDNY
15


178
NSDNYKEEP
15


258
LNDNVFATP
15


















TABLE XXII





Pos
123456789
score















V5-HLA-A1-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









 7
ISEKSPRSP
14


 3
ASSCISEKS
 7


 4
SSCISEKSP
 6







V6-HLA-A1-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









 2
KSEEAIDAE
14


 6
AIDAESRLN
13


 3
SEEAIDAES
12


 8
DAESRLNDN
10


 7
IDAESRLND
 7







V10-HLA-A1-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO: 21; each start position


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


position for each peptide is the start position plus eight.









 6
ILQKFQWIY
16


 2
IPEDILQKF
12


 3
PEDILQKFQ
10


13
IYPTQKLNK
 8


15
PTQKLNKMR
 8







V12-HLA-A1-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO: 25; each start position


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


position for each peptide is the start position plus eight.









 5
GEESLLSKY
27


 4
DGEESLLSK
16


 2
ALDGEESLL
15


















TABLE XXIII





Pos
123456789
score















V1-HLA-A0201-9mers-193P1E1B Each peptide is a portion


of SEQ ID NO:3; each start position is specified, the


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


for each peptide is the start position plus eight.









43
ILYDLHSEV
27


366
KIPEDILQL
26


46
DLHSEVQTL
25


10
KLRSLASTL
24


17
TLDCETARL
24


224
GISEYTMCL
24


111
VLPNPPQAV
23


304
ALVSTNYPL
23


374
LLSKYNSNL
23


200
VLKTPKCAL
22


6
SFCGKLRSL
21


257
RLNDNVFAT
21


298
STKNSIALV
21


295
KIPSTKNSI
20


348
TISSYENLL
20


383
ATPIAIKAV
20


102
GLERYIVSQ
19


189
VTPPTKQSL
19


341
LTDPSSPTI
19


381
NLATPIAIK
19


3
PIRSFCGKL
18


13
SLASTLDCE
18


39
YPMRILYDL
18


55
KDDVNIPEL
18


192
PTKQSLVKV
18


280
YTNSPLVPT
18


390
AVPPSKRFL
18


142
LMEKNSMDI
17


196
SLVKVLKTP
17


307
STNYPLSKT
17


314
KTNSSSNDL
17


359
PTPPEVTKI
17


42
RILYDLHSE
16


52
QTLKDDVNI
16


133
IDFIKATKV
16


163
SPRVKKNSV
16


232
LNEDYTMGL
16


265
TPSPIIQQL
16


322
LEVEDRTSL
16


355
LLRTPTPPE
16


356
LRTPTPPEV
16


367
IPEDILQLL
16


370
DILQLLSKY
16


373
QLLSKYNSN
16


9
GKLRSLAST
15


21
ETARLQRAL
15


24
RLQRALDGE
15


53
TLKDDVNIP
15


97
QLSDFGLER
15


103
LERYIVSQV
15


106
YIVSQVLPN
15


114
NPPQAVNLL
15


117
QAVNLLDKA
15


119
VNLLDKARL
15


121
LLDKARLEN
15


125
ARLENQEGI
15


141
VLMEKNSMD
15


145
KNSMDIMKI
15


158
QKYGYSPRV
15


209
KMDDFECVT
15


340
NLTDPSSPT
15


364
VTKIPEDIL
15


399
KHGQNIRDV
15


28
ALDGEESDF
14


77
DLSDPPVAS
14


90
GKSPRSPQL
14


99
SDFGLERYI
14


120
NLLDKARLE
14


135
FIKATKVLM
14


140
KVLMEKNSM
14


152
KIREYFQKY
14


176
AINSDNYKE
14


193
TKQSLVKVL
14


195
QSLVKVLKT
14


208
LKMDDFECV
14


250
EAIDTESRL
14


278
AEYTNSPLV
14


329
SLVLNSDTC
14


331
VLNSDTCFE
14


350
SSYENLLRT
14


14
LASTLDCET
13


78
LSDPPVASS
13


95
SPQLSDFGL
13


110
QVLPNPPQA
13


128
ENQEGIDFI
13


179
SDNYKEEPV
13


181
NYKEEPVIV
13


207
ALKMDDFEC
13


212
DFECVTPKL
13


219
KLEHFGISE
13


231
CLNEDYTMG
13


261
NVFATPSPI
13


262
VFATPSPII
13


268
PIIQQLEKS
13


272
QLEKSDAEY
13


286
VPTFCTPGL
13


293
GLKIPSTKN
13


300
KNSIALVST
13


316
NSSSNDLEV
13


323
EVEDRTSLV
13


347
PTISSYENL
13


380
SNLATPIAI
13


382
LATPIAIKA
13


386
IAIKAVPPS
13


397
FLKHGQNIR
13


50
EVQTLKDDV
12


59
NIPELSNCE
12


75
KDDLSDPPV
12


87
CISGKSPRS
12


132
GIDFIKATK
12


134
DFIKATKVL
12


182
YKEEPVIVT
12


187
VIVTPPTKQ
12


202
KTPKCALKM
12


229
TMCLNEDYT
12


236
YTMGLKNAR
12


269
IIQQLEKSD
12


276
SDAEYTNSP
12


288
TFCTPGLKI
12


291
TPGLKIPST
12


302
SIALVSTNY
12


324
VEDRTSLVL
12


354
NLLRTPTPP
12


371
ILQLLSKYN
12


387
AIKAVPPSK
12


403
NIRDVSNKE
12







V5-HLA-A0201-9mers-193P1E1B Each peptide is a portion


of SEQ ID NO:11; each start position is specified, the


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


for each peptide is the start position plus eight.









6
CISEKSPRS
12


2
VASSCISEK
11


9
EKSPRSPQL
10


1
PVASSCISE
5







V6-HLA-A0201-9mers-193P1E1B Each peptide is a portion


of SEQ ID NO:13; each start position is specified, the


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


for each peptide is the start position plus eight.









5
EAIDAESRL
14


9
AESRLNDNV
13


6
AIDAESRLN
10


2
KSEEAIDAE
7


7
IDAESRLND
7


8
DAESRLNDN
7


1
NKSEEAIDA
6







V10-HLA-A0201-9mers-193P1E1B Each peptide is a portion


is SEQ ID NO:21; each start position is specified, the


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


for each peptide is the start position plus eight.









5
DILQKFQWI
18


1
KIPEDILQK
16


11
QWIYPTQKL
16


6
ILQKFQWIY
13


14
YPTQKLNKM
13


8
QKFQWIYPT
10


12
WIYPTQKLN
10


2
IPEDILQKF
8







V12-HLA-A0201-9mers-193P1E1B Each peptide is a portion


of SEQ ID NO:25; each start position is specified, the


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


for each peptide is the start position plus eight.









2
ALDGEESLL
24


1
RALDGEESL
21


8
SLLSKYNSN
18


















TABLE XXIV





Pos
123456789
score















V1-HLA-A0203-9mers-193P1E1B


NoResultsFound.


V5-HLA-A0203-9mers-193P1E1B


NoResultsFound.


V6-HLA-A0203-9mers-193P1E1B


NoResultsFound.


V10-HLA-A0203-9mers-193P1E1B


NoResultsFound.


V12-HLA-A0203-9mers-193P1E1B


NoResultsFound.


















TABLE XXV





Pos
123456789
score















V1-HLA-A3-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









186
PVIVTPPTK
28


387
AIKAVPPSK
28


10
KLRSLASTL
26


132
GIDFIKATK
25


381
NLATPIAIK
24


97
QLSDFGLER
23


239
GLKNARNNK
23


28
ALDGEESDF
22


197
LVKVLKTPK
22


110
QVLPNPPQA
21


152
KIREYFQKY
21


272
QLEKSDAEY
21


358
TPTPPEVTK
21


402
QNIRDVSNK
21


43
ILYDLHSEV
20


292
PGLKIPSTK
20


102
GLERYIVSQ
19


118
AVNLLDKAR
19


151
MKIREYFQK
19


160
YGYSPRVKK
19


165
RVKKNSVHE
19


194
KQSLVKVLK
19


302
SIALVSTNY
19


369
EDILQLLSK
19


370
DILQLLSKY
19


159
KYGYSPRVK
18


188
IVTPPTKQS
18


215
CVTPKLEHF
18


219
KLEHFGISE
18


267
SPIIQQLEK
18


330
LVLNSDTCF
18


366
KIPEDILQL
18


385
PIAIKAVPP
18


24
RLQRALDGE
17


77
DLSDPPVAS
17


120
NLLDKARLE
17


140
KVLMEKNSM
17


191
PPTKQSLVK
17


201
LKTPKCALK
17


284
PLVPTFCTP
17


306
VSTNYPLSK
17


354
NLLRTPTPP
17


373
QLLSKYNSN
17


397
FLKHGQNIR
17


2
DPIRSFCGK
16


42
RILYDLHSE
16


57
DVNIPELSN
16


126
RLENQEGID
16


141
VLMEKNSMD
16


196
SLVKVLKTP
16


199
KVLKTPKCA
16


257
RLNDNVFAT
16


261
NVFATPSPI
16


323
EVEDRTSLV
16


329
SLVLNSDTC
16


390
AVPPSKRFL
16


67
ENFQKTDVK
15


73
DVKDDLSDP
15


135
FIKATKVLM
15


137
KATKVLMEK
15


170
SVHEQEAIN
15


183
KEEPVIVTP
15


311
PLSKTNSSS
15


37
EDYPMRILY
14


46
DLHSEVQTL
14


116
PQAVNLLDK
14


121
LLDKARLEN
14


154
REYFQKYGY
14


175
EAINSDNYK
14


207
ALKMDDFEC
14


321
DLEVEDRTS
14


340
NLTDPSSPT
14


344
PSSPTISSY
14


17
TLDCETARL
13


23
ARLQRALDG
13


47
LHSEVQTLK
13


63
LSNCENFQK
13


79
SDPPVASSC
13


82
PVASSCISG
13


83
VASSCISGK
13


86
SCISGKSPR
13


107
IVSQVLPNP
13


111
VLPNPPQAV
13


149
DIMKIREYF
13


231
CLNEDYTMG
13


293
GLKIPSTKN
13


295
KIPSTKNSI
13


304
ALVSTNYPL
13


355
LLRTPTPPE
13


371
ILQLLSKYN
13


374
LLSKYNSNL
13


403
NIRDVSNKE
13







V5-HLA-A3-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









1
PVASSCISE
13


2
VASSCISEK
13


5
SCISEKSPR
11


6
CISEKSPRS
10


9
EKSPRSPQL
8


8
SEKSPRSPQ
6







V6-HLA-A3-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









6
AIDAESRLN
13


4
EEAIDAESR
11


7
IDAESRLND
8


5
EAIDAESRL
7


3
SEEAIDAES
6


9
AESRLNDNV
6







V10-HLA-A3-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus eight.









1
KIPEDILQK
28


6
ILQKFQWIY
18


10
FQWIYPTQK
16


12
WIYPTQKLN
15


13
IYPTQKLNK
15







V12-HLA-A3-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus eight.









2
ALDGEESLL
18


4
DGEESLLSK
16


8
SLLSKYNSN
16


1
RALDGEESL
11


5
GEESLLSKY
9


















TABLE XXVI





Pos
123456789
score















V1-HLA-A26-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









370
DILQLLSKY
29


21
ETARLQRAL
28


215
CVTPKLEHF
25


73
DVKDDLSDP
24


250
EAIDTESRL
24


363
EVTKIPEDI
23


37
EDYPMRILY
22


46
DLHSEVQTL
22


149
DIMKIREYF
22


323
EVEDRTSLV
22


50
EVQTLKDDV
21


253
DTESRLNDN
21


347
PTISSYENL
21


57
DVNIPELSN
20


131
EGIDFIKAT
20


134
DFIKATKVL
20


366
KIPEDILQL
20


369
EDILQLLSK
20


148
MDIMKIREY
19


228
YTMCLNEDY
19


255
ESRLNDNVF
19


390
AVPPSKRFL
19


104
ERYIVSQVL
18


189
VTPPTKQSL
18


211
DDFECVTPK
18


330
LVLNSDTCF
18


335
DTCFENLTD
18


152
KIREYFQKY
17


212
DFECVTPKL
17


265
TPSPIIQQL
17


277
DAEYTNSPL
17


314
KTNSSSNDL
17


344
PSSPTISSY
17


128
ENQEGIDFI
16


155
EYFQKYGYS
16


184
EEPVIVTPP
16


221
EHFGISEYT
16


281
TNSPLVPTF
16


326
DRTSLVLNS
16


364
VTKIPEDIL
16


3
PIRSFCGKL
15


6
SFCGKLRSL
15


67
ENFQKTDVK
15


186
PVIVTPPTK
15


193
TKQSLVKVL
15


214
ECVTPKLEH
15


220
LEHFGISEY
15


227
EYTMCLNED
15


261
NVFATPSPI
15


264
ATPSPIIQQ
15


302
SIALVSTNY
15


307
STNYPLSKT
15


322
LEVEDRTSL
15


367
IPEDILQLL
15


39
YPMRILYDL
14


56
DDVNIPELS
14


93
PRSPQLSDF
14


98
LSDFGLERY
14


106
YIVSQVLPN
14


107
IVSQVLPNP
14


175
EAINSDNYK
14


185
EPVIVTPPT
14


224
GISEYTMCL
14


325
EDRTSLVLN
14


348
TISSYENLL
14


359
PTPPEVTKI
14







V5-HLA-A26-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









9
EKSPRSPQL
20


1
PVASSCISE
13







V6-HLA-A26-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









5
EAIDAESRL
24


8
DAESRLNDN
13


4
EEAIDAESR
11







V10-HLA-A26-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus eight.









2
IPEDILQKF
16


4
EDILQKFQW
15


5
DILQKFQWI
13


11
QWIYPTQKL
13


1
KIPEDILQK
12


6
ILQKFQWIY
10


15
PTQKLNKMR
10


8
QKFQWIYPT
8


14
YPTQKLNKM
7







V12-HLA-A26-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus eight.









5
GEESLLSKY
18


4
DGEESLLSK
16


6
EESLLSKYN
11


1
RALDGEESL
10


7
ESLLSKYNS
10


2
ALDGEESLL
9


















TABLE XXVII





Pos
123456789
score















V1-HLA-B0702-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









265
TPSPIIQQL
23


286
VPTFCTPGL
22


39
YPMRILYDL
21


114
NPPQAVNLL
21


367
IPEDILQLL
21


95
SPQLSDFGL
20


185
EPVIVTPPT
20


283
SPLVPTFCT
20


296
IPSTKNSIA
19


163
SPRVKKNSV
18


291
TPGLKIPST
18


92
SPRSPQLSD
17


80
DPPVASSCI
16


112
LPNPPQAVN
16


190
TPPTKQSLV
16


217
TPKLEHFGI
16


343
DPSSPTISS
16


358
TPTPPEVTK
16


392
PPSKRFLKH
16


36
FEDYPMRIL
14


90
GKSPRSPQL
14


191
PPTKQSLVK
14


200
VLKTPKCAL
14


324
VEDRTSLVL
14


390
AVPPSKRFL
14


10
KLRSLASTL
13


17
TLDCETARL
13


21
ETARLQRAL
13


55
KDDVNIPEL
13


113
PNPPQAVNL
13


115
PPQAVNLLD
13


134
DFIKATKVL
13


224
GISEYTMCL
13


304
ALVSTNYPL
13


364
VTKIPEDIL
13


366
KIPEDILQL
13


374
LLSKYNSNL
13


384
TPIAIKAVP
13


3
PIRSFCGKL
12


6
SFCGKLRSL
12


104
ERYIVSQVL
12


193
TKQSLVKVL
12


212
DFECVTPKL
12


267
SPIIQQLEK
12


280
YTNSPLVPT
12


288
TFCTPGLKI
12


297
PSTKNSIAL
12


322
LEVEDRTSL
12


333
NSDTCFENL
12


348
TISSYENLL
12


2
DPIRSFCGK
11


28
ALDGEESDF
11


46
DLHSEVQTL
11


60
IPELSNCEN
11


81
PPVASSCIS
11


119
VNLLDKARL
11


182
YKEEPVIVT
11


189
VTPPTKQSL
11


232
LNEDYTMGL
11


250
EAIDTESRL
11


262
VFATPSPII
11


277
DAEYTNSPL
11


281
TNSPLVPTF
11


300
KNSIALVST
11


310
YPLSKTNSS
11


314
KTNSSSNDL
11


357
RTPTPPEVT
11


360
TPPEVTKIP
11


361
PPEVTKIPE
11


389
KAVPPSKRF
11


391
VPPSKRFLK
11







V5-HLA-B0702-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









9
EKSPRSPQL
15







V6-HLA-B0702-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









5
EAIDAESRL
11


9
AESRLNDNV
10


1
NKSEEAIDA
8


7
IDAESRLND
5







V10-HLA-B0702-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus eight.









2
IPEDILQKF
17


14
YPTQKLNKM
16


11
QWIYPTQKL
14







V12-HLA-B0702-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus eight.









2
ALDGEESLL
15


1
RALDGEESL
11


















TABLE XXVIII





Pos
123456789
score















V1-HLA-B08-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









200
VLKTPKCAL
28


163
SPRVKKNSV
25


217
TPKLEHFGI
23


6
SFCGKLRSL
22


10
KLRSLASTL
22


364
VTKIPEDIL
21


3
PIRSFCGKL
20


8
CGKLRSLAS
20


141
VLMEKNSMD
20


90
GKSPRSPQL
19


95
SPQLSDFGL
19


150
IMKIREYFQ
19


122
LDKARLENQ
18


291
TPGLKIPST
18


296
IPSTKNSIA
18


46
DLHSEVQTL
17


53
TLKDDVNIP
17


114
NPPQAVNLL
17


203
TPKCALKMD
17


205
KCALKMDDF
17


207
ALKMDDFEC
17


224
GISEYTMCL
17


239
GLKNARNNK
17


265
TPSPIIQQL
17


286
VPTFCTPGL
17


293
GLKIPSTKN
17


304
ALVSTNYPL
17


366
KIPEDILQL
17


367
IPEDILQLL
17


374
LLSKYNSNL
17


391
VPPSKRFLK
17


397
FLKHGQNIR
17


17
TLDCETARL
16


39
YPMRILYDL
16


120
NLLDKARLE
16


135
FIKATKVLM
16


190
TPPTKQSLV
16


215
CVTPKLEHF
16


250
EAIDTESRL
16


277
DAEYTNSPL
16


310
YPLSKTNSS
16


373
QLLSKYNSN
16


255
ESRLNDNVF
15


385
PIAIKAVPP
15


87
CISGKSPRS
14


92
SPRSPQLSD
14


348
TISSYENLL
14


387
AIKAVPPSK
14


392
PPSKRFLKH
14


21
ETARLQRAL
13


55
KDDVNIPEL
13


69
FQKTDVKDD
13


80
DPPVASSCI
13


124
KARLENQEG
13


166
VKKNSVHEQ
13


179
SDNYKEEPV
13


181
NYKEEPVIV
13


271
QQLEKSDAE
13


298
STKNSIALV
13







V5-HLA-B08-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









9
EKSPRSPQL
20


6
CISEKSPRS
16


8
SEKSPRSPQ
12







V6-HLA-B08-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









5
EAIDAESRL
16


8
DAESRLNDN
12







V10-HLA-B08-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus eight.









5
DILQKFQWI
20


14
YPTQKLNKM
16


2
IPEDILQKF
13


11
QWIYPTQKL
11


7
LQKFQWIYP
10







V12-HLA-B08-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus eight.









8
SLLSKYNSN
18


2
ALDGEESLL
16


1
RALDGEESL
14


















TABLE XXIX





Pos
123456789
score















V1-HLA-B1510-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









21
ETARLQRAL
15


55
KDDVNIPEL
15


90
GKSPRSPQL
15


265
TPSPIIQQL
15


390
AVPPSKRFL
15


399
KHGQNIRDV
15


36
FEDYPMRIL
14


113
PNPPQAVNL
14


193
TKQSLVKVL
14


250
EAIDTESRL
14


367
IPEDILQLL
14


6
SFCGKLRSL
13


17
TLDCETARL
13


104
ERYIVSQVL
13


119
VNLLDKARL
13


134
DFIKATKVL
13


189
VTPPTKQSL
13


200
VLKTPKCAL
13


224
GISEYTMCL
13


281
TNSPLVPTF
13


297
PSTKNSIAL
13


46
DLHSEVQTL
12


47
LHSEVQTLK
12


70
QKTDVKDDL
12


114
NPPQAVNLL
12


171
VHEQEAINS
12


212
DFECVTPKL
12


221
EHFGISEYT
12


232
LNEDYTMGL
12


322
LEVEDRTSL
12


324
VEDRTSLVL
12


348
TISSYENLL
12


366
KIPEDILQL
12


374
LLSKYNSNL
12


10
KLRSLASTL
11


39
YPMRILYDL
11


277
DAEYTNSPL
11


286
VPTFCTPGL
11


364
VTKIPEDIL
11


389
KAVPPSKRF
11


3
PIRSFCGKL
10


95
SPQLSDFGL
10


255
ESRLNDNVF
10


304
ALVSTNYPL
10


314
KTNSSSNDL
10


333
NSDTCFENL
10


347
PTISSYENL
10


93
PRSPQLSDF
9


135
FIKATKVLM
9


149
DIMKIREYF
8


205
KCALKMDDF
8


215
CVTPKLEHF
8


28
ALDGEESDF
7


33
ESDFEDYPM
7


61
PELSNCENF
7


77
DLSDPPVAS
7


127
LENQEGIDF
7


140
KVLMEKNSM
7


143
MEKNSMDIM
7


182
YKEEPVIVT
7


183
KEEPVIVTP
7


202
KTPKCALKM
7


222
HFGISEYTM
7


230
MCLNEDYTM
7


358
TPTPPEVTK
7







V5-HLA-B1510-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









9
EKSPRSPQL
15


7
ISEKSPRSP
7







V6-HLA-B1510-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









5
EAIDAESRL
14







V10-HLA-B1510-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus eight.









11
QWIYPTQKL
11


2
IPEDILQKF
10


14
YPTQKLNKM
8







V12-HLA-B1510-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus eight.









1
RALDGEESL
12


2
ALDGEESLL
11


















TABLE XXX





Pos
123456789
score















V1-HLA-B2705-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









104
ERYIVSQVL
25


164
PRVKKNSVH
25


93
PRSPQLSDF
23


125
ARLENQEGI
22


4
IRSFCGKLR
21


137
KATKVLMEK
18


366
KIPEDILQL
18


389
KAVPPSKRF
18


395
KRFLKHGQN
18


23
ARLQRALDG
17


55
KDDVNIPEL
17


67
ENFQKTDVK
17


90
GKSPRSPQL
17


119
VNLLDKARL
17


132
GIDFIKATK
17


211
DDFECVTPK
17


292
PGLKIPSTK
17


370
DILQLLSKY
17


10
KLRSLASTL
16


47
LHSEVQTLK
16


86
SCISGKSPR
16


140
KVLMEKNSM
16


154
REYFQKYGY
16


160
YGYSPRVKK
16


194
KQSLVKVLK
16


202
KTPKCALKM
16


239
GLKNARNNK
16


265
TPSPIIQQL
16


267
SPIIQQLEK
16


330
LVLNSDTCF
16


369
EDILQLLSK
16


374
LLSKYNSNL
16


396
RFLKHGQNI
16


402
QNIRDVSNK
16


6
SFCGKLRSL
15


28
ALDGEESDF
15


34
SDFEDYPMR
15


41
MRILYDLHS
15


113
PNPPQAVNL
15


134
DFIKATKVL
15


148
MDIMKIREY
15


175
EAINSDNYK
15


191
PPTKQSLVK
15


220
LEHFGISEY
15


224
GISEYTMCL
15


236
YTMGLKNAR
15


250
EAIDTESRL
15


281
TNSPLVPTF
15


302
SIALVSTNY
15


314
KTNSSSNDL
15


322
LEVEDRTSL
15


326
DRTSLVLNS
15


347
PTISSYENL
15


381
NLATPIAIK
15


388
IKAVPPSKR
15


397
FLKHGQNIR
15


11
LRSLASTLD
14


16
STLDCETAR
14


17
TLDCETARL
14


52
QTLKDDVNI
14


61
PELSNCENF
14


83
VASSCISGK
14


114
NPPQAVNLL
14


118
AVNLLDKAR
14


127
LENQEGIDF
14


129
NQEGIDFIK
14


145
KNSMDIMKI
14


151
MKIREYFQK
14


159
KYGYSPRVK
14


186
PVIVTPPTK
14


197
LVKVLKTPK
14


205
KCALKMDDF
14


212
DFECVTPKL
14


230
MCLNEDYTM
14


243
ARNNKSEEA
14


255
ESRLNDNVF
14


256
SRLNDNVFA
14


272
QLEKSDAEY
14


297
PSTKNSIAL
14


304
ALVSTNYPL
14


344
PSSPTISSY
14


349
ISSYENLLR
14


358
TPTPPEVTK
14


387
AIKAVPPSK
14


390
AVPPSKRFL
14


404
IRDVSNKEN
14


2
DPIRSFCGK
13


5
RSFCGKLRS
13


21
ETARLQRAL
13


39
YPMRILYDL
13


46
DLHSEVQTL
13


63
LSNCENFQK
13


95
SPQLSDFGL
13


98
LSDFGLERY
13


149
DIMKIREYF
13


152
KIREYFQKY
13


153
IREYFQKYG
13


157
FQKYGYSPR
13


180
DNYKEEPVI
13


189
VTPPTKQSL
13


193
TKQSLVKVL
13


201
LKTPKCALK
13


214
ECVTPKLEH
13


215
CVTPKLEHF
13


244
RNNKSEEAI
13


287
PTFCTPGLK
13


319
SNDLEVEDR
13


324
VEDRTSLVL
13


356
LRTPTPPEV
13


359
PTPPEVTKI
13


367
IPEDILQLL
13


392
PPSKRFLKH
13


3
PIRSFCGKL
12


19
DCETARLQR
12


26
QRALDGEES
12


37
EDYPMRILY
12


70
QKTDVKDDL
12


97
QLSDFGLER
12


99
SDFGLERYI
12


116
PQAVNLLDK
12


128
ENQEGIDFI
12


144
EKNSMDIMK
12


200
VLKTPKCAL
12


277
DAEYTNSPL
12


306
VSTNYPLSK
12


333
NSDTCFENL
12







V5-B2705-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









2
VASSCISEK
15


5
SCISEKSPR
15


9
EKSPRSPQL
14







V6-B2705-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









5
EAIDAESRL
15


4
EEAIDAESR
12







V10-B2705-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start positon plus eight.









1
KIPEDILQK
18


2
IPEDILQKF
15


11
QWIYPTQKL
15


13
IYPTQKLNK
15


14
YPTQKLNKM
15


6
ILQKFQWIY
14


15
PTQKLNKMR
14


10
FQWIYPTQK
13


5
DILQKFQWI
11


8
QKFQWIYPT
9







V12-B2705-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus eight.









1
RALDGEESL
19


5
GEESLLSKY
16


2
ALDGEESLL
15


4
DGEESLLSK
15


















TABLE XXXI





Pos
123456789
score















V1-HLA-B2709-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









104
ERYIVSQVL
22


125
ARLENQEGI
21


356
LRTPTPPEV
21


93
PRSPQLSDF
19


90
GKSPRSPQL
16


23
ARLQRALDG
15


326
DRTSLVLNS
15


366
KIPEDILQL
15


395
KRFLKHGQN
15


396
RFLKHGQNI
15


10
KLRSLASTL
14


113
PNPPQAVNL
14


119
VNLLDKARL
14


256
SRLNDNVFA
14


304
ALVSTNYPL
14


52
QTLKDDVNI
13


55
KDDVNIPEL
13


224
GISEYTMCL
13


265
TPSPIIQQL
13


314
KTNSSSNDL
13


347
PTISSYENL
13


389
KAVPPSKRF
13


39
YPMRILYDL
12


41
MRILYDLHS
12


46
DLHSEVQTL
12


61
PELSNCENF
12


133
IDFIKATKV
12


140
KVLMEKNSM
12


158
QKYGYSPRV
12


193
TKQSLVKVL
12


202
KTPKCALKM
12


244
RNNKSEEAI
12


250
EAIDTESRL
12


278
AEYTNSPLV
12


286
VPTFCTPGL
12


322
LEVEDRTSL
12


367
IPEDILQLL
12


390
AVPPSKRFL
12


3
PIRSFCGKL
11


4
IRSFCGKLR
11


17
TLDCETARL
11


26
QRALDGEES
11


43
ILYDLHSEV
11


70
QKTDVKDDL
11


75
KDDLSDPPV
11


103
LERYIVSQV
11


114
NPPQAVNLL
11


134
DFIKATKVL
11


145
KNSMDIMKI
11


153
IREYFQKYG
11


164
PRVKKNSVH
11


180
DNYKEEPVI
11


189
VTPPTKQSL
11


212
DFECVTPKL
11


230
MCLNEDYTM
11


243
ARNNKSEEA
11


281
TNSPLVPTF
11


295
KIPSTKNSI
11


297
PSTKNSIAL
11


324
VEDRTSLVL
11


333
NSDTCFENL
11


348
TISSYENLL
11


374
LLSKYNSNL
11


404
IRDVSNKEN
11


6
SFCGKLRSL
10


11
LRSLASTLD
10


21
ETARLQRAL
10


36
FEDYPMRIL
10


95
SPQLSDFGL
10


99
SDFGLERYI
10


200
VLKTPKCAL
10


205
KCALKMDDF
10


215
CVTPKLEHF
10


232
LNEDYTMGL
10


261
NVFATPSPI
10


277
DAEYTNSPL
10


316
NSSSNDLEV
10


330
LVLNSDTCF
10


341
LTDPSSPTI
10


359
PTPPEVTKI
10


363
EVTKIPEDI
10


364
VTKIPEDIL
10


380
SNLATPIAI
10


399
KHGQNIRDV
10







V5-HLA-B2709-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









9
EKSPRSPQL
13







V6-HLA-B2709-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









5
EAIDAESRL
12


9
AESRLNDNV
10







V10-HLA-B2709-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus eight.









11
QWIYPTQKL
11


2
IPEDILQKF
10


5
DILQKFQWI
10


14
YPTQKLNKM
9


1
KIPEDILQK
5







V12-HLA-B2709-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus eight.









1
RALDGEESL
16


2
ALDGEESLL
11


















TABLE XXXII





Pos
123456789
score















V1-HLA-B4402-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









324
VEDRTSLVL
24


220
LEHFGISEY
23


36
FEDYPMRIL
22


61
PELSNCENF
22


31
GEESDFEDY
21


127
LENQEGIDF
21


174
QEAINSDNY
21


322
LEVEDRTSL
21


154
REYFQKYGY
20


183
KEEPVIVTP
19


265
TPSPIIQQL
19


37
EDYPMRILY
18


390
AVPPSKRFL
18


134
DFIKATKVL
17


148
MDIMKIREY
17


344
PSSPTISSY
17


366
KIPEDILQL
17


21
ETARLQRAL
16


55
KDDVNIPEL
16


90
GKSPRSPQL
16


113
PNPPQAVNL
16


250
EAIDTESRL
16


255
ESRLNDNVF
16


278
AEYTNSPLV
16


281
TNSPLVPTF
16


389
KAVPPSKRF
16


28
ALDGEESDF
15


93
PRSPQLSDF
15


114
NPPQAVNLL
15


184
EEPVIVTPP
15


6
SFCGKLRSL
14


32
EESDFEDYP
14


39
YPMRILYDL
14


49
SEVQTLKDD
14


145
KNSMDIMKI
14


172
HEQEAINSD
14


189
VTPPTKQSL
14


193
TKQSLVKVL
14


213
FECVTPKLE
14


215
CVTPKLEHF
14


297
PSTKNSIAL
14


333
NSDTCFENL
14


359
PTPPEVTKI
14


362
PEVTKIPED
14


367
IPEDILQLL
14


380
SNLATPIAI
14


10
KLRSLASTL
13


99
SDFGLERYI
13


103
LERYIVSQV
13


104
ERYIVSQVL
13


125
ARLENQEGI
13


128
ENQEGIDFI
13


130
QEGIDFIKA
13


131
EGIDFIKAT
13


149
DIMKIREYF
13


152
KIREYFQKY
13


200
VLKTPKCAL
13


226
SEYTMCLNE
13


233
NEDYTMGLK
13


249
EEAIDTESR
13


304
ALVSTNYPL
13


341
LTDPSSPTI
13


347
PTISSYENL
13


348
TISSYENLL
13


368
PEDILQLLS
13


370
DILQLLSKY
13


17
TLDCETARL
12


20
CETARLQRA
12


46
DLHSEVQTL
12


95
SPQLSDFGL
12


98
LSDFGLERY
12


119
VNLLDKARL
12


205
KCALKMDDF
12


212
DFECVTPKL
12


224
GISEYTMCL
12


232
LNEDYTMGL
12


248
SEEAIDTES
12


254
TESRLNDNV
12


261
NVFATPSPI
12


302
SIALVSTNY
12


314
KTNSSSNDL
12


330
LVLNSDTCF
12


352
YENLLRTPT
12


363
EVTKIPEDI
12


3
PIRSFCGKL
11


70
QKTDVKDDL
11


143
MEKNSMDIM
11


169
NSVHEQEAI
11


228
YTMCLNEDY
11


273
LEKSDAEYT
11


286
VPTFCTPGL
11


288
TFCTPGLKI
11


295
KIPSTKNSI
11


338
FENLTDPSS
11


374
LLSKYNSNL
11


378
YNSNLATPI
11


383
ATPIAIKAV
11







V5-HLA-B4402-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









9
EKSPRSPQL
18


8
SEKSPRSPQ
12







V6-HLA-B4402-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









5
EAIDAESRL
16


9
AESRLNDNV
15


4
EEAIDAESR
13


3
SEEAIDAES
12







V10-HLA-B4402-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus eight.









4
EDILQKFQW
17


11
QWIYPTQKL
15


2
IPEDILQKF
14


3
PEDILQKFQ
13


5
DILQKFQWI
10


6
ILQKFQWIY
10







V12-HLA-B4402-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus eight.









5
GEESLLSKY
22


2
ALDGEESLL
16


6
EESLLSKYN
16


1
RALDGEESL
12


















TABLE XXXIII





Pos
123456789
score















V1-HLA-B5101-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









80
DPPVASSCI
25


180
DNYKEEPVI
22


190
TPPTKQSLV
22


277
DAEYTNSPL
22


114
NPPQAVNLL
21


217
TPKLEHFGI
21


163
SPRVKKNSV
20


367
IPEDILQLL
20


39
YPMRILYDL
19


250
EAIDTESRL
19


35
DFEDYPMRI
18


265
TPSPIIQQL
18


95
SPQLSDFGL
17


286
VPTFCTPGL
17


360
TPPEVTKIP
17


2
DPIRSFCGK
16


133
IDFIKATKV
16


303
IALVSTNYP
16


310
YPLSKTNSS
16


359
PTPPEVTKI
16


380
SNLATPIAI
16


382
LATPIAIKA
16


43
ILYDLHSEV
15


101
FGLERYIVS
15


104
ERYIVSQVL
15


112
LPNPPQAVN
15


115
PPQAVNLLD
15


134
DFIKATKVL
15


158
QKYGYSPRV
15


160
YGYSPRVKK
15


191
PPTKQSLVK
15


261
NVFATPSPI
15


288
TFCTPGLKI
15


296
IPSTKNSIA
15


341
LTDPSSPTI
15


343
DPSSPTISS
15


384
TPIAIKAVP
15


386
IAIKAVPPS
15


392
PPSKRFLKH
15


46
DLHSEVQTL
14


52
QTLKDDVNI
14


60
IPELSNCEN
14


125
ARLENQEGI
14


137
KATKVLMEK
14


206
CALKMDDFE
14


212
DFECVTPKL
14


263
FATPSPIIQ
14


358
TPTPPEVTK
14


378
YNSNLATPI
14


14
LASTLDCET
13


27
RALDGEESD
13


30
DGEESDFED
13


99
SDFGLERYI
13


117
QAVNLLDKA
13


128
ENQEGIDFI
13


142
LMEKNSMDI
13


145
KNSMDIMKI
13


181
NYKEEPVIV
13


192
PTKQSLVKV
13


193
TKQSLVKVL
13


203
TPKCALKMD
13


278
AEYTNSPLV
13


291
TPGLKIPST
13


295
KIPSTKNSI
13


391
VPPSKRFLK
13


396
RFLKHGQNI
13


81
PPVASSCIS
12


83
VASSCISGK
12


100
DFGLERYIV
12


103
LERYIVSQV
12


175
EAINSDNYK
12


185
EPVIVTPPT
12


208
LKMDDFECV
12


238
MGLKNARNN
12


244
RNNKSEEAI
12


262
VFATPSPII
12


283
SPLVPTFCT
12


292
PGLKIPSTK
12


324
VEDRTSLVL
12


356
LRTPTPPEV
12


361
PPEVIKIPE
12


363
EVTKIPEDI
12


389
KAVPPSKRF
12







V5-HLA-B5101-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









2
VASSCISEK
12


7
ISEKSPRSP
6


9
EKSPRSPQL
6







V6-HLA-B5101-9mers-193P1E1B


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


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


position for each peptide is the start position plus eight.









5
EAIDAESRL
18


8
DAESRLNDN
16


9
AESRLNDNV
9







V10-HLA-B5101-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus eight.









5
DILQKFQWI
18


14
YPTQKLNKM
17


2
IPEDILQKF
16


11
QWIYPTQKL
8







V12-HLA-B5101-9mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus eight.









1
RALDGEESL
19


4
DGEESLLSK
15


















TABLE XXXIV





Pos
1234567890
score















V1-HLA-A1-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









36
FEDYPMRILY
33


30
DGEESDFEDY
28


147
SMDIMKIREY
27


219
KLEHFGISEY
26


153
IREYFQKYGY
25


173
EQEAINSDNY
25


71
KTDVKDDLSD
22


129
NQEGIDFIKA
20


151
MKIREYFQKY
20


225
ISEYTMCLNE
20


341
LTDPSSPTIS
20


233
NEDYTMGLKN
19


301
NSIALVSTNY
19


78
LSDPPVASSC
18


251
AIDTESRLND
18


367
IPEDILQLLS
18


253
DTESRLNDNV
17


323
EVEDRTSLVL
17


369
EDILQLLSKY
17


97
QLSDFGLERY
16


126
RLENQEGIDF
16


227
EYTMCLNEDY
16


333
NSDTCFENLT
16


368
PEDILQLLSK
16







V5-HLA-A1-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









8
ISEKSPRSPQ
15







V6-HLA-A1-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









7
AIDAESRLND
18


3
KSEEAIDAES
14


4
SEEAIDAESR
12


9
DAESRLNDNV
11







V10-HLA-A1-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus nine.









6
DILQKFQWIY
15


1
TKIPEDILQK
10


3
IPEDILQKFQ
10


4
PEDILQKFQW
10


13
WIYPTQKLNK
10







V12-HLA-A1-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus nine.









5
DGEESLLSKY
27


3
ALDGEESLLS
21


















TABLE XXXV





Pos
1234567890
score















V1-HLA-A0201-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









355
LLRTPTPPEV
25


366
KIPEDILQLL
25


102
GLERYIVSQV
24


231
CLNEDYTMGL
24


141
VLMEKNSMDI
22


373
QLLSKYNSNL
22


16
STLDCETARL
21


42
RILYDLHSEV
21


207
ALKMDDFECV
21


340
NLTDPSSPTI
21


382
LATPIAIKAV
21


13
SLASTLDCET
20


45
YDLHSEVQTL
20


54
LKDDVNIPEL
20


132
GIDFIKATKV
20


264
ATPSPIIQQL
20


321
DLEVEDRTSL
20


110
QVLPNPPQAV
19


118
AVNLLDKARL
19


188
IVTPPTKQSL
19


303
IALVSTNYPL
19


365
TKIPEDILQL
19


77
DLSDPPVASS
18


199
KVLKTPKCAL
18


285
LVPTFCTPGL
18


381
NLATPIAIKA
18


398
LKHGQNIRDV
18


5
RSFCGKLRSL
17


112
LPNPPQAVNL
17


189
VTPPTKQSLV
17


290
CTPGLKIPST
17


294
LKIPSTKNSI
17


374
LLSKYNSNLA
17


389
KAVPPSKRFL
17


99
SDFGLERYIV
16


120
NLLDKARLEN
16


121
LLDKARLENQ
16


147
SMDIMKIREY
16


219
KLEHFGISEY
16


257
RLNDNVFATP
16


276
SDAEYTNSPL
16


9
GKLRSLASTL
15


97
QLSDFGLERY
15


106
YIVSQVLPNP
15


127
LENQEGIDFI
15


162
YSPRVKKNSV
15


211
DDFECVTPKL
15


229
TMCLNEDYTM
15


358
TPTPPEVTKI
15


28
ALDGEESDFE
14


38
DYPMRILYDL
14


43
ILYDLHSEVQ
14


113
PNPPQAVNLL
14


124
KARLENQEGI
14


191
PPTKQSLVKV
14


209
KMDDFECVTP
14


216
VTPKLEHFGI
14


261
NVFATPSPII
14


269
IIQQLEKSDA
14


280
YTNSPLVPTF
14


322
LEVEDRTSLV
14


331
VLNSDTCFEN
14


347
PTISSYENLL
14


2
DPIRSFCGKL
13


34
SDFEDYPMRI
13


74
VKDDLSDPPV
13


101
FGLERYIVSQ
13


111
VLPNPPQAVN
13


135
FIKATKVLME
13


142
LMEKNSMDIM
13


192
PTKQSLVKVL
13


223
FGISEYTMCL
13


239
GLKNARNNKS
13


256
SRLNDNVFAT
13


272
QLEKSDAEYT
13


287
PTFCTPGLKI
13


295
KIPSTKNSIA
13


296
IPSTKNSIAL
13


299
TKNSIALVST
13


302
SIALVSTNYP
13


304
ALVSTNYPLS
13


315
TNSSSNDLEV
13


332
LNSDTCFENL
13


354
NLLRTPTPPE
13


371
ILQLLSKYNS
13


14
LASTLDCETA
12


24
RLQRALDGEE
12


49
SEVQTLKDDV
12


59
NIPELSNCEN
12


79
SDPPVASSCI
12


87
CISGKSPRSP
12


89
SGKSPRSPQL
12


133
IDFIKATKVL
12


144
EKNSMDIMKI
12


176
AINSDNYKEE
12


180
DNYKEEPVIV
12


194
KQSLVKVLKT
12


196
SLVKVLKTPK
12


200
VLKTPKCALK
12


243
ARNNKSEEAI
12


253
DTESRLNDNV
12


277
DAEYTNSPLV
12


297
PSTKNSIALV
12


313
SKTNSSSNDL
12


323
EVEDRTSLVL
12


329
SLVLNSDTCF
12


385
PIAIKAVPPS
12







V5-HLA-A0201-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









7
CISEKSPRSP
12


9
SEKSPRSPQL
12


2
PVASSCISEK
9


3
VASSCISEKS
7


6
SCISEKSPRS
5







V6-HLA-A0201-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









9
DAESRLNDNV
12


7
AIDAESRLND
11


8
IDAESRLNDN
11


5
EEAIDAESRL
9


2
NKSEEAIDAE
7


3
KSEEAIDAES
6







V10-HLA-A0201-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus nine.









2
KIPEDILQKF
17


11
FQWIYPTQKL
14


14
IYPTQKLNKM
13


7
ILQKFQWIYP
12


13
WIYPTQKLNK
12







V12-HLA-A0201-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus nine.









9
SLLSKYNSNL
24


2
RALDGEESLL
17


3
ALDGEESLLS
15


1
QRALDGEESL
14


4
LDGEESLLSK
11


















TABLE XXXVI





Pos
1234567890
score















V1-HLA-A0203-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









6
SFCGKLRSLA
10


14
LASTLDCETA
10


19
DCETARLQRA
10


75
KDDLSDPPVA
10


109
SQVLPNPPQA
10


116
PQAVNLLDKA
10


129
NQEGIDFIKA
10


167
KKNSVHEQEA
10


198
VKVLKTPKCA
10


234
EDYTMGLKNA
10


242
NARNNKSEEA
10


255
ESRLNDNVFA
10


269
IIQQLEKSDA
10


295
KIPSTKNSIA
10


374
LLSKYNSNLA
10


378
YNSNLATPIA
10


381
NLATPIAIKA
10


7
FCGKLRSLAS
9


15
ASTLDCETAR
9


20
CETARLQRAL
9


76
DDLSDPPVAS
9


110
QVLPNPPQAV
9


117
QAVNLLDKAR
9


130
QEGIDFIKAT
9


168
KNSVHEQEAI
9


199
KVLKTPKCAL
9


235
DYTMGLKNAR
9


243
ARNNKSEEAI
9


256
SRLNDNVFAT
9


270
IQQLEKSDAE
9


296
IPSTKNSIAL
9


375
LSKYNSNLAT
9


379
NSNLATPIAI
9


382
LATPIAIKAV
9


8
CGKLRSLAST
8


16
STLDCETARL
8


21
ETARLQRALD
8


77
DLSDPPVASS
8


111
VLPNPPQAVN
8


118
AVNLLDKARL
8


131
EGIDFIKATK
8


169
NSVHEQEAIN
8


200
VLKTPKCALK
8


236
YTMGLKNARN
8


244
RNNKSEEAID
8


257
RLNDNVFATP
8


271
QQLEKSDAEY
8


297
PSTKNSIALV
8


376
SKYNSNLATP
8


380
SNLATPIAIK
8


383
ATPIAIKAVP
8







V5-HLA-A0203-10mers-193P1E1B


NoResultsFound.


V6-HLA-A0203-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









1
NNKSEEAIDA
10


2
NKSEEAIDAE
9


3
KSEEAIDAES
8







V10-HLA-A0203-10mers-193P1E1B


NoResultsFound.


V12-HLA-A0203-10mers-193P1E1B


NoResultsFound.


















TABLE XXXVII





Pos
1234567890
score















V1-HLA-A3-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









390
AVPPSKRFLK
27


305
LVSTNYPLSK
26


62
ELSNCENFQK
24


82
PVASSCISGK
24


200
VLKTPKCALK
24


158
QKYGYSPRVK
23


126
RLENQEGIDF
22


196
SLVKVLKTPK
22


219
KLEHFGISEY
22


257
RLNDNVFATP
22


357
RTPTPPEVTK
22


387
AIKAVPPSKR
22


43
ILYDLHSEVQ
21


46
DLHSEVQTLK
21


131
EGIDFIKATK
21


97
QLSDFGLERY
19


102
GLERYIVSQV
19


110
QVLPNPPQAV
19


140
KVLMEKNSMD
19


291
TPGLKIPSTK
19


3
PIRSFCGKLR
18


24
RLQRALDGEE
18


111
VLPNPPQAVN
18


159
KYGYSPRVKK
18


323
EVEDRTSLVL
18


380
SNLATPIAIK
18


386
IAIKAVPPSK
18


10
KLRSLASTLD
17


42
RILYDLHSEV
17


118
AVNLLDKARL
17


120
NLLDKARLEN
17


150
IMKIREYFQK
17


188
IVTPPTKQSL
17


190
TPPTKQSLVK
17


329
SLVLNSDTCF
17


373
QLLSKYNSNL
17


77
DLSDPPVASS
16


185
EPVIVTPPTK
16


199
KVLKTPKCAL
16


368
PEDILQLLSK
16


9
GKLRSLASTL
15


28
ALDGEESDFE
15


115
PPQAVNLLDK
15


135
FIKATKVLME
15


152
KIREYFQKYG
15


163
SPRVKKNSVH
15


165
RVKKNSVHEQ
15


170
SVHEQEAINS
15


186
PVIVTPPTKQ
15


251
AIDTESRLND
15


272
QLEKSDAEYT
15


278
AEYTNSPLVP
15


311
PLSKTNSSSN
15


340
NLTDPSSPTI
15


348
TISSYENLLR
15


354
NLLRTPTPPE
15


27
RALDGEESDF
14


50
EVQTLKDDVN
14


57
DVNIPELSNC
14


66
CENFQKTDVK
14


136
IKATKVLMEK
14


174
QEAINSDNYK
14


193
TKQSLVKVLK
14


207
ALKMDDFECV
14


266
PSPIIQQLEK
14


271
QQLEKSDAEY
14


284
PLVPTFCTPG
14


295
KIPSTKNSIA
14


321
DLEVEDRTSL
14


355
LLRTPTPPEV
14


381
NLATPIAIKA
14


401
GQNIRDVSNK
14


17
TLDCETARLQ
13


87
CISGKSPRSP
13


107
IVSQVLPNPP
13


143
MEKNSMDIMK
13


215
CVTPKLEHFG
13


238
MGLKNARNNK
13


268
PIIQQLEKSD
13


269
IIQQLEKSDA
13


298
STKNSIALVS
13


304
ALVSTNYPLS
13


330
LVLNSDTCFE
13


369
EDILQLLSKY
13


371
ILQLLSKYNS
13


376
SKYNSNLATP
13







V5-HLA-A3-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









2
PVASSCISEK
24


7
CISEKSPRSP
12







V6-HLA-A3-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









7
AIDAESRLND
17


10
AESRLNDNVF
14


4
SEEAIDAESR
12


3
KSEEAIDAES







V10-HLA-A3-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus nine.









13
WIYPTQKLNK
28


1
TKIPEDILQK
22


10
KFQWIYPTQK
18


2
KIPEDILQKF
16


6
DILQKFQWIY
16







V12-HLA-A3-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus nine.









3
ALDGEESLLS
18


4
LDGEESLLSK
16


9
SLLSKYNSNL
16


2
RALDGEESLL
10


5
DGEESLLSKY
10


















TABLE XXXVIII





Pos
1234567890
score















V1-HLA-A26-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









323
EVEDRTSLVL
30


369
EDILQLLSKY
30


363
EVTKIPEDIL
29


214
ECVTPKLEHF
26


343
DPSSPTISSY
25


57
DVNIPELSNC
23


211
DDFECVTPKL
23


2
DPIRSFCGKL
22


264
ATPSPIIQQL
22


280
YTNSPLVPTF
22


188
IVTPPTKQSL
21


227
EYTMCLNEDY
21


347
PTISSYENLL
21


30
DGEESDFEDY
20


38
DYPMRILYDL
20


50
EVQTLKDDVN
20


173
EQEAINSDNY
20


192
PTKQSLVKVL
20


335
DTCFENLTDP
20


365
TKIPEDILQL
20


73
DVKDDLSDPP
19


199
KVLKTPKCAL
19


249
EEAIDTESRL
19


16
STLDCETARL
18


21
ETARLQRALD
18


118
AVNLLDKARL
18


285
LVPTFCTPGL
18


366
KIPEDILQLL
18


5
RSFCGKLRSL
17


35
DFEDYPMRIL
17


253
DTESRLNDNV
17


321
DLEVEDRTSL
17


37
EDYPMRILYD
16


82
PVASSCISGK
16


131
EGIDFIKATK
16


144
EKNSMDIMKI
16


155
EYFQKYGYSP
16


67
ENFQKTDVKD
15


92
SPRSPQLSDF
15


97
QLSDFGLERY
15


147
SMDIMKIREY
15


151
MKIREYFQKY
15


175
EAINSDNYKE
15


185
EPVIVTPPTK
15


186
PVIVTPPTKQ
15


219
KLEHFGISEY
15


221
EHFGISEYTM
15


250
EAIDTESRLN
15


279
EYTNSPLVPT
15


287
PTFCTPGLKI
15


325
EDRTSLVLNS
15


326
DRTSLVLNSD
15


56
DDVNIPELSN
14


77
DLSDPPVASS
14


165
RVKKNSVHEQ
14


170
SVHEQEAINS
14


234
EDYTMGLKNA
14


261
NVFATPSPII
14


301
NSIALVSTNY
14







V5-HLA-A26-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









2
PVASSCISEK
16


9
SEKSPRSPQL
11


10
EKSPRSPQLS
11


7
CISEKSPRSP
7







V6-HLA-A26-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









5
EEAIDAESRL
19


6
EAIDAESRLN
15


9
DAESRLNDNV
9







V10-HLA-A26-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus nine.









6
DILQKFQWIY
22


2
KIPEDILQKF
19


5
EDILQKFQWI
14


1
TKIPEDILQK
12







V12-HLA-A26-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus nine.









5
DGEESLLSKY
26


















TABLE XXXIX





Pos
1234567890
score















V1-HLA-B0702-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









296
IPSTKNSIAL
24


112
LPNPPQAVNL
23


2
DPIRSFCGKL
20


346
SPTISSYENL
20


191
PPTKQSLVKV
19


358
TPTPPEVTKI
19


92
SPRSPQLSDF
18


60
IPELSNCENF
17


323
EVEDRTSLVL
14


384
TPIAIKAVPP
14


103
LERYIVSQVL
13


115
PPQAVNLLDK
13


118
AVNLLDKARL
13


133
IDFIKATKVL
13


163
SPRVKKNSVH
13


190
TPPTKQSLVK
13


199
KVLKTPKCAL
13


265
TPSPIIQQLE
13


332
LNSDTCFENL
13


365
TKIPEDILQL
13


367
IPEDILQLLS
13


389
KAVPPSKRFL
13


391
VPPSKRFLKH
13


392
PPSKRFLKHG
13


16
STLDCETARL
12


20
CETARLQRAL
12


54
LKDDVNIPEL
12


113
PNPPQAVNLL
12


114
NPPQAVNLLD
12


185
EPVIVTPPTK
12


188
IVTPPTKQSL
12


192
PTKQSLVKVL
12


194
KQSLVKVLKT
12


211
DDFECVTPKL
12


249
EEAIDTESRL
12


255
ESRLNDNVFA
12


264
ATPSPIIQQL
12


276
SDAEYTNSPL
12


285
LVPTFCTPGL
12


303
IALVSTNYPL
12


321
DLEVEDRTSL
12


343
DPSSPTISSY
12


363
EVTKIPEDIL
12


5
RSFCGKLRSL
11


39
YPMRILYDLH
11


45
YDLHSEVQTL
11


81
PPVASSCISG
11


89
SGKSPRSPQL
11


94
RSPQLSDFGL
11


95
SPQLSDFGLE
11


217
TPKLEHFGIS
11


223
FGISEYTMCL
11


231
CLNEDYTMGL
11


283
SPLVPTFCTP
11


291
TPGLKIPSTK
11


349
ISSYENLLRT
11


360
TPPEVTKIPE
11


361
PPEVTKIPED
11


366
KIPEDILQLL
11







V5-HLA-B0702-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









1
PPVASSCISE
11


9
SEKSPRSPQL
11







V6-HLA-B0702-10mers-193P1E1B


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


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


position for each peptide is the start position plus nine.









5
EEAIDAESRL
12


10
AESRLNDNVF
11


7
AIDAESRLND
7


1
NNKSEEAIDA
6


9
DAESRLNDNV
6







V10-HLA-B0702-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21; each start position is


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


position for each peptide is the start position plus nine.









3
IPEDILQKFQ
12


11
FQWIYPTQKL
11


15
YPTQKLNKMR
10


5
EDILQKFQWI
8


2
KIPEDILQKF
7


8
LQKFQWIYPT
7


14
IYPTQKLNKM
7







V12-HLA-B0702-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25; each start position is


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


position for each peptide is the start position plus nine.









1
QRALDGEESL
11


2
RALDGEESLL
11


9
SLLSKYNSNL
10


3
ALDGEESLLS
7


















TABLE XL





Pos
1234567890
score















V1-HLA-B08-10mers-193P1E1B


NoResultsFound.


V5-HLA-B08-10mers-193P1E1B


NoResultsFound.


V6-HLA-B08-10mers-193P1E1B


NoResultsFound.


V10-HLA-B08-10mers-193P1E1B


NoResultsFound.


V12-HLA-B08-10mers-193P1E1B


NoResultsFound.


















TABLE XLI





Pos
1234567890
score















V1-HLA-B1510-10mers-193P1E1B


NoResultsFound.


V5-HLA-B1510-10mers-193P1E1B


NoResultsFound.


V6-HLA-B1510-10mers-193P1E1B


NoResultsFound.


V10-HLA-B1510-10mers-193P1E1B


NoResultsFound.


V12-HLA-B1510-10mers-193P1E1B


NoResultsFound.


















TABLE XLII





Pos
1234567890
score















V1-HLA-B2705-10mers-193P1E1B


NoResultsFound.


V5-HLA-B2705-10mers-193P1E1B


NoResultsFound.


V6-HLA-B2705-10mers-193P1E1B


NoResultsFound.


V10-HLA-B2705-10mers-193P1E1B


NoResultsFound.


V12-HLA-B2705-10mers-193P1E1B


NoResultsFound.


















TABLE XLIII





Pos
1234567890
score















V1-HLA-B2709-10mers-193P1E1B


NoResultsFound.


V5-HLA-B2709-10mers-193P1E1B


NoResultsFound.


V6-HLA-B2709-10mers-193P1E1B


NoResultsFound.


V10-HLA-B2709-10mers-193P1E1B


NoResultsFound.


V12-HLA-B2709-10mers-193P1E1B


NoResultsFound.


















TABLE XLIV





Pos
1234567890
score















V1-HLA-B4402-10mers-193P1E1B


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


each start position is specified, the length of


peptide is 10 amino acids, and the end postion


for each peptide is the start position plus nine.









36
FEDYPMRILY
24


254
TESRLNDNVF
24


20
CETARLQRAL
23


249
EEAIDTESRL
22


103
LERYIVSQVL
21


127
LENQEGIDFI
21


365
TKIPEDILQL
21


362
PEVTKIPEDI
20


264
ATPSPIIQQL
18


369
EDILQLLSKY
18


113
PNPPQAVNLL
17


130
QEGIDFIKAT
17


278
AEYTNSPLVP
17


133
IDFIKATKVL
16


147
SMDIMKIREY
16


151
MKIREYFQKY
16


2
DPIRSFCGKL
15


54
LKDDVNIPEL
15


112
LPNPPQAVNL
15


183
KEEPVIVTPP
15


294
LKIPSTKNSI
15


296
IPSTKNSIAL
15


323
EVEDRTSLVL
15


324
VEDRTSLVLN
15


347
PTISSYENLL
15


389
KAVPPSKRFL
15


5
RSFCGKLRSL
14


9
GKLRSLASTL
14


16
STLDCETARL
14


32
EESDFEDYPM
14


118
AVNLLDKARL
14


144
EKNSMDIMKI
14


148
MDIMKIREYF
14


184
EEPVIVTPPT
14


192
PTKQSLVKVL
14


199
KVLKTPKCAL
14


211
DDFECVTPKL
14


214
ECVTPKLEHF
14


219
KLEHFGISEY
14


223
FGISEYTMCL
14


226
SEYTMCLNED
14


233
NEDYTMGLKN
14


243
ARNNKSEEAI
14


301
NSIALVSTNY
14


343
DPSSPTISSY
14


366
KIPEDILQLL
14


379
NSNLATPIAI
14


45
YDLHSEVQTL
13


79
SDPPVASSCI
13


97
QLSDFGLERY
13


188
IVTPPTKQSL
13


285
LVPTFCTPGL
13


313
SKTNSSSNDL
13


332
LNSDTCFENL
13


352
YENLLRTPTP
13


358
TPTPPEVTKI
13


368
PEDILQLLSK
13


27
RALDGEESDF
12


34
SDFEDYPMRI
12


35
DFEDYPMRIL
12


38
DYPMRILYDL
12


61
PELSNCENFQ
12


66
CENFQKTDVK
12


89
SGKSPRSPQL
12


92
SPRSPQLSDF
12


126
RLENQEGIDF
12


143
MEKNSMDIMK
12


168
KNSVHEQEAI
12


173
EQEAINSDNY
12


227
EYTMCLNEDY
12


248
SEEAIDTESR
12


280
YTNSPLVPTF
12


287
PTFCTPGLKI
12


322
LEVEDRTSLV
12


329
SLVLNSDTCF
12


338
FENLTDPSSP
12


363
EVTKIPEDIL
12


388
IKAVPPSKRF
12


395
KRFLKHGQNI
12


30
DGEESDFEDY
11


49
SEVQTLKDDV
11


60
IPELSNCENF
11


69
FQKTDVKDDL
11


94
RSPQLSDFGL
11


174
QEAINSDNYK
11


213
FECVTPKLEH
11


231
CLNEDYTMGL
11


261
NVFATPSPII
11


271
QQLEKSDAEY
11


273
LEKSDAEYTN
11


276
SDAEYTNSPL
11


303
IALVSTNYPL
11


321
DLEVEDRTSL
11


340
NLTDPSSPTI
11


346
SPTISSYENL
11


373
QLLSKYNSNL
11







V5-HLA-B4402-10mers-193P1E1B


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


each start position is specified, the length of


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









9
SEKSPRSPQL
22







V6-HLA-B4402-10mers-193P1E1B


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


each start position is specified, the length of


peptide is 10 amino acids, and the end position


for each peptide is the start postion plus nine.









10
AESRLNDNVF
27


5
EEAIDAESRL
22


4
SEEAIDAESR
12







V10-HLA-B4402-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21;


each start position is specified, the length of


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









4
PEDILQKFQW
22


5
EDILQKFQWI
15


2
KIPEDILQKF
14


11
FQWIYPTQKL
12


1
TKIPEDILQK
11


6
DILQKFQWIY
11







V12-HLA-B4402-10mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25;


each start postition is specified, the length of


peptide is 10 amino acids, and the end position


for each peptide is the start position plus nine.









7
EESLLSKYNS
14


2
RALDGEESLL
13


5
DGEESLLSKY
12


6
GEESLLSKYN
12


9
SLLSKYNSNL
12


1
QRALDGEESL
11


3
ALDGEESLLS
7


















TABLE XLV





Pos
123456789012345
score















V1-HLA-B5101-10mers-193P1E1B


NoResultsFound.


V5-HLA-B5101-10mers-193P1E1B


NoResultsFound.


V6-HLA-B5101-10mers-193P1E1B


NoResultsFound.


V10-HLA-B5101-10mers-193P1E1B


NoResultsFound.


V12-HLA-B5101-10mers-193P1E1B


NoResultsFound.


















TABLE XLVI





Pos
123456789012345
score















V1-HLA-DRB1-0101-15mers-193P1E1B


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


each start position is specified, the length of


peptide is 15 amino acids, and the end position


for each peptide is the start position plus fourteen.









130
QEGIDFIKATKVLME
33


124
KARLENQEGIDFIKA
31


168
KNSVHEQEAINSDNY
31


38
DYPMRILYDLHSEVQ
29


300
KNSIALVSTNYPLSK
29


105
RYIVSQVLPNPPQAV
28


375
LSKYNSNLATPIAIK
28


291
TPGLKIPSTKNSIAL
27


23
ARLQRALDGEESDFE
26


116
PQAVNLLDKARLENQ
26


153
IREYFQKYGYSPRVK
26


335
DTCFENLTDPSSPTI
26


8
CGKLRSLASTLDCET
25


229
TMCLNEDYTMGLKNA
25


267
SPIIQQLEKSDAEYT
25


307
STNYPLSKTNSSSND
25


361
PPEVTKIPEDILQLL
25


369
EDILQLLSKYNSNLA
25


379
NSNLATPIAIKAVPP
25


5
RSFCGKLRSLASTLD
24


185
EPVIVTPPTKQSLVK
24


195
QSLVKVLKTPKCALK
24


283
SPLVPTFCTPGLKIP
24


350
SSYENLLRTPTPPEV
24


353
ENLLRTPTPPEVTKI
24


372
LQLLSKYNSNLATPI
24


71
KTDVKDDLSDPPVAS
23


82
PVASSCISGKSPRSP
23


85
SSCISGKSPRSPQLS
23


104
ERYIVSQVLPNPPQA
23


210
MDDFECVTPKLEHFG
23


256
SRLNDNVFATPSPII
23


338
FENLTDPSSPTISSY
23


368
PEDILQLLSKYNSNL
23


41
MRILYDLHSEVQTLK
22


101
FGLERYIVSQVLPNP
22


108
VSQVLPNPPQAVNLL
22


139
TKVLMEKNSMDIMKI
22


259
NDNVFATPSPIIQQL
22


321
DLEVEDRTSLVLNSD
22


376
SKYNSNLATPIAIKA
22


382
LATPIAIKAVPPSKR
22


385
PIAIKAVPPSKRFLK
22


214
ECVTPKLEHFGISEY
21


77
DLSDPPVASSCISGK
20


132
GIDFIKATKVLMEKN
20


286
VPTFCTPGLKIPSTK
20


1
MDPIRSFCGKLRSLA
19


33
ESDFEDYPMRILYDL
19


36
FEDYPMRILYDLHSE
19


98
LSDFGLERYIVSQVL
19


159
KYGYSPRVKKNSVHE
19


327
RTSLVLNSDTCFENL
19


349
ISSYENLLRTPTPPE
19


383
ATPIAIKAVPPSKRF
19


4
IRSFCGKLRSLASTL
18


92
SPRSPQLSDFGLERY
18


131
EGIDFIKATKVLMEK
18


136
IKATKVLMEKNSMDI
18


140
KVLMEKNSMDIMKIR
18


145
KNSMDIMKIREYFQK
18


189
VTPPTKQSLVKVLKT
18


194
KQSLVKVLKTPKCAL
18


197
LVKVLKTPKCALKMD
18


199
KVLKTPKCALKMDDF
18


205
KCALKMDDFECVTPK
18


227
EYTMCLNEDYTMGLK
18


235
DYTMGLKNARNNKSE
18


237
TMGLKNARNNKSEEA
18


265
TPSPIIQQLEKSDAE
18


270
IQQLEKSDAEYTNSP
18


15
ASTLDCETARLQRAL
17


20
CETARLQRALDGEES
17


97
QLSDFGLERYIVSQV
17


102
GLERYIVSQVLPNPP
17


110
QVLPNPPQAVNLLDK
17


137
KATKVLMEKNSMDIM
17


178
NSDNYKEEPVIVTPP
17


186
PVIVTPPTKQSLVKV
17


196
SLVKVLKTPKCALKM
17


207
ALKMDDFECVTPKLE
17


255
ESRLNDNVFATPSPI
17


285
LVPTFCTPGLKIPST
17


296
IPSTKNSIALVSTNY
17


302
SIALVSTNYPLSKTN
17


306
VSTNYPLSKTNSSSN
17


319
SNDLEVEDRTSLVLN
17


393
PSKRFLKHGQNIRDV
17


7
FCGKLRSLASTLDCE
16


12
RSLASTLDCETARLQ
16


40
PMRILYDLHSEVQTL
16


48
HSEVQTLKDDVNIPE
16


57
DVNIPELSNCENFQK
16


72
TDVKDDLSDPPVASS
16


75
KDDLSDPPVASSCIS
16


100
DFGLERYIVSQVLPN
16


107
IVSQVLPNPPQAVNL
16


142
LMEKNSMDIMKIREY
16


147
SMDIMKIREYFQKYG
16


183
KEEPVIVTPPTKQSL
16


184
EEPVIVTPPTKQSLV
16


202
KTPKCALKMDDFECV
16


232
LNEDYTMGLKNARNN
16


240
LKNARNNKSEEAIDT
16


252
IDTESRLNDNVFATP
16


258
LNDNVFATPSPIIQQ
16


260
DNVFATPSPIIQQLE
16


290
CTPGLKIPSTKNSIA
16


293
GLKIPSTKNSIALVS
16


309
NYPLSKTNSSSNDLE
16


318
SSNDLEVEDRTSLVL
16


336
TCFENLTDPSSPTIS
16


351
SYENLLRTPTPPEVT
16


364
VTKIPEDILQLLSKY
16


371
ILQLLSKYNSNLATP
16


377
KYNSNLATPIAIKAV
16


380
SNLATPIAIKAVPPS
16







V5-HLA-DRB1-0101-15mers-193P1E1B


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


each start position is specified, the length of


peptide is 15 amino acids, and the end position


for each peptide is the start position plus fourteen.









10
SSCISEKSPRSPQLS
23


2
DLSDPPVASSCISEK
20


6
PPVASSCISEKSPRS
15


7
PVASSCISEKSPRSP
15


12
CISEKSPRSPQLSDF
15


3
LSDPPVASSCISEKS
14


9
ASSCISEKSPRSPQL
14


13
ISEKSPRSPQLSDFG
14







V6-HLA-DRB1-0101-15mers-193P1E1B


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


each start position is specified, the length of


peptide is 15 amino acids, and the end position


for each peptide is the start position plus fourteen.









4
ARNNKSEEAIDAESR
18


1
LKNARNNKSEEAIDA
16


13
IDAESRLNDNVFATP
16


7
NKSEEAIDAESRLND
14


8
KSEEAIDAESRLNDN
11


14
DAESRLNDNVFATPS
11


2
KNARNNKSEEAIDAE
9


6
NNKSEEAIDAESRLN
8


9
SEEAIDAESRLNDNV
8


10
EEAIDAESRLNDNVF
8


12
AIDAESRLNDNVFAT
8







V10-HLA-DRB1-0101-15mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21;


each start position is specified, the length of


peptide is 15 amino acids, and the end position


for each peptide is the start position plus fourteen.









2
PPEVTKIPEDILQKF
25


13
LQKFQWIYPTQKLNK
23


10
EDILQKFQWIYPTQK
18


14
QKFQWIYPTQKLNKM
18


5
VTKIPEDILQKFQWI
17


1
TPPEVTKIPEDILQK
15


8
IPEDILQKFQWIYPT
15







V12-HLA-DRB1-0101-15mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25;


each start position is specified, the length of


peptide is 15 amino acids, and the end position


for each peptide is the start position plus fourteen.









3
ARLQRALDGEESLLS
26


13
ESLLSKYNSNLATPI
24


6
QRALDGEESLLSKYN
23


10
DGEESLLSKYNSNLA
17


12
EESLLSKYNSNLATP
16


9
LDGEESLLSKYNSNL
15


















TABLE XLVII





Pos
123456789012345
score















V1-HLA-DRB1-0301-15mers-193P1E1B


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


each start position is specified, the length of


peptide is 15 amino acids, and the end position


for each peptide is the start position plus fourteen.









364
VTKIPEDILQLLSKY
31


229
TMCLNEDYTMGLKNA
29


197
LVKVLKTPKCALKMD
28


371
ILQLLSKYNSNLATP
28


51
VQTLKDDVNIPELSN
27


186
PVIVTPPTKQSLVKV
27


116
PQAVNLLDKARLENQ
26


361
PPEVTKIPEDILQLL
26


67
ENFQKTDVKDDLSDP
25


247
KSEEAIDTESRLNDN
24


319
SNDLEVEDRTSLVLN
24


40
PMRILYDLHSEVQTL
23


71
KTDVKDDLSDPPVAS
22


205
KCALKMDDFECVTPK
22


174
QEAINSDNYKEEPVI
21


327
RTSLVLNSDTCFENL
21


329
SLVLNSDTCFENLTD
21


138
ATKVLMEKNSMDIMK
20


346
SPTISSYENLLRTPT
20


13
SLASTLDCETARLQR
19


95
SPQLSDFGLERYIVS
19


118
AVNLLDKARLENQEG
19


124
KARLENQEGIDFIKA
19


145
KNSMDIMKIREYFQK
19


213
FECVTPKLEHFGISE
19


217
TPKLEHFGISEYTMC
19


237
TMGLKNARNNKSEEA
19


249
EEAIDTESRLNDNVF
19


321
DLEVEDRTSLVLNSD
19


369
EDILQLLSKYNSNLA
19


388
IKAVPPSKRFLKHGQ
19


24
RLQRALDGEESDFED
18


44
LYDLHSEVQTLKDDV
18


57
DVNIPELSNCENFQK
18


109
SQVLPNPPQAVNLLD
18


150
IMKIREYFQKYGYSP
18


194
KQSLVKVLKTPKCAL
18


266
PSPIIQQLEKSDAEY
18


271
QQLEKSDAEYTNSPL
18


283
SPLVPTFCTPGLKIP
18


293
GLKIPSTKNSIALVS
18


315
TNSSSNDLEVEDRTS
18


345
SSPTISSYENLLRTP
18


394
SKRFLKHGQNIRDVS
18


18
LDCETARLQRALDGE
17


25
LQRALDGEESDFEDY
17


29
LDGEESDFEDYPMRI
17


33
ESDFEDYPMRILYDL
17


60
IPELSNCENFQKTDV
17


132
GIDFIKATKVLMEKN
17


225
ISEYTMCLNEDYTMG
17


267
SPIIQQLEKSDAEYT
17


387
AIKAVPPSKRFLKHG
17


395
KRFLKHGQNIRDVSN
17


4
IRSFCGKLRSLASTL
16


74
VKDDLSDPPVASSCI
16


108
VSQVLPNPPQAVNLL
16


146
NSMDIMKIREYFQKY
16


147
SMDIMKIREYFQKYG
16


206
CALKMDDFECVTPKL
16


301
NSIALVSTNYPLSKT
16


34
SDFEDYPMRILYDLH
15


117
QAVNLLDKARLENQE
15


274
EKSDAEYTNSPLVPT
15







V5-HLA-DRB1-0301-15mers


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


each start position is specified, the length of


peptide is 15 amino acids, and the end position


for each peptide is the start position plus fourteen.









10
SSCISEKSPRSPQLS
12


5
DPPVASSCISEKSPR
11


12
CISEKSPRSPQLSDF
10


15
EKSPRSPQLSDFGLE
10


14
SEKSPRSPQLSDFGL
8


7
PVASSCISEKSPRSP
7


8
VASSCISEKSPRSPQ
7


11
SCISEKSPRSPQLSD
7







V6-HLA-DRB1-0301-15mers


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


each start position is specified, the length of


peptide is 15 amino acids, and the end position


for each peptide is the start position plus fourteen.









8
KSEEAIDAESRLNDN
24


10
EEAIDAESRLNDNVF
19


15
AESRLNDNVFATPSP
13







V10-HLA-DRB1-0301-15mers


Each peptide is a portion of SEQ ID NO:21;


each start position is specified, the length of


peptide is 15 amino acids, and the end position


for each peptide is the start position plus fourteen.









5
VTKIPEDILQKFQWI
31


2
PPEVTKIPEDILQKF
26


9
PEDILQKFQWIYPTQ
26







V12-HLA-DRB1-0301-15mers


Each peptide is a portion of SEQ ID NO:25;


each start position is specified, the length of


peptide is 15 amino acids, and the end position


for each peptide is the start position plus fourteen.









12
EESLLSKYNSNLATP
28


4
RLQRALDGEESLLSK
26


5
LQRALDGEESLLSKY
18


6
QRALDGEESLLSKYN
13


















TABLE XLVIII





Pos
123456789012345
score















V1-HLA-DR1-0401-15mers-193P1E1B


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


each start position is specified, the length of


peptide is 15 amino acids, and the end position


for each peptide is the start position plus fourteen.









349
ISSYENLLRTPTPPE
28


40
PMRILYDLHSEVQTL
26


41
MRILYDLHSEVQTLK
26


44
LYDLHSEVQTLKDDV
26


57
DVNIPELSNCENFQK
26


229
TMCLNEDYTMGLKNA
26


237
TMGLKNARNNKSEEA
26


283
SPLVPTFCTPGLKIP
26


319
SNDLEVEDRTSLVLN
26


368
PEDILQLLSKYNSNL
26


98
LSDFGLERYIVSQVL
22


132
GIDFIKATKVLMEKN
22


157
FQKYGYSPRVKKNSV
22


179
SDNYKEEPVIVTPPT
22


307
STNYPLSKTNSSSND
22


335
DTCFENLTDPSSPTI
22


8
CGKLRSLASTLDCET
20


15
ASTLDCETARLQRAL
20


26
QRALDGEESDFEDYP
20


38
DYPMRILYDLHSEVQ
20


48
HSEVQTLKDDVNIPE
20


51
VQTLKDDVNIPELSN
20


60
IPELSNCENFQKTDV
20


71
KTDVKDDLSDPPVAS
20


109
SQVLPNPPQAVNLLD
20


116
PQAVNLLDKARLENQ
20


119
VNLLDKARLENQEGI
20


130
QEGIDFIKATKVLME
20


138
ATKVLMEKNSMDIMK
20


147
SMDIMKIREYFQKYG
20


185
EPVIVTPPTKQSLVK
20


194
KQSLVKVLKTPKCAL
20


195
QSLVKVLKTPKCALK
20


205
KCALKMDDFECVTPK
20


249
EEAIDTESRLNDNVF
20


259
NDNVFATPSPIIQQL
20


267
SPIIQQLEKSDAEYT
20


291
TPGLKIPSTKNSIAL
20


293
GLKIPSTKNSIALVS
20


300
KNSIALVSTNYPLSK
20


309
NYPLSKTNSSSNDLE
20


329
SLVLNSDTCFENLTD
20


338
FENLTDPSSPTISSY
20


361
PPEVTKIPEDILQLL
20


364
VTKIPEDILQLLSKY
20


369
EDILQLLSKYNSNLA
20


372
LQLLSKYNSNLATPI
20


388
IKAVPPSKRFLKHGQ
20


5
RSFCGKLRSLASTLD
18


77
DLSDPPVASSCISGK
18


78
LSDPPVASSCISGKS
18


97
QLSDFGLERYIVSQV
18


101
FGLERYIVSQVLPNP
18


106
YIVSQVLPNPPQAVN
18


122
LDKARLENQEGIDFI
18


170
SVHEQEAINSDNYKE
18


182
YKEEPVIVTPPTKQS
18


214
ECVTPKLEHFGISEY
18


221
EHFGISEYTMCLNED
18


234
EDYTMGLKNARNNKS
18


264
ATPSPIIQQLEKSDA
18


280
YTNSPLVPTFCTPGL
18


290
CTPGLKIPSTKNSIA
18


320
NDLEVEDRTSLVLNS
18


325
EDRTSLVLNSDTCFE
18


337
CFENLTDPSSPTISS
18


343
DPSSPTISSYENLLR
18


365
TKIPEDILQLLSKYN
18


376
SKYNSNLATPIAIKA
18


392
PPSKRFLKHGQNIRD
18


4
IRSFCGKLRSLASTL
17


33
ESDFEDYPMRILYDL
16


42
RILYDLHSEVQTLKD
16


103
LERYIVSQVLPNPPQ
16


210
MDDFECVTPKLEHFG
16


225
ISEYTMCLNEDYTMG
16


260
DNVFATPSPIIQQLE
16


277
DAEYTNSPLVPTFCT
16


375
LSKYNSNLATPIAIK
16


394
SKRFLKHGQNIRDVS
16


118
AVNLLDKARLENQEG
15


139
TKVLMEKNSMDIMKI
15


321
DLEVEDRTSLVLNSD
15


371
ILQLLSKYNSNLATP
15


1
MDPIRSFCGKLRSLA
14


11
LRSLASTLDCETARL
14


22
TARLQRALDGEESDF
14


75
KDDLSDPPVASSCIS
14


80
DPPVASSCISGKSPR
14


95
SPQLSDFGLERYIVS
14


100
DFGLERYIVSQVLPN
14


105
RYIVSQVLPNPPQAV
14


108
VSQVLPNPPQAVNLL
14


140
KVLMEKNSMDIMKIR
14


150
IMKIREYFQKYGYSP
14


163
SPRVKKNSVHEQEAI
14


168
KNSVHEQEAINSDNY
14


174
QEAINSDNYKEEPVI
14


184
EEPVIVTPPTKQSLV
14


186
PVIVTPPTKQSLVKV
14


197
LVKVLKTPKCALKMD
14


198
VKVLKTPKCALKMDD
14


207
ALKMDDFECVTPKLE
14


217
TPKLEHFGISEYTMC
14


222
HFGISEYTMCLNEDY
14


227
EYTMCLNEDYTMGLK
14


270
IQQLEKSDAEYTNSP
14


302
SIALVSTNYPLSKTN
14


303
IALVSTNYPLSKTNS
14


328
TSLVLNSDTCFENLT
14


346
SPTISSYENLLRTPT
14


352
YENLLRTPTPPEVTK
14


353
ENLLRTPTPPEVTKI
14


379
NSNLATPIAIKAVPP
14


385
PIAIKAVPPSKRFLK
14


395
KRFLKHGQNIRDVSN
14







V5-HLA-DR1-0401-15mers-193P1E1B


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


each start position is specified, the length of


peptide is 15 amino acids, and the end position


for each peptide is the start position plus fourteen.









2
DLSDPPVASSCISEK
18


3
LSDPPVASSCISEKS
18


9
ASSCISEKSPRSPQL
18


5
DPPVASSCISEKSPR
14


6
PPVASSCISEKSPRS
12


11
SCISEKSPRSPQLSD
12


12
CISEKSPRSPQLSDF
12







V6-HLA-DR1-0401-15mers-193P1E1B


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


each start position is specified, the length of


peptide is 15 amino acids, and the end position


for each peptide is the start position plus fourteen.









10
EEAIDAESRLNDNVF
20


9
SEEAIDAESRLNDNV
18


1
LKNARNNKSEEAIDA
12


4
ARNNKSEEAIDAESR
12


6
NNKSEEAIDAESRLN
12


8
KSEEAIDAESRLNDN
12


14
DAESRLNDNVFATPS
12


15
AESRLNDNVFATPSP
12







V10-HLA-DR1-0401-15mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21;


each start position is specified, the length of


peptide is 15 amino acids, and the end position


for each peptide is the start position plus fourteen.









13
LQKFQWIYPTQKLNK
22


15
KFQWIYPTQKLNKMR
22


2
PPEVTKIPEDILQKF
20


6
TKIPEDILQKFQWIY
18


5
VTKIPEDILQKFQWI
14


10
EDILQKFQWIYPTQK
14


4
EVTKIPEDILQKFQW
12


14
QKFQWIYPTQKLNKM
12







V12-HLA-DR1-0401-15mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25;


each start position is specified, the length of


peptide is 15 amino acids, and the end position


for each peptide is the start position plus fourteen.









6
QRALDGEESLLSKYN
26


13
ESLLSKYNSNLATPI
20


9
LDGEESLLSKYNSNL
18


12
EESLLSKYNSNLATP
15


2
TARLQRALDGEESLL
14


3
ARLQRALDGEESLLS
12


4
RLQRALDGEESLLSK
12


7
RALDGEESLLSKYNS
12


10
DGEESLLSKYNSNLA
12


14
SLLSKYNSNLATPIA
12


















TABLE XLIX





Pos
123456789012345
score















V1-HLA-DRB1-1101-15mers-193P1E1B


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


each start position is specified, the length of


peptide is 15 amino acids, and the end position for


each peptide is the start position plus fourteen.









159
KYGYSPRVKKNSVHE
26


267
SPIIQQLEKSDAEYT
26


369
EDILQLLSKYNSNLA
26


4
IRSFCGKLRSLASTL
25


349
ISSYENLLRTPTPPE
24


116
PQAVNLLDKARLENQ
22


335
DTCFENLTDPSSPTI
22


194
KQSLVKVLKTPKCAL
20


233
NEDYTMGLKNARNNK
20


306
VSTNYPLSKTNSSSN
20


132
GIDFIKATKVLMEKN
19


157
FQKYGYSPRVKKNSV
19


38
DYPMRILYDLHSEVQ
18


105
RYIVSQVLPNPPQAV
18


300
KNSIALVSTNYPLSK
18


98
LSDFGLERYIVSQVL
17


153
IREYFQKYGYSPRVK
17


191
PPTKQSLVKVLKTPK
17


210
MDDFECVTPKLEHFG
17


286
VPTFCTPGLKIPSTK
17


2
DPIRSFCGKLRSLAS
16


97
QLSDFGLERYIVSQV
16


144
EKNSMDIMKIREYFQ
16


186
PVIVTPPTKQSLVKV
16


287
PTFCTPGLKIPSTKN
16


307
STNYPLSKTNSSSND
16


19
DCETARLQRALDGEE
15


68
NFQKTDVKDDLSDPP
15


319
SNDLEVEDRTSLVLN
15


361
PPEVTKIPEDILQLL
15


381
NLATPIAIKAVPPSK
15


388
IKAVPPSKRFLKHGQ
15


397
FLKHGQNIRDVSNKE
15


40
PMRILYDLHSEVQTL
14


104
ERYIVSQVLPNPPQA
14


118
AVNLLDKARLENQEG
14


137
KATKVLMEKNSMDIM
14


160
YGYSPRVKKNSVHEQ
14


175
EAINSDNYKEEPVIV
14


195
QSLVKVLKTPKCALK
14


197
LVKVLKTPKCALKMD
14


213
FECVTPKLEHFGISE
14


214
ECVTPKLEHFGISEY
14


249
EEAIDTESRLNDNVF
14


255
ESRLNDNVFATPSPI
14


358
TPTPPEVTKIPEDIL
14


392
PPSKRFLKHGQNIRD
14


8
CGKLRSLASTLDCET
13


41
MRILYDLHSEVQTLK
13


48
HSEVQTLKDDVNIPE
13


85
SSCISGKSPRSPQLS
13


102
GLERYIVSQVLPNPP
13


127
LENQEGIDFIKATKV
13


130
QEGIDFIKATKVLME
13


136
IKATKVLMEKNSMDI
13


145
KNSMDIMKIREYFQK
13


237
TMGLKNARNNKSEEA
13


293
GLKIPSTKNSIALVS
13


302
SIALVSTNYPLSKTN
13


318
SSNDLEVEDRTSLVL
13


365
TKIPEDILQLLSKYN
13


372
LQLLSKYNSNLATPI
13


376
SKYNSNLATPIAIKA
13


379
NSNLATPIAIKAVPP
13


385
PIAIKAVPPSKRFLK
13


5
RSFCGKLRSLASTLD
12


12
RSLASTLDCETARLQ
12


23
ARLQRALDGEESDFE
12


33
ESDFEDYPMRILYDL
12


57
DVNIPELSNCENFQK
12


71
KTDVKDDLSDPPVAS
12


75
KDDLSDPPVASSCIS
12


82
PVASSCISGKSPRSP
12


121
LLDKARLENQEGIDF
12


147
SMDIMKIREYFQKYG
12


150
IMKIREYFQKYGYSP
12


165
RVKKNSVHEQEAINS
12


168
KNSVHEQEAINSDNY
12


181
NYKEEPVIVTPPTKQ
12


185
EPVIVTPPTKQSLVK
12


207
ALKMDDFECVTPKLE
12


225
ISEYTMCLNEDYTMG
12


232
LNEDYTMGLKNARNN
12


256
SRLNDNVFATPSPII
12


277
DAEYTNSPLVPTFCT
12


282
NSPLVPTFCTPGLKI
12


291
TPGLKIPSTKNSIAL
12


350
SSYENLLRTPTPPEV
12


368
PEDILQLLSKYNSNL
12


382
LATPIAIKAVPPSKR
12


383
ATPIAIKAVPPSKRF
12







V5-HLA-DRB1-1101-15mers-193P1E1B


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


each start position is specified, the length of


peptide is 15 amino acids, and the end position


for each peptide is the start position plus fourteen.









10
SSCISEKSPRSPQLS
13


7
PVASSCISEKSPRSP
12


3
LSDPPVASSCISEKS
8


8
VASSCISEKSPRSPQ
8


9
ASSCISEKSPRSPQL
8


11
SCISEKSPRSPQLSD
8


6
PPVASSCISEKSPRS
7


13
ISEKSPRSPQLSDFG
7


2
DLSDPPVASSCISEK
6


5
DPPVASSCISEKSPR
6







V6-HLA-DRB1-1101-15mers-193P1E1B


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


each start position is specified, the length of


peptide is 15 amino acids, and the end position


for each peptide is the start position plus fourteen.









10
EEAIDAESRLNDNVF
14


13
IDAESRLNDNVFATP
7


1
LKNARNNKSEEAIDA
6


4
ARNNKSEEAIDAESR
6


6
NNKSEEAIDAESRLN
6


7
NKSEEAIDAESRLND
6


14
DAESRLNDNVFATPS
6







V10-HLA-DRB1-1101-15mers-193P1E1B


Each peptide is a portion of SEQ ID NO:21;


each start position is specified, the length of


peptide is 15 amino acids, and the end position


for each peptide is the start position plus fourteen.









13
LQKFQWIYPTQKLNK
16


2
PPEVTKIPEDILQKF
15


7
KIPEDILQKFQWIYP
14


10
EDILQKFQWIYPTQK
12


15
KFQWIYPTQKLNKMR
11


5
VTKIPEDILQKFQWI
7


9
PEDILQKFQWIYPTQ
7







V12-HLA-DRB1-1101-15mers-193P1E1B


Each peptide is a portion of SEQ ID NO:25;


each start position is specified, the length of


peptide is 15 amino acids, and the end position


for each peptide is the start position plus fourteen.









10
DGEESLLSKYNSNLA
20


6
QRALDGEESLLSKYN
13


13
ESLLSKYNSNLATPI
13


3
ARLQRALDGEESLLS
12
















TABLE L







Properties of 193P1E1B










Bioinformatic Program
Outcome













Variants 1, 5, 6




ORF
ORF Finder
805-2043


Protein Length
n/a
412 amino acids


Transmembrane region
TM Pred
No TM



HMMTop
No TM



Sosui
No TM, soluble



TMHMM
No TM


Signal Peptide
Signal P
indicates no signal


pI
pI/MW tool
pI 5.03


Molecular weight
pI/MW tool
46.2 kDa


Localization
PSORT
Mitochondrial 48%



PSORT II
Nuclear 60.9%



iPSORT
No signal motif


Motifs
Pfam
No motif



Prints
Rhodopsin



Blocks
No motif



Prosite
No motif


Variant 9


ORF
ORF Finder
989-1981


Protein Length
n/a
330 amino acids


Transmembrane region
TM Pred
No TM



HMMTop
No TM



Sosui
No TM, soluble



TMHMM
No TM


Signal Peptide
Signal P
indicates no signal


pI
pI/MW tool
pI 5.17


Molecular weight
pI/MW tool
16.5 kDa


Localization
PSORT
Cytoplasmic 45%



PSORT II
Nuclear 60.9%



iPSORT
No signal motif


Motifs
Pfam
No motif



Prints
No motif



Blocks
No motif



Prosite
No motif


Variant 10


ORF
ORF Finder
805-1971


Protein Length
n/a
388 amino acids


Transmembrane region
TM Pred
No TM



HMMTop
No TM



Sosui
No TM, soluble



TMHMM
No TM


Signal Peptide
Signal P
indicates no signal


pI
pl/MW tool
pl 4.8


Molecular weight
pl/MW tool
34.5 kDa


Localization
PSORT
Mitochondrial 48%



PSORT II
Nuclear 60.9%



iPSORT
No signal motif


Motifs
Pfam
No motif



Prints
No motif



Blocks
No motif



Prosite
No motif


Variant 12


ORF
ORF Finder
805-1026


Protein Length
n/a
73 amino acids


Transmembrane region
TM Pred
No TM



HMMTop
No TM



Sosui
No TM, soluble



TMHMM
No TM


Signal Peptide
Signal P
indicates no signal


pI
pl/MW tool
pl 9.4


Molecular weight
pl/MW tool
8.1 kDa


Localization
PSORT
Mitochondrial 48%



PSORT II
Nuclear 60.9%



iPSORT
No signal motif


Motifs
Pfam
No motif



Prints
No motif



Blocks
No motif



Prosite
No motif
















TABLE LI





Nucleotide sequence of transcript variant 193P1E1B v.9
















(SEQ ID NO: 93)










1
tatcatctgt gactgaggaa atccctatct tcctatcaga ctaatgaaac cacaggacag



 


61
caattagact tttaagtatt ggggggttta gagctctaga tattcgatat gcagactact


 


121
catgtttgtt tgttttaata aagactggtc caaaggctca ttttcacaca agctacagtt


 


181
tttcagttcc aggaccaggt aaagatggtc agctccgtga tccataaaat ccaagggtga


 


241
cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctgg tccgcaatga


 


301
atctatgcgg ggggaacttg gaagtggcgg ccgcctttat ggcctcgaag gcctccctcc


 


361
tgcgcaccgc ggcgtggccg cgctcctgct cccgggtcat gtagggcatg ctcagccagt


 


421
aatggttctc cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca


 


481
cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg ccgcgccaga


 


541
ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgt ctccgcggcc


 


601
gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg gtctccgggg gcctcggcga


 


661
gagacttcgg ctctcgcgag agaggactgc gcctgcgcag agccgaggac gcgtccggcg


 


721
ccgagattca aactagtggc gggaggctgt gagctgagcg gtggggtctg cgtacgcctg


 


781
gagtccttcc ccgctgtgct cagcatggac cctatccgga gcttctgcgg gaagctgcgg


 


841
tctctggcca gcacgctgga ctgcgagacg gcccggctgc agcgagcgct ggacggagag


 


901
gaaagcggat gatgttaata ttcttcttga taaagcaaga ttggaaaatc aagaaggcat


 


961
tgatttcata aaggcaacaa aagtactaat ggaaaaaaat tcaatggata ttatgaaaat


 


1021
aagagagtat ttccagaagt atggatatag tccacgtgtc aagaaaaatt cagtacacga


 


1081
gcaagaagcc attaactctg acccagagtt gtctaattgt gaaaattttc agaagactga


 


1141
tgtgaaagat gatctgtctg atcctcctgt tgcaagcagt tgtatttctg ggaagtctcc


 


1201
acgtagtcca caactttcag attttggact tgagcggtac atcgtatccc aagttctacc


 


1261
aaaccctcca caggcagtga acaactataa ggaagagccc gtaattgtaa ccccacctac


 


1321
caaacaatca ctagtaaaag tactaaaaac tccaaaatgt gcactaaaaa tggatgattt


 


1381
tgagtgtgta actcctaaat tagaacactt tggtatctct gaatatacta tgtgtttaaa


 


1441
tgaagattac acaatgggac ttaaaaatgc gaggaataat aaaagtgagg aggccataga


 


1501
tacagaatcc aggctcaatg ataatgtttt tgccactccc agccccatca tccagcagtt


 


1561
ggaaaaaagt gatgccgaat ataccaactc tcctttggta cctacattct gtactcctgg


 


1621
tttgaaaatt ccatctacaa agaacagcat agctttggta tccacaaatt acccattatc


 


1681
aaaaacaaat agttcatcaa atgatttgga agttgaagat cgtacttcgt tggttttaaa


 


1741
ttcagacaca tgctttgaga atttaacaga tccctcttca cctacgattt cttcttatga


 


1801
gaatctgctc agaacaccta cacctccaga agtaactaaa attccagaag atattctcca


 


1861
gcttttatca aaatacaact caaacctagc tactccaata gcaattaaag cagtgccacc


 


1921
cagtaaaagg ttccttaaac atggacagaa catccgagat gtcagcaaca aagaaaactg


 


1981
aaattccagt ggatctatcc aacacagaaa ctgaacaaaa tgagatgaaa gccgagctgg


 


2041
accgatttta acattcacat tgccctgcct ctgtccccct ttaaacgttg acccatttta


 


2101
aagacaaaca tgaacattaa catcataata tgctttttat gaagtttcaa taaggtttaa


 


2161
ccttagtctt gttgacatgt agcccagtca ttcactcttt aaggactatt agtgtttcat


 


2221
tgatactaaa ttacccagct taatcaacag aatggtttaa gtagtaccag gaagtaggac


 


2281
aagtaatttc aaaaatataa aggtgtttgc tactcagatg aggccgcccc tgaccttctg


 


2341
gccagagaga cattgctgcc agccagctct gccttcccat catctccttt caggaccgtc


 


2401
ccacaccttt tacttgctca gtgctgtctg aagatgcagt tgctgtttgc aaacaacagg


 


2461
aacaccagtt aaactaatta ggaaacagag ggagatttcc aggcctgggt aactatatac


 


2521
tgtgaccatt ggcggttgag accggtcttc aaccagtgga accccgaact ctgctgtcag


 


2581
ggtgtggact tcggtgctct tccaagtttt cacctggggg ggggagctaa ccccctatgt


 


2641
tcacgccttc tattcccatt ggcgctgaac tcttaaggtc actctggtcg cttgtgaccc


 


2701
cgtaaccctg atgtacccct ctaaaaggtg aggggc
















TABLE LII





Nuclerotide sequence alignment of 193P1E1B v.1 (SEQ ID NO: 94) and 193P1E1B v.9 (SEQ ID NO: 95)















Score = 1744 bits (907), Expect = 0.0Identities = 907/907 (100%) Strand = Plus/Plus








 








 








 








Score = 3519 bits (1830), Expect = 0.0 Identities = 1830/1830 (100%) Strand = Plus/Plus








 








 








 








 








 








 








 






















TABLE LIII





Peptide sequences of protein coded by 193P1E1B v.9
















(SEQ ID NO: 96)









MEKNSMDIMK IREYFQKYGY SPRVKKNSVH EQEAINSDPE LSNCENFQKT DVKDDLSDPP  60



 


VASSCISGKS PRSPQLSDFG LERYIVSQVL PNPPQAVNNY KEEPVIVTPP TKQSLVKVLK 120


 


TPKCALKMDD FECVTPKLEH FGISEYTMCL NEDYTMGLKN ARNNKSEEAI DTESRLNDNV 180


 


FATPSPIIQQ LEKSDAEYTN SPLVPTFCTP GLKIPSTKNS IALVSTNYPL SKTNSSSNDL 240


 


EVEDRTSLVL NSDTCFENLT DPSSPTISSY ENLLRTPTPP EVTKIPEDIL QLLSKYNSNL 300


 


ATPIAIKAVP PSKRFLKHGQ NIRDVSNKEN                                  330

















TABLE LIV





Amino acid sequence alignment of 193P1E1B v.1 (SEQ ID NO: 97)



and 193P1E1B v.9 (SEQ ID NO: 98)
















Score = 665 bits (1716), Expect = 0.0Identities = 330/330(100%),



Positives = 330/330 (100%)


 










v.1:
83
MEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPP 142





MEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPP


v.9:
1
MEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPP  60


 


v.1:
143
VASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLK 202




VASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLK


v.9:
61
VASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLK 120


 


v.1:
203
TPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNV 262




TPKCALKMDDFECVTPKLEHEGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNV


v.9:
121
TPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNV 180


 


v.1:
263
FATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDL 322




FATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDL


v.9:
181
FATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDL 240


 


v.1:
323
EVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPTPPEVTKIPEDILQLLSKYNSNL 382




EVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPTPPEVTKIPEDILQLLSKYNSNL


v.9:
241
EVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPTPPEVTKIPEDILQLLSKYNSNL 300


 


v.1:
383
ATPIAIKAVPPSKRFLKHGQNIRDVSNKEN                               412




ATPIAIKAVPPSKRFLKHGQNIRDVSNKEN


v.9:
301
ATPIAIKAVPPSKRELKHGQNIRDVSNKEN                               330
















TABLE LV





Nucleotide sequence of transcript variant 193P1E1B v.10
















(SEQ ID NO: 99)









   1 tatcatctgt gactgaggaa atccctatct tcctatcaga ctaatgaaac cacaggacag



 


  61 caattagact tttaagtatt ggggggttta gagctctaga tattcgatat gcagactact


 


 121 catgtttgtt tgttttaata aagactggtc caaaggctca ttttcacaca agctacagtt


 


 181 tttcagttcc aggaccaggt aaagatggtc agctccgtga tccataaaat ccaagggtga


 


 241 cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctgg tccgcaatga


 


 301 atctatgcgg ggggaacttg gaagtggcgg ccgcctttat ggcctcgaag gcctccctcc


 


 361 tgcgcaccgc ggcgtggccg cgctcctgct cccgggtcat gtagggcatg ctcagccagt


 


 421 aatggttctc cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca


 


 481 cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg ccgcgccaga


 


 541 ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgt ctccgcggcc


 


 601 gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg gtctccgggg gcctcggcga


 


 661 gagacttcgg ctctcgcgag agaggactgc gcctgcgcag agccgaggac gcgtccggcg


 


 721 ccgagattca aactagtggc gggaggctgt gagctgagcg gtggggtctg cgtacgcctg


 


 781 gagtccttcc ccgctgtgct cagcatggac cctatccgga gcttctgcgg gaagctgcgg


 


 841 tctctggcca gcacgctgga ctgcgagacg gcccggctgc agcgagcgct ggacggagag


 


 901 gaaagcgact ttgaagatta tccaatgaga attttatatg accttcattc agaagttcag


 


 961 actctaaagg atgatgttaa tattcttctt gataaagcaa gattggaaaa tcaagaaggc


 


1021 attgatttca taaaggcaac aaaagtacta atggaaaaaa attcaatgga tattatgaaa


 


1081 ataagagagt atttccagaa gtatggatat agtccacgtg tcaagaaaaa ttcagtacac


 


1141 gagcaagaag ccattaactc tgacccagag ttgtctaatt gtgaaaattt tcagaagact


 


1201 gatgtgaaag atgatctgtc tgatcctcct gttgcaagca gttgtatttc tgggaagtct


 


1261 ccacgtagtc cacaactttc agattttgga cttgagcggt acatcgtatc ccaagttcta


 


1321 ccaaaccctc cacaggcagt gaacaactat aaggaagagc ccgtaattgt aaccccacct


 


1381 accaaacaat cactagtaaa agtactaaaa actccaaaat gtgcactaaa aatggatgat


 


1441 tttgagtgtg taactcctaa attagaacac tttggtatct ctgaatatac tatgtgttta


 


1501 aatgaagatt acacaatggg acttaaaaat gcgaggaata ataaaagtga ggaggccata


 


1561 gatacagaat ccaggctcaa tgataatgtt tttgccactc ccagccccat catccagcag


 


1621 ttggaaaaaa gtgatgccga atataccaac tctcctttgg tacctacatt ctgtactcct


 


1681 ggtttgaaaa ttccatctac aaagaacagc atagctttgg tatccacaaa ttacccatta


 


1741 tcaaaaacaa atagttcatc aaatgatttg gaagttgaag atcgtacttc gttggtttta


 


1801 aattcagaca catgctttga gaatttaaca gatccctctt cacctacgat ttcttcttat


 


1861 gagaatctgc tcagaacacc tacacctcca gaagtaacta aaattccaga agatattctc


 


1921 cagaaattcc agtggatcta tccaacacag aaactgaaca aaatgagatg aaagccgagc


 


1981 tggaccgatt ttaacattca cattgccctg cctctgtccc cctttaaacg ttgacccatt


 


2041 ttaaagacaa acatgaacat taacatcata atatgctttt tatgaagttt caataaggtt


 


2101 taaccttagt cttgttgaca tgtagcccag tcattcactc tttaaggact attagtgttt


 


2161 cattgatact aaattaccca gcttaatcaa cagaatggtt taagtagtac caggaagtag


 


2221 gacaagtaat ttcaaaaata taaaggtgtt tgctactcag atgaggccgc ccctgacctt


 


2281 ctggccagag agacattgct gccagccagc tctgccttcc catcatctcc tttcaggacc


 


2341 gtcccacacc ttttacttgc tcagtgctgt ctgaagatgc agttgctgtt tgcaaacaac


 


2401 aggaacacca gttaaactaa ttaggaaaca gagggagatt tccaggcctg ggtaactata


 


2461 tactgtgacc attggcggtt gagaccggtc ttcaaccagt ggaaccccga actctgctgt


 


2521 cagggtgtgg acttcggtgc tcttccaagt tttcacctgg gggggggagc taacccccta


 


2581 tgttcacgcc ttctattccc attggcgctg aactcttaag gtcactctgg tcgcttgtga


 


2641 ccccgtaacc ctgatgtacc cctctaaaag gtgaggggc
















TABLE LVI





Nuclerotide sequence alignment of 193P1E1B v.1 (SEQ ID NO: 100) and 193P1E1B v.10 (SEQ ID NO: 101)















Score = 3698 bits (1923), Expect = 0.0Identities = 1923/1923 (100%) Strand = Plus/Plus








 








 








 








 








 








 








 








Score = 1456 bits (757), Expect = 0.0 Identities = 757/757 (100%) Strand = Plus/Plus








 








 






















TABLE LVII





Peptide sequences of protein coded by 193P1E1B v.10
















(SEQ ID NO: 102)









MDPIRSFCGK LRSLASTLDC ETARLQRALD GEESDFEDYP MRILYDLHSE VQTLKDDVNI  60



 


LLDKARLENQ EGIDFIKATK VLMEKNSMDI MKIREYFQKY GYSPRVKKNS VHEQEAINSD 120


 


PELSNCENFQ KTDVKDDLSD PPVASSCISG KSPRSPQLSD FGLERYIVSQ VLPNPPQAVN 180


 


NYKEEPVIVT PPTKQSLVKV LKTPKCALKM DDFECVTPKL EHFGISEYTM CLNEDYTMGL 240


 


KNARNNKSEE AIDTESRLND NVFATPSPII QQLEKSDAEY TNSPLVPTFC TPGLKIPSTK 300


 


NSIALVETNY PLSKTNSSSN DLEVEDRTSL VLNSDTCFEN LTDPSSPTIS SYENLLRTPT 360


 


PPEVTKIPED ILQKFQWIYP TQKLNKMR                                    388

















TABLE LVIII





Amino acid sequence alignment of 193P1E1B v.1 (SEQ ID NO: 103)



and 193P1E1B v.10 (SEQ ID NO: 104)
















Score = 749 bits (1935), Expect = 0.0Identities = 373/373 (100%),



Positives = 373/373(100%)


 


v.1:    1 MDPIRSFCGKLRSLASTLDCETARLQRALDGEESDFEDYPMRILYDLHSEVQTLKDDVNI  60


           MDPIRSFCGKLRSLASTLDCETARLQRALDGEESDFEDYPMRILYDLHSEVQTLKDDVNI


v.10:   1 MDPIRSFCGKLRSLASTLDCETARLQRALDGEESDFEDYPMRILYDLHSEVQTLKDDVNI  60


 


v.1:   61 LLDKARLENQEGIDFIKATKVLMEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSD 120


          LLDKARLENQEGIDFIKATKVLMEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSD


v.10:  61 LLDKARLENQEGIDFIKATKVLMEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSD 120


 


v.1:  121 PELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVN 180


          PELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVN


v.10: 121 PELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVN 180


 


v.1:  181 NYKEEPVIVTPPTKQSLVKVLKTPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGL 240


          NYKEEPVIVTPPTKQSLVKVLKTPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGL


v.10: 181 NYKEEPVIVTPPTKQSLVKVLKTPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGL 240


 


v.1:  241 KNARNNKSEEAIDTESRLNDNVFATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTK                                               300


          KNARNNKSEEAIDTESRLNDNVFATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTK


v.10: 241 KNARNNKSEEAIDTESRLNDNVFATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTK                                                300


 


v.1:  301 NSIALVSTNYPLSKTNSSSNDLEVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPT 360


          NSIALVSTNYPLSKTNSSSNDLEVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPT


v.10: 301 NSIALVSTNYPLSKTNSSSNDLEVEDRTSLVLNSDTCFENLTDPSSPTTSSYENLLRTPT 360


 


v.1:  361 PPEVTKIPEDILQ 373


          PPEVTKIPEDILQ


v.10: 361 PPEVTKIPEDILQ 373
















TABLE LIX





Nucleotide sequence of transcript variant 193P1E1Bv.11
















(SEQ ID NO: 105)









   1 tatcatctgt gactgaggaa atccctatct tcctatcaga ctaatgaaac cacaggacag



 


  61 caattagact tttaagtatt ggggggttta gagctctaga tattcgatat gcagactact


 


 121 catgtttgtt tgttttaata aagactggtc caaaggctca ttttcacaca agctacagtt


 


 181 tttcagttcc aggaccaggt aaagatggtc agctccgtga tccataaaat ccaagggtga


 


 241 cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctgg tccgcaatga


 


 301 atctatgcgg ggggaacttg gaagtggcgg ccgcctttat ggcctcgaag gcctccctcc


 


 361 tgcgcaccgc ggcgtggccg cgctcctgct cccgggtcat gtagggcatg ctcagccagt


 


 421 aatggttctc cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca


 


 481 cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg ccgcgccaga


 


 541 ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgt ctccgcggcc


 


 601 gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg gtctccgggg gcctcggcga


 


 661 gagacttcgg ctctcgcgag agaggactgc gcctgcgcag agccgaggac gcgtccggcg


 


 721 ccgagattca aactagtggc gggaggctgt gagctgagcg gtggggtctg cgtacgcctg


 


 781 gagtccttcc ccgctgtgct cagcatggac catatccgga gcttctgcgg gaagctgcgg


 


 841 tctctggcca gcacgctgga ctgcgagacg gcccggctgc agcgagcgct ggacggagag


 


 901 gaaagcggat gatgttaata ttcttcttga taaagcaaga ttggaaaatc aagaaggcat


 


 961 tgatttcata aaggcaacaa aagtactaat ggaaaaaaat tcaatggata ttatgaaaat


 


1021 aagagagtat ttccagaagt atggatatag tccacgtgtc aagaaaaatt cagtacacga


 


1081 gcaagaagcc attaactctg acccagagtt gtctaattgt gaaaattttc agaagactga


 


1141 tgtgaaagat gatctgtctg atcctcctgt tgcaagcagt tgtatttctg ggaagtctcc


 


1201 acgtagtcca caactttcag attttggact tgagcggtac atcgtatccc aagttctacc


 


1261 aaaccctcca caggcagtga acaactataa ggaagagccc gtaattgtaa ccccacctac


 


1321 caaacaatca ctagtaaaag tactaaaaac tccaaaatgt gcactaaaaa tggatgattt


 


1381 tgagtgtgta actcctaaat tagaacactt tggtatctct gaatatacta tgtgtttaaa


 


1441 tgaagattac acaatgggac ttaaaaatgc gaggaataat aaaagtgagg aggccataga


 


1501 tacagaatcc aggctcaatg ataatgtttt tgccactccc agccccatca tccagcagtt


 


1561 ggaaaaaagt gatgccgaat ataccaactc tcctttggta cctacattct gtactcctgg


 


1621 tttgaaaatt ccatctacaa agaacagcat agctttggta tccacaaatt acccattatc


 


1681 aaaaacaaat agttcatcaa atgatttgga agttgaagat cgtacttcgt tggttttaaa


 


1741 ttcagacaca tgctttgaga atttaacaga tccctcttca cctacgattt cttcttatga


 


1801 gaatctgctc agaacaccta cacctccaga agtaactaaa attccagaag atattctcca


 


1861 gaaattccag tggatctatc caacacagaa actgaacaaa atgagatgaa agccgagctg


 


1921 gaccgatttt aacattcaca ttgccctgcc tctgtccccc tttaaacgtt gacccatttt


 


1981 aaagacaaac atgaacatta acatcataat atgcttttta tgaagtttca ataaggttta


 


2041 accttagtct tgttgacatg tagcccagtc attcactctt taaggactat tagtgtttca


 


2101 ttgatactaa attacccagc ttaatcaaca gaatggttta agtagtacca ggaagtagga


 


2161 caagtaattt caaaaatata aaggtgtttg ctactcagat gaggccgccc ctgaccttct


 


2221 ggccagagag acattgctgc cagccagctc tgccttccca tcatctcctt tcaggaccgt


 


2281 cccacacctt ttacttgctc agtgctgtct gaagatgcag ttgctgtttg caaacaacag


 


2341 gaacaccagt taaactaatt aggaaacaga gggagatttc caggcctggg taactatata


 


2401 ctgtgaccat tggcggttga gaccggtctt caaccagtgg aaccccgaac tctgctgtca


 


2461 gggtgtggac ttcggtgctc ttccaagttt tcacctgggg gggggagcta accccctatg


 


2521 ttcacgcctt ctattcccat tggcgctgaa ctcttaaggt cactctggtc gcttgtgacc


 


2581 ccgtaaccct gatgtacccc tctaaaaggt gaggggc
















TABLE LX





Nucteotide sequence alignment of 193P1E1B v.1 (SEQ ID NO: 106) and 193P1E1B v.11


(SEQ ID NO: 107)















Score = 1744 bits (907), Expect = 0.0Identities = 907/907 (100%) Strand =


Plus/Plus


 








 








 








 








 


Score = 1836 bits (955), Expect = 0.0Identities = 955/955 (100%) Strand =


Plus/Plus


 








 








 








 








 


Score = 1456 bits (757), Expect = 0.0Identities = 757/757 (100%) Strand =


Plus/Plus


 








 








 























TABLE LXI





Peptide sequences of protein coded by 193P1E1B v.11

















(SEQ ID NO: 108)









MEKNSMDIMK IREYFQKYGY SPRVKKNSVH EQEATNSDPE LSNCENFQKT DVKDDLSDPP  60



 


VASSCISGKS PRSPQLSDFG LERYIVSQVL PNPPQAVNNY KEEPVIVTPP TKQSLVKVLK 120


 


TPKCALKMDD FECVTPKLEH FGISEYTMCL NEDYTMGLKN ARNNKSEEAI DTESRLNDNV 180


 


FATPSPIIQQ LEKSDAEYTN SPLVPTFCTP GLKIPSTKNS IALVSTNYPL SKTNSSSNDL 240


 


EVEDRTSLVL NSDTCFENLT DPSSPTISSY ENLLRTPTPP EVTKIPEDIL QKFQWIYPTQ 300


 


KLNKMR                                                            306

















TABLE LXII





Amino acid sequence alignment of 193P1E1B v.1 (SEQ ID NO: 109) and



193P1E1B v.11 (SEQ ID NO: 110)
















Score = 589 bits (1518), Expect = e-167Identities = 291/291 (100%),



Positives = 291/291 (100%)


 


v.1:   83 MEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPP 142


          MEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPP


v.11:   1 MEKNSMDIMKIREYFQKYGYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPP  60


 


v.1:  143 VASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLK 202


          VASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLK


v.11:  61 VASSCISGKSPRSPQLSDFGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLK 120


 


v.1:  203 TPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNV 262


          TPKCALKMDDFECVTPKLEHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNV


v.11: 121 TPKCALKMDDFECVTPKLEHFGTSEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNV 180


 


v.1:  263 FATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDL 322


          FATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKTPSTKNSIALVSTNYPLSKTNSSSNDL


v.11: 181 FATPSPIIQQLEKSDAEYTNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDL 240


 


v.1:  323 EVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPTPPEVTKIPEDILQ          373


          EVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLRTPTPPEVTKIPEDILQ


v.11: 241 EVEDRTSLVLNSDTCFENLTDPSSPTISSYENLLITPTPPEVTKIPEDILQ          291
















TABLE LXIII





Nucleotide sequence of transcript variant 193P1E1B v.12
















(SEQ ID NO: 111)









   1 tatcatctgt gactgaggaa atccctatct tcctatcaga ctaatgaaac cacaggacag



 


  61 caattagact tttaagtatt ggggggttta gagctctaga tattcgatat gcagactact


 


 121 catgtttgtt tgttttaata aagactggtc caaaggctca ttttcacaca agctacagtt


 


 181 tttcagttcc aggaccaggt aaagatggtc agctccgtga tccataaaat ccaagggtga


 


 241 cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctgg tccgcaatga


 


 301 atctatgcgg ggggaacttg gaagtggcgg ccgcctttat ggcctcgaag gcctccctcc


 


 361 tgcgcaccgc ggcgtggccg cgctcctgct cccgggtcat gtagggcatg ctcagccagt


 


 421 aatggttctc cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca


 


 481 cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg ccgcgccaga


 


 541 ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgt ctccgcggcc


 


 601 gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg gtctccgggg gcctcggcga


 


 661 gagacttcgg ctctcgcgag agaggactgc gcctgcgcag agccgaggac gcgtccggcg


 


 721 ccgagattca aactagtggc gggaggctgt gagctgagcg gtggggtctg cgtacgcctg


 


 781 gagtccttcc ccgctgtgct cagcatggac cctatccgga gcttctgcgg gaagctgcgg


 


 841 tctctggcca gcacgctgga ctgcgagacg gcccggctgc agcgagcgct ggacggagag


 


 901 gaaagccttt tatcaaaata caactcaaac ctagctactc caatagcaat taaagcagtg


 


 961 ccacccagta aaaggttcct taaacatgga cagaacatcc gagatgtcag caacaaagaa


 


1021 aactgaaatt ccagtggatc tatccaacac agaaactgaa caaaatgaga tgaaagccga


 


1081 gctggaccga ttttaacatt cacattgccc tgcctctgtc cccctttaaa cgttgaccca


 


1141 ttttaaagac aaacatgaac attaacatca taatatgctt tttatgaagt ttcaataagg


 


1201 tttaacctta gtcttgttga catgtagccc agtcattcac tctttaagga ctattagtgt


 


1261 ttcattgata ctaaattacc cagcttaatc aacagaatgg tttaagtagt accaggaagt


 


1321 aggacaagta atttcaaaaa tataaaggtg tttgctactc agatgaggcc gcccctgacc


 


1381 ttctggccag agagacattg ctgccagcca gctctgcctt cccatcatct cctttcagga


 


1441 ccgtcccaca ccttttactt gctcagtgct gtctgaagat gcagttgctg tttgcaaaca


 


1501 acaggaacac cagttaaact aattaggaaa cagagggaga tttccaggcc tgggtaacta


 


1561 tatactgtga ccattggcgg ttgagaccgg tcttcaacca gtggaacccc gaactctgct


 


1621 gtcagggtgt ggacttcggt gctcttccaa gttttcacct ggggggggga gctaaccccc


 


1681 tatgttcacg ccttctattc ccattggcgc tgaactctta aggtcactct ggtcgcttgt


 


1741 gaccccgtaa ccctgatgta cccctctaaa aggtgagggg c
















TABLE LXIV





Nucleotide sequence alignment of 193P1E1B v.1 (SEQ ID NO: 112) and 193P1E1B v.12


(SEQ ID NO: 113)















Score = 1742 bits (906), Expect = 0.0Identities = 906/906 (100%) Strand =


Plus/Plus


 








 








 








 








 


Score = 1683 bits (875), Expect = 0.0Identities = 875/875 (100%) Strand =


Plus/Plus


 








 








 








 






















TABLE LXV





Peptide sequences of protein coded by 193P1E1B v.12
















(SEQ ID NO: 114)









MDPIRSFCGK LRSLASTLDC ETARLQRALD GEESLLSKYN SNLATPIAIK AVPPSKRFLK 60



 


HGQNIRDVSN KEN                                                          73

















TABLE LXVI





Amino acid sequence alignment of 193P1E1B v.1



(SEQ ID NO: 115) and 193P1E1B v.12 (SEQ ID NO: 116)
















Score = 72.0 bits (175), Expect = 2e-12Identities = 35/39



(89%), Positives = 35/39 (89%)


 


v.1:        1      MDPIRSFCGKLRSLASTLDCETARLQRALDGEESDFEDY 39


                   MDPIRSECGKLRSLASTLDCETARLQRALDGEES    Y


v.12:       1      MDPIRSFCGKLRSLASTLDCETARLQRALDGEESLLSKY 39


 


Score = 80.9 bits (198), Expect = 4e-15Identities = 39/39


(100%), Positives = 39/39 (100%)


 


v.1:      374      LLSKYNSNLATPIAIKAVPPSKRFLKHGQNIRDVSNKEN 412


                   LLSKYNSNLATPIAIKAVPPSKRFLKHGQNIRDVSNKEN


v.12:      35      LLSKYNSNLATPIAIKAVPPSKRFLKHGQNIRDVSNKEN  73
















TABLE LXVII





Nucleotide sequence of transcript variant 193P1E1B v.13
















(SEQ ID NO: 117)









   1 tatcatctgt gactgaggaa atccctatct tcctatcaga ctaatgaaac cacaggacag



 


  61 caattagact tttaagtatt ggggggttta gagctctaga tattcgatat gcagactact


 


 121 catgtttgtt tgttttaata aagactggtc caaaggctca ttttcacaca agctacagtt


 


 181 tttcagttcc aggaccaggt aaagatggtc agctccgtga tccataaaat ccaagggtga


 


 241 cgactcagga ttaggaccat ttcttggtga cattgagatg gtcgagctgg tccgcaatga


 


 301 atctatgcgg ggggaacttg gaagtggcgg ccgcctttat ggcctcgaag gcctccctcc


 


 361 tgcgcaccgc ggcgtggccg cgctcctgct cccgggtcat gtagggcatg ctcagccagt


 


 421 aatggttctc cgcctcgatc tccaggcggc ggatcatgtt ctgcttggcg cgcaacgaca


 


 481 cgaaccgcgg ccgccggtgc ttcccgatcc actgacggcc gggaatgcgg ccgcgccaga


 


 541 ggagcgcagt caggaacatg gtgcctgccg cgctgctcaa gactctgcgt ctccgcggcc


 


 601 gccagcagac gccgtggcgt aagcgcaccc gtctcgcggg gtctccgggg gcctcggcga


 


 661 gagacttcgg ctctcgcgag agaggactgc gcctgcgcag agccgaggac gcgtccggcg


 


 721 ccgagattca aactagtggc gggaggctgt gagctgagcg gtggggtctg cgtacgcctg


 


 781 gagtccttcc ccgctgtgct cagcatggac cctatccgga gcttctgcgg gaagctgcgg


 


 841 tctctggcca gcacgctgga ctgcgagacg gcccggctgc agcgagcgct ggacggagag


 


 901 gaaagcggtg cgtgaggcgg gcggccaggg cacgactttg aagattatcc aatgagaatt


 


 961 ttatatgacc ttcattcaga agttcagact ctaaaggatg atgttaatat tcttcttgat


 


1021 aaagcaagat tggaaaatca agaaggcatt gatttcataa aggcaacaaa agtactaatg


 


1081 gaaaaaaatt caatggatat tatgaaaata agagagtatt tccagaagta tggatatagt


 


1141 ccacgtgtca agaaaaattc agtacacgag caagaagcca ttaactctga cccagagttg


 


1201 tctaattgtg aaaattttca gaagactgat gtgaaagatg atctgtctga tcctcctgtt


 


1261 gcaagcagtt gtatttctgg gaagtctcca cgtagtccac aactttcaga ttttggactt


 


1321 gagcggtaca tcgtatccca agttctacca aaccctccac aggcagtgaa caactataag


 


1381 gaagagcccg taattgtaac cccacctacc aaacaatcac tagtaaaagt actaaaaact


 


1441 ccaaaatgtg cactaaaaat ggatgatttt gagtgtgtaa ctcctaaatt agaacacttt


 


1501 ggtatctctg aatatactat gtgtttaaat gaagattaca caatgggact taaaaatgcg


 


1561 aggaataata aaagtgagga ggccatagat acagaatcca ggctcaatga taatgttttt


 


1621 gccactccca gccccatcat ccagcagttg gaaaaaagtg atgccgaata taccaactct


 


1681 cctttggtac ctacattctg tactcctggt ttgaaaattc catctacaaa gaacagcata


 


1741 gctttggtat ccacaaatta cccattatca aaaacaaata gttcatcaaa tgatttggaa


 


1801 gttgaagatc gtacttcgtt ggttttaaat tcagacacat gctttgagaa tttaacagat


 


1861 ccctcttcac ctacgatttc ttcttatgag aatctgctca gaacacctac acctccagaa


 


1921 gtaactaaaa ttccagaaga tattctccag cttttatcaa aatacaactc aaacctagct


 


1981 actccaatag caattaaagc agtgccaccc agtaaaaggt tccttaaaca tggacagaac


 


2041 atccgagatg tcagcaacaa agaaaactga aattccagtg gatctatcca acacagaaac


 


2101 tgaacaaaat gagatgaaag ccgagctgga ccgattttaa cattcacatt gccctgcctc


 


2161 tgtccccctt taaacgttga cccattttaa agacaaacat gaacattaac atcataatat


 


2221 gctttttatg aagtttcaat aaggtttaac cttagtcttg ttgacatgta gcccagtcat


 


2281 tcactcttta aggactatta gtgtttcatt gatactaaat tacccagctt aatcaacaga


 


2341 atggtttaag tagtaccagg aagtaggaca agtaatttca aaaatataaa ggtgtttgct


 


2401 actcagatga ggccgcccct gaccttctgg ccagagagac attgctgcca gccagctctg


 


2461 ccttcccatc atctcctttc aggaccgtcc cacacctttt acttgctcag tgctgtctga


 


2521 agatgcagtt gctgtttgca aacaacagga acaccagtta aactaattag gaaacagagg


 


2581 gagatttcca ggcctgggta actatatact gtgaccattg gcggttgaga ccggtcttca


 


2641 accagtggaa ccccgaactc tgctgtcagg gtgtggactt cggtgctctt ccaagttttc


 


2701 acctgggggg gggagctaac cccctatgtt cacgccttct attcccattg gcgctgaact


 


2761 cttaaggtca ctctggtcgc ttgtgacccc gtaaccctga tgtacccctc taaaaggtga


 


2821 ggggc
















TABLE LXVIII





Nucleotide sequence alignment of 193P1E1B v.1 (SEQ ID NO: 118) and 193P1E1B v.13


(SEQ ID NO: 119)















Score 1744 bits (907), Expect = 0.0Identities = 907/907 (100%) Strand =


Plus/Plus


 








 








 








 








 


Score = 3640 bits (1893), Expect = 0.0Identities = 1893/1893 (100%) Strand =


Plus/Plus


 








 








 








 








 








 








 








 








Score = 1744 bits (907), Expect = 0.0Identities = 907/907 (100%) Strand =


Plus/Plus


 








 








 








 






















TABLE LXIX





Peptide sequences of protein coded by 193P1E1B v.13















(SEQ ID NO:120)








MRILYDLHSE VQTLKDDVNI LLDKARLENQ EGIDFIKATK VLMEKNSMDI MKIREYFQKY
 60


 


GYSPRVKKNS VHEQEAINSD PELSNCENFQ KTDVKDDLSD PPVASSCISG KSPRSPQLSD
120


 


FGLERYIVSQ VLPNPPQAVN NYKEEPVIVT PPTKQSLVKV LKTPKCALKM DDFECVTPKL
180


 


EHFGISEYTM CLNEDYTMGL KNARNNKSEE AIDTESRLND NVFATPSPII QQLEKSDAEY
240


 


TNSPLVPTFC TPGLKIPSTK NSIALVSTNY PLSKTNSSSN DLEVEDRTSL VLNSDTCFEN
300


 


LTDPSSPTIS SYENLLRTPT PPEVTKIPED ILQLLSKYNS NLATPIAIKA VPPSKRFLKH
360


 


GQNIRDVSNK EN
372
















TABLE LXX





Amino acid sequence alignment of 193P1E1B v.1 (SEQ ID NO:121) and 193P1E1B


v.13 (SEQ ID NO:122)















Score = 745 bits (1923), Expect = 0.0Identities = 372/372 (100%),


Positives = 372/372 (100%)










V.1:
41
MRILYDLHSEVQTLKDDVNILLDKARLENQEGIDFIKATKVLMEKNSMDIMKIREYFQKY
100




MRILYDLHSEVQTLKDDVNILLDKARLENQEGIDFIKATKVLMEKNSMDIMKIREYFQKY


V.13:
1
MRILYDLHSEVQTLKDDVNILLDKARLENQEGIDFIKATKVLMEKNSMDIMKIREYFQKY
 60


 


V.1:
101
GYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSD
160




GYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSD


V.13:
61
GYSPRVKKNSVHEQEAINSDPELSNCENFQKTDVKDDLSDPPVASSCISGKSPRSPQLSD
120


 


V.1:
161
FGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLKTPKCALKMDDFECVTPKL
220




FGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLKTPKCALKMDDFECVTPKL


V.13:
121
FGLERYIVSQVLPNPPQAVNNYKEEPVIVTPPTKQSLVKVLKTPKCALKMDDFECVTPKL
180


 


V.1:
221
EHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNVFATPSPIIQQLEKSDAEY
280




EHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNVFATPSPIIQQLEKSDAEY


V.13:
181
EHFGISEYTMCLNEDYTMGLKNARNNKSEEAIDTESRLNDNVFATPSPIIQQLEKSDAEY
240


 


V.1:
281
TNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDLEVEDRTSLVLNSDTCFEN
340




TNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDLEVEDRTSLVLNSDTCFEN


V.13:
241
TNSPLVPTFCTPGLKIPSTKNSIALVSTNYPLSKTNSSSNDLEVEDRTSLVLNSDTCFEN
300


 


V.1:
341
LTDPSSPTISSYENLLRTPTPPEVTKIPEDILQLLSKYNSNLATPIAIKAVPPSKRFLKH
400




LTDPSSPTISSYENLLRTPTPPEVTKIPEDILQLLSKYNSNLATPIAIKAVPPSKRFLKH


V.13:
301
LTDPSSPTISSYENLLRTPTPPEVTKIPEDILQLLSKYNSNLATPIAIKAVPPSKRFLKH
360


 


V.1:
401
GQNIRDVSNKEN
412




GQNIRDVSNKEN


V.13:
361
GQNIRDVSNKEN
372

## Claims

1. A method for detecting, in a sample, the presence of a protein, comprising:
contacting the sample with an antibody or fragment thereof that specifically binds to a protein comprising the amino acid sequence of SEQ ID NO:3, SEQ ID NO: 11 or SEQ ID NO: 13; and,
detecting the formation and/or presence of a complex comprising the antibody and the protein, wherein the protein comprises an amino acid sequence, and wherein the amino acid sequence is selected from group consisting of SEQ ID NO:3, SEQ ID NO: 11, and SEQ ID NO: 13.

2. The method of claim 1, wherein the protein comprises the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 13.