# Patent

## Title
Nucleic acid and corresponding protein entitled STEAP-1 useful in treatment and detection of cancer

## Abstract

A novel gene 08P1D4 (also designated STEAP-1) and its encoded protein, and variants thereof, are described wherein STEAP-1 exhibits tissue specific expression in normal adult tissue, and is aberrantly expressed in the cancers listed in Table I. Consequently, STEAP-1 provides a diagnostic, prognostic, prophylactic and/or therapeutic target for cancer. The STEAP-1 gene or fragment thereof, or its encoded protein, or variants thereof, or a fragment thereof, can be used to elicit a humoral or cellular immune response; antibodies or T cells reactive with STEAP-1 can be used in active or passive immunization.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 10/236,878, filed Sep. 6, 2002 now abandoned, which claims the benefit of U.S. Provisional Application No. 60/370,387, filed Apr. 5, 2002, and U.S. Provisional Application No. 60/317,840, filed Sep. 6, 2001. This application relates to U.S. Provisional Application No. 60/087,520, filed Jun. 1, 1998 and U.S. Provisional Application No. 60/091,183, filed Jun. 30, 1998 and to U.S. patent application Ser. No. 09/323,873, filed Jun. 1, 1999, now issued as U.S. Pat. No. 6,329,503 on Dec. 11, 2001, U.S. patent application Ser. No. 10/011,095, filed Dec. 6, 2001, now issued as U.S. Pat. No. 7,053,186 on May 30, 2006, U.S. patent application Ser. No. 10/010,667, filed Dec. 6, 2001, now issued as U.S. Pat. No. 6,887,975 on May 3, 2005, U.S. patent application Ser. No. 10/750,262, filed Dec. 31, 2003; U.S. patent application Ser. No. 10/858,887, filed Jun. 1, 2004, U.S. patent application Ser. No. 11/225,661, filed Sep. 12, 2005, U.S. patent application Ser. No. 10/830,899, filed Apr. 23, 2004, and U.S. patent application Ser. No. 10/861,662, filed Jun. 4, 2004. The contents of each application listed in this paragraph are fully incorporated by reference herein.


STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH
Not applicable.
FIELD OF THE INVENTION
The invention described herein relates to a gene and its encoded protein, termed 08P1D4 or STEAP-1, expressed in certain cancers, and to diagnostic and therapeutic methods and compositions useful in the management of cancers that express STEAP-1.
BACKGROUND OF THE INVENTION
Cancer is the second leading cause of human death next to coronary disease. Worldwide, millions of people die from cancer every year. In the United States alone, as reported by the American Cancer Society, cancer causes the death of well over a half-million people annually, with over 1.2 million new cases diagnosed per year. While deaths from heart disease have been declining significantly, those resulting from cancer generally are on the rise. In the early part of the next century, cancer is predicted to become the leading cause of death.
Worldwide, several cancers stand out as the leading killers. In particular, carcinomas of the lung, prostate, breast, colon, pancreas, and ovary represent the primary causes of cancer death. These and virtually all other carcinomas share a common lethal feature. With very few exceptions, metastatic disease from a carcinoma is fatal. Moreover, even for those cancer patients who initially survive their primary cancers, common experience has shown that their lives are dramatically altered. Many cancer patients experience strong anxieties driven by the awareness of the potential for recurrence or treatment failure. Many cancer patients experience physical debilitations following treatment. Furthermore, many cancer patients experience a recurrence.
Worldwide, prostate cancer is the fourth most prevalent cancer in men. In North America and Northern Europe, it is by far the most common cancer in males and is the second leading cause of cancer death in men. In the United States alone, well over 30,000 men die annually of this disease—second only to lung cancer. Despite the magnitude of these figures, there is still no effective treatment for metastatic prostate cancer. Surgical prostatectomy, radiation therapy, hormone ablation therapy, surgical castration and chemotherapy continue to be the main treatment modalities. Unfortunately, these treatments are ineffective for many and are often associated with undesirable consequences.
On the diagnostic front, the lack of a prostate tumor marker that can accurately detect early-stage, localized tumors remains a significant limitation in the diagnosis and management of this disease. Although the serum prostate specific antigen (PSA) assay has been a very useful tool, however its specificity and general utility is widely regarded as lacking in several important respects.
Progress in identifying additional specific markers for prostate cancer has been improved by the generation of prostate cancer xenografts that can recapitulate different stages of the disease in mice. The LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer xenografts that have survived passage in severe combined immune deficient (SCID) mice and have exhibited the capacity to mimic the transition from androgen dependence to androgen independence (Klein et al., 1997, Nat. Med. 3:402). More recently identified prostate cancer markers include PCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res 1996 Sep. 2 (9): 1445-51), STEAP (Hubert, et al., Proc Natl. Acad. Sci USA. 1999 Dec. 7; 96(25): 14523-8) and prostate stem cell antigen (PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).
While previously identified markers such as PSA, PSM, PCTA and PSCA have facilitated efforts to diagnose and treat prostate cancer, there is need for the identification of additional markers and therapeutic targets for prostate and related cancers in order to further improve diagnosis and therapy.
Renal cell carcinoma (RCC) accounts for approximately 3 percent of adult malignancies. Once adenomas reach a diameter of 2 to 3 cm, malignant potential exists. In the adult, the two principal malignant renal tumors are renal cell adenocarcinoma and transitional cell carcinoma of the renal pelvis or ureter. The incidence of renal cell adenocarcinoma is estimated at more than 29,000 cases in the United States, and more than 11,600 patients died of this disease in 1998. Transitional cell carcinoma is less frequent, with an incidence of approximately 500 cases per year in the United States.
Surgery has been the primary therapy for renal cell adenocarcinoma for many decades. Until recently, metastatic disease has been refractory to any systemic therapy. With recent developments in systemic therapies, particularly immunotherapies, metastatic renal cell carcinoma may be approached aggressively in appropriate patients with a possibility of durable responses. Nevertheless, there is a remaining need for effective therapies for these patients.
Of all new cases of cancer in the United States, bladder cancer represents approximately 5 percent in men (fifth most common neoplasm) and 3 percent in women (eighth most common neoplasm). The incidence is increasing slowly, concurrent with an increasing older population. In 1998, there was an estimated 54,500 cases, including 39,500 in men and 15,000 in women. The age-adjusted incidence in the United States is 32 per 100,000 for men and eight per 100,000 in women. The historic male/female ratio of 3:1 may be decreasing related to smoking patterns in women. There were an estimated 11,000 deaths from bladder cancer in 1998 (7,800 in men and 3,900 in women). Bladder cancer incidence and mortality strongly increase with age and will be an increasing problem as the population becomes more elderly.
Most bladder cancers recur in the bladder. Bladder cancer is managed with a combination of transurethral resection of the bladder (TUR) and intravesical chemotherapy or immunotherapy. The multifocal and recurrent nature of bladder cancer points out the limitations of TUR. Most muscle-invasive cancers are not cured by TUR alone. Radical cystectomy and urinary diversion is the most effective means to eliminate the cancer but carry an undeniable impact on urinary and sexual function. There continues to be a significant need for treatment modalities that are beneficial for bladder cancer patients.
An estimated 130,200 cases of colorectal cancer occurred in 2000 in the United States, including 93,800 cases of colon cancer and 36,400 of rectal cancer. Colorectal cancers are the third most common cancers in men and women. Incidence rates declined significantly during 1992-1996 (−2.1% per year). Research suggests that these declines have been due to increased screening and polyp removal, preventing progression of polyps to invasive cancers. There were an estimated 56,300 deaths (47,700 from colon cancer, 8,600 from rectal cancer) in 2000, accounting for about 11% of all U.S. cancer deaths.
At present, surgery is the most common form of therapy for colorectal cancer, and for cancers that have not spread, it is frequently curative. Chemotherapy, or chemotherapy plus radiation, is given before or after surgery to most patients whose cancer has deeply perforated the bowel wall or has spread to the lymph nodes. A permanent colostomy (creation of an abdominal opening for elimination of body wastes) is occasionally needed for colon cancer and is infrequently required for rectal cancer. There continues to be a need for effective diagnostic and treatment modalities for colorectal cancer.
There were an estimated 164,100 new cases of lung and bronchial cancer in 2000, accounting for 14% of all U.S. cancer diagnoses. The incidence rate of lung and bronchial cancer is declining significantly in men, from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s, the rate of increase among women began to slow. In 1996, the incidence rate in women was 42.3 per 100,000.
Lung and bronchial cancer caused an estimated 156,900 deaths in 2000, accounting for 28% of all cancer deaths. During 1992-1996, mortality from lung cancer declined significantly among men (−1.7% per year) while rates for women were still significantly increasing (0.9% per year). Since 1987, more women have died each year of lung cancer than breast cancer, which, for over 40 years, was the major cause of cancer death in women. Decreasing lung cancer incidence and mortality rates most likely resulted from decreased smoking rates over the previous 30 years; however, decreasing smoking patterns among women lag behind those of men. Of concern, although the declines in adult tobacco use have slowed, tobacco use in youth is increasing again.
Treatment options for lung and bronchial cancer are determined by the type and stage of the cancer and include surgery, radiation therapy, and chemotherapy. For many localized cancers, surgery is usually the treatment of choice. Because the disease has usually spread by the time it is discovered, radiation therapy and chemotherapy are often needed in combination with surgery. Chemotherapy alone or combined with radiation is the treatment of choice for small cell lung cancer; on this regimen, a large percentage of patients experience remission, which in some cases is long lasting. There is however, an ongoing need for effective treatment and diagnostic approaches for lung and bronchial cancers.
An estimated 182,800 new invasive cases of breast cancer were expected to occur among women in the United States during 2000. Additionally, about 1,400 new cases of breast cancer were expected to be diagnosed in men in 2000. After increasing about 4% per year in the 1980s, breast cancer incidence rates in women have leveled off in the 1990s to about 110.6 cases per 100,000.
In the U.S. alone, there were an estimated 41,200 deaths (40,800 women, 400 men) in 2000 due to breast cancer. Breast cancer ranks second among cancer deaths in women. According to the most recent data, mortality rates declined significantly during 1992-1996 with the largest decreases in younger women, both white and black. These decreases were probably the result of earlier detection and improved treatment.
Taking into account the medical circumstances and the patient's preferences, treatment of breast cancer may involve lumpectomy (local removal of the tumor) and removal of the lymph nodes under the arm; mastectomy (surgical removal of the breast) and removal of the lymph nodes under the arm; radiation therapy; chemotherapy; or hormone therapy. Often, two or more methods are used in combination. Numerous studies have shown that, for early stage disease, long-term survival rates after lumpectomy plus radiotherapy are similar to survival rates after modified radical mastectomy. Significant advances in reconstruction techniques provide several options for breast reconstruction after mastectomy. Recently, such reconstruction has been done at the same time as the mastectomy.
Local excision of ductal carcinoma in situ (DCIS) with adequate amounts of surrounding normal breast tissue may prevent the local recurrence of the DCIS. Radiation to the breast and/or tamoxifen may reduce the chance of DCIS occurring in the remaining breast tissue. This is important because DCIS, if left untreated, may develop into invasive breast cancer. Nevertheless, there are serious side effects or sequalae 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 STEAP-1, that has now been found to be over-expressed in the cancer(s) listed in Table I. Northern blot expression analysis of STEAP-1 gene expression in normal tissues shows a restricted expression pattern in adult tissues. The nucleotide (FIG. 2) and amino acid (FIG. 2, and FIG. 3) sequences of STEAP-1 are provided. The tissue-related profile of STEAP-1 in normal adult tissues, combined with the over-expression observed in the tissues listed in Table I, shows that STEAP-1 is aberrantly over-expressed in at least some cancers, and thus serves as a useful diagnostic, prophylactic, prognostic, and/or therapeutic target for cancers of the tissue(s) such as those listed in Table I.
The invention provides polynucleotides corresponding or complementary to all or part of the STEAP-1 genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding STEAP-1-related proteins and fragments of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 contiguous amino acids; at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100 or more than 100 contiguous amino acids of a STEAP-1-related protein, as well as the peptides/proteins themselves; DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides or oligonucleotides complementary or having at least a 90% homology to the STEAP-1 genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the STEAP-1 genes, mRNAs, or to STEAP-1-encoding polynucleotides. Also provided are means for isolating cDNAs and the genes encoding STEAP-1. Recombinant DNA molecules containing STEAP-1 polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of STEAP-1 gene products are also provided. The invention further provides antibodies that bind to STEAP-1 proteins and polypeptide fragments thereof, including polyclonal and monoclonal antibodies, murine and other mammalian antibodies, chimeric antibodies, humanized and fully human antibodies, and antibodies labeled with a detectable marker or therapeutic agent. In certain embodiments there is a proviso that the entire nucleic acid sequence of FIG. 2 is not encoded and/or the entire amino acid sequence of FIG. 2 is not prepared. In certain embodiments, the entire nucleic acid sequence of FIG. 2 is encoded and/or the entire amino acid sequence of FIG. 2 is prepared, either of which are in respective human unit dose forms.
The invention further provides methods for detecting the presence and status of STEAP-1 polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express STEAP-1. A typical embodiment of this invention provides methods for monitoring STEAP-1 gene products in a tissue or hematology sample having or suspected of having some form of growth dysregulation such as cancer.
The invention further provides various immunogenic or therapeutic compositions and strategies for treating cancers that express STEAP-1 such as cancers of tissues listed in Table I, including therapies aimed at inhibiting the transcription, translation, processing or function of STEAP-1 as well as cancer vaccines. In one aspect, the invention provides compositions, and methods comprising them, for treating a cancer that expresses STEAP-1 in a human subject wherein the composition comprises a carrier suitable for human use and a human unit dose of one or more than one agent that inhibits the production or function of STEAP-1. Preferably, the carrier is a uniquely human carrier. In another aspect of the invention, the agent is a moiety that is immunoreactive with STEAP-1 protein. Non-limiting examples of such moieties include, but are not limited to, antibodies (such as single chain, monoclonal, polyclonal, humanized, chimeric, or human antibodies), functional equivalents thereof (whether naturally occurring or synthetic), and combinations thereof. The antibodies can be conjugated to a diagnostic or therapeutic moiety. In another aspect, the agent is a small molecule as defined herein.
In another aspect, the agent comprises one or more than one peptide which comprises a cytotoxic T lymphocyte (CTL) epitope that binds an HLA class I molecule in a human to elicit a CTL response to STEAP-1 and/or one or more than one peptide which comprises a helper T lymphocyte (HTL) epitope which binds an HLA class II molecule in a human to elicit an HTL response. The peptides of the invention may be on the same or on one or more separate polypeptide molecules. In a further aspect of the invention, the agent comprises one or more than one nucleic acid molecule that expresses one or more than one of the CTL or HTL response stimulating peptides as described above. In yet another aspect of the invention, the one or more than one nucleic acid molecule may express a moiety that is immunologically reactive with STEAP-1 as described above. The one or more than one nucleic acid molecule may also be, or encodes, a molecule that inhibits production of STEAP-1. Non-limiting examples of such molecules include, but are not limited to, those complementary to a nucleotide sequence essential for production of STEAP-1 (e.g. antisense sequences or molecules that form a triple helix with a nucleotide double helix essential for STEAP-1 production) or a ribozyme effective to lyse STEAP-1 mRNA.
Note that to determine the starting position of any peptide set forth in Tables V-XVIII and XXII to LI (collectively HLA Peptide Tables) respective to its parental protein, e.g., variant 1, variant 2, etc., reference is made to three factors: the particular variant, the length of the peptide in an HLA Peptide Table, and the Search Peptides in Table LXI. Generally, a unique Search Peptide is used to obtain HLA peptides of a particular for a particular variant. The position of each Search Peptide relative to its respective parent molecule is listed in Table LXI. Accordingly, if a Search Peptide begins at position “X”, one must add the value “X−1” to each position in Tables V-XVIII and XXII to LI to obtain the actual position of the HLA peptides in their parental molecule. For example, if a particular Search Peptide begins at position 150 of its parental molecule, one must add 150−1, i.e., 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.
One embodiment of the invention comprises an HLA peptide, that occurs at least twice in Tables V-XVIII and XXII to LI collectively, or an oligonucleotide that encodes the HLA peptide. Another embodiment of the invention comprises an HLA peptide that occurs at least once in Tables V-XVIII and at least once in tables XXII to LI, or an oligonucleotide that encodes the HLA peptide.
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 STEAP-1 SSH sequence of 436 nucleotides.
FIG. 2. The cDNA and amino acid sequence of STEAP-1 variant 1 (also called “STEAP-1 v.1” or “STEAP-1 variant 1”) is shown in FIG. 2A. The start methionine is underlined. The open reading frame extends from nucleic acid 66-1085 including the stop codon. The cDNA and amino acid sequence of STEAP-1 variant 2 (also called “STEAP-1 v.2”) is shown in FIGS. 2B-1-2B-2. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 96-872 including the stop codon. The cDNA and amino acid sequence of STEAP-1 variant 3 (also called “STEAP-1 v.3”) is shown in FIGS. 2C-1-2C-2. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 96-944 including the stop codon. The cDNA and amino acid sequence of STEAP-1 variant 4 (also called “STEAP-1 v.4”) is shown in FIGS. 2D-1-2D-2. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 96-872 including the stop codon. The cDNA and amino acid sequence of STEAP-1 variant 5 (also called “STEAP-1 v.5”) is shown in FIGS. 2E-1-2E-2. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 96-872 including the stop codon. The cDNA and amino acid sequence of STEAP-1 variant 6 (also called “STEAP-1 v.6”) is shown in FIGS. 2F-1-2F-2. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 96-872 including the stop codon. The cDNA and amino acid sequence of STEAP-1 variant 7 (also called “STEAP-1 v.7”) is shown in FIGS. 2G-1-2G-2. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 96-872 including the stop codon. The cDNA and amino acid sequence of STEAP-1 variant 8 (also called “STEAP-1 v.8”) is shown in FIGS. 2H-1-2H-2. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 96-872 including the stop codon. The cDNA and amino acid sequence of STEAP-1 variant 9 (also called “STEAP-1 v.9”) is shown in FIGS. 2I-1-2I-2. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 96-872 including the stop codon. The cDNA and amino acid sequence of STEAP-1 variant 10 (also called “STEAP-1 v.10”) is shown in FIGS. 2J-1-2J-2. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 96-872 including the stop codon. The cDNA and amino acid sequence of STEAP-1 variant 11 (also called “STEAP-1 v.11”) is shown in FIGS. 2K-1-2K-2. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 96-872 including the stop codon. The cDNA and amino acid sequence of STEAP-1 variant 12 (also called “STEAP-1 v.12”) is shown in FIGS. 2L-1-2L-2. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 96-872 including the stop codon. The cDNA and amino acid sequence of STEAP-1 variant 13 (also called “STEAP-1 v.13”) is shown in FIGS. 2M-1-2M-2. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 96-872 including the stop codon. The cDNA and amino acid sequence of STEAP-1 variant 14 (also called “STEAP-1 v.14”) is shown in FIGS. 2N-1-2N-2. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 96-872 including the stop codon. The cDNA and amino acid sequence of STEAP-1 variant 15 (also called “STEAP-1 v.15”) is shown in FIGS. 2O-1-2O-2. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 96-872 including the stop codon. The cDNA and amino acid sequence of STEAP-1 variant 16 (also called “STEAP-1 v.16”) is shown in FIGS. 2P-1-2P-2. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 96-872 including the stop codon. The cDNA and amino acid sequence of STEAP-1 variant 17 (also called “STEAP-1 v.17”) is shown in FIGS. 2Q-1-2Q-2. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 96-872 including the stop codon. As used herein, a reference to STEAP-1 includes all variants thereof, including those shown in FIGS. 10 and 12.
FIG. 3. Amino acid sequence of STEAP-1 v.1 is shown in FIG. 3A; it has 339 amino acids. The amino acid sequence of STEAP-1 v.2 is shown in FIG. 3B; it has 258 amino acids. The amino acid sequence of STEAP-1 v.3 is shown in FIG. 3C; it has 282 amino acids. The amino acid sequence of STEAP-1 v.4 is shown in FIG. 3D; it has 258 amino acids. As used herein, a reference to STEAP-1 includes all variants thereof, including those shown in FIG. 11.
FIG. 4. The amino acid sequence alignment of STEAP-1 v.1 with mouse TNFα- induced adipose-related protein (gi/16905133) is shown in FIG. 4A. The amino acid sequence alignment of STEAP-1 v.1 with rat pHyde protein (gi/21717665) is shown in FIG. 4B. FIG. 4C shows Homology of STEAP-1 to mouse six transmembrane epithelial antigen of the prostate (gi/20820492).
FIGS. 5A and 5B. (5A) Hydrophilicity amino acid profile of STEAP-1 variant 1. (5B) Hydrophilicity amino acid profile of STEAP-1 variant 3, determined by computer algorithm sequence analysis using the method of Hopp and Woods (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828) accessed on the ProtScale website through the ExPasy molecular biology server.
FIGS. 6a and 6B. (6A) Hydropathicity amino acid profile of STEAP-1 variant 1. (6B) Hydropathicity amino acid profile of STEAP-1 variant 3, determined by computer algorithm sequence analysis using the method of Kyte and Doolittle (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132) accessed on the ProtScale website through the ExPasy molecular biology server.
FIGS. 7A and 7B. (7A) Percent accessible residues amino acid profile of STEAP-1 variant 1. (7B) Percent accessible residues amino acid profile of STEAP-1 variant 3, determined by computer algorithm sequence analysis using the method of Janin (Janin J., 1979 Nature 277:491-492) accessed on the ProtScale website through the ExPasy molecular biology server.
FIGS. 8A and 8B. (8A) Average flexibility amino acid profile of STEAP-1 variant 1. (8B) Average flexibility amino acid profile of STEAP-1 variant 3, determined by computer algorithm sequence analysis using the method of Bhaskaran and Ponnuswamy (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255) accessed on the ProtScale website through the ExPasy molecular biology server.
FIGS. 9A, and 9B. (9A) Beta-turn amino acid profile of STEAP-1 variant 1. (9B) Beta-turn amino acid profile of STEAP-1 variant 3, determined by computer algorithm sequence analysis using the method of Deleage and Roux (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294) accessed on the ProtScale website through the ExPasy molecular biology server.
FIGS. 10A and 10B. Schematic alignment of SNP variants of 8P1D4. Variants 8P1D4 v.4 through v.17 are variants with single nucleotide differences as compared to 8P1D4 v.2. Though these SNP variants are shown separately, they could also occur in any combinations and in any transcript variants that contains the base pairs, e.g., 8P1D4 v.1 and v.3. Numbers correspond to those of 8P1D4 v.2. Black box shows the same sequence as 8P1D4 v.2. SNPs are indicated above the box.
FIG. 11. Exon compositions of transcript variants of 8P1D4. This figure shows the structure of the transcript variants without poly A tail. Variants 8P1D4 v.1, v.2 and v.3 are transcript variants that share the same exons 2 and 3. The first exon of 8P1D4 v.1 is 30 bases shorter at 5′ end than the first exons of the other two transcript variants. The fourth exon of 8P1D4 v.2 is the same as the combined exon 4, intron 4 and exon 5 of 8P1D4 v.1. Compared with 8P1D4 v.1, variant 8P1D4 v.3 has an additional exon spliced out from intron 4 of 8P1D4 v.1. Lengths of introns and exons are not proportional.
FIG. 12. Schematic alignment of protein variants of 8P1D4. Protein variants correspond to nucleotide variants. Nucleotide variants 8P1D4 v.5 through v.17 in FIG. 10 code for the same protein as 8P 1D4 v.2. Proteins translated from transcript variants 8P1D4 v.1 and v.3 as shown in FIG. 11 may contain amino acid F (Phe) or L (Leu) at position 169. Single amino acid differences were indicated above the boxes. Black boxes represent the same sequence as 8P1D4 v.1. Boxes with different patterns of filling show different sequences. Numbers underneath the box correspond to 8P1D4 v.1
FIG. 13. FIGS. 13(a)-(c). Secondary structure and transmembrane domains prediction for 8P1D4 protein variants. The secondary structure of 8P1D4 protein variants 1 (SEQ ID NO: 93), 2 (SEQ ID NO: 94), and 3 (SEQ ID NO: 95); (FIGS. 13a-13c, respectively) were predicted using the HNN—Hierarchical Neural Network method, 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 also listed FIGS. 13(d), 13(f), and 13(h): Schematic representations of the probability of existence of transmembrane regions and orientation of 8P1D4 variant 1-3, (FIGS. 13(d), 13(f) and 13(h) respectively, based on the TMpred algorithm of Hofmann and Stoffel which utilizes TMBASE (K. Hofmann, W. Stoffel. TMBASE—A database of membrane spanning protein segments Biol. Chem. Hoppe-Seyler 374:166, 1993). FIGS. 13(e), 13(g), and 13(i): Schematic representations of the probability of the existence of transmembrane regions and the extracellular and intracellular orientation of 8P1D4 variants 1-3, FIGS. 13(e), 13(g), and 13(i) respectively, based on the TMHMM algorithm of Sonnhammer, von Heijne, and Krogh (Erik L. L. Sonnhammer, Gunnar von Heijne, and Anders Krogh: A hidden Markov model for predicting transmembrane helices in protein sequences. In Proc. of Sixth Int. Conf. on Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, Calif.: AAAI Press, 1998). The TMpred and TMHMM algorithms are accessed from the ExPasy molecular biology server.
FIG. 14. Expression of STEAP-1 in stomach cancer patient specimen. RNA was extracted from normal stomach (N) and from 10 different stomach cancer patient specimens (T). Northern blot with 10 μg of total RNA/lane was probed with STEAP-1 sequence. Results show strong expression of an approximately 1.6 kb STEAP-1 in the stomach tumor tissues. The lower panel represents ethidium bromide staining of the blot showing quality of the RNA samples.
FIG. 15 STEAP-1 expression in rectum cancer patient tissues. RNA was extracted from normal rectum (N), rectum cancer patient tumors (T), and rectum cancer metastasis (M). Northern blots with 10 μg of total RNA were probed with the STEAP-1 sequence. Results show strong expression of STEAP-1 in the rectum cancer patient tissues. The lower panel represents ethidium bromide staining of the blot showing quality of the RNA samples.
FIG. 16. Expression of STEAP-1 in human umbilical vein endothelial cells (HUVEC). First strand cDNA was prepared from HUVEC cells, LAPC-4AD and LAPC-9AD prostate cancer xenografts, as well as from human brain tissues. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to STEAP-1, was performed at 27 and 30 cycles of amplification (A). As a control, PCR using primers to actin is shown in (B). Results show strong expression of STEAP-1 in HUVEC cells similar to the expression detected in prostate cancer xenograft tissues. Expression of STEAP-1 in HUVEC cells indicates that targeting STEAP-1 may also target endothelial cells of the neovasculature of the tumors.
FIG. 17. Shows that STEAP-1 increased calcium flux in response to LPA.
FIG. 18. Shows that STEAP-1-mediated calcium transport regulates prostate cancer growth by regulating intracellular levels of calcium.
FIG. 19. The figure demonstrates that STEAP-1 mediates the transfer of the small molecule calcein between adjacent cells, and thereby regulates cell-cell communication in prostate cancer cells. The results demonstrated that while PC3 control cells (no detectable STEAP-1 protein expression) exhibit little calcein transfer, the expression of STEAP-1 allows the transfer of small molecules between cells, whereby the initially red recipient cells take on a brownish color, and co-localize the red and green molecules.
FIG. 20. The figure demonstrates that STEAP-1 mediates the transfer of the small molecule calcein between adjacent cells, and thereby regulates cell-cell communication in prostate cancer cells. The figure shows the time-dependent manner of cell-cell communication mediated by STEAP-1, where little transfer in PC3-STEAP-1 cells is seen at 6 hours, and much transfer is seen at 24 hours.
FIG. 21. This figure shows whether expression of STEAP-1 is sufficient to regulate specific signaling pathways not otherwise active in resting PC3 cells, the effect of these genes on the activation of the p38 MAPK cascade was investigated in the prostate cancer cell line PC3. FIG. 21A shows that while expression of the control neo gene has no effect on p38 phosphorylation, expression of STEAP-1 in PC3 cells is sufficient to induce the activation of the p38 pathway. FIG. 21B shows the results were verified using western blotting with an anti-p38 Ab, which shows equal protein loading on the gels.
FIG. 22. This figure shows the sufficiency of expression of STEAP-1 in the prostate cancer cell line PC3 to activate the mitogenic MAPK pathway, namely the ERK cascade. FIG. 22A shows that while expression of the control neo gene has no effect on ERK phosphorylation, expression of STEAP-1 in PC3 cells is sufficient to induce an increase in ERK phosphorylation. FIG. 22B shows these results were verified using anti-ERK western blotting and confirm the activation of the ERK pathway by STEAP-1 and STEAP-2.
FIG. 23. Shows necessary expression of STEAP-1 on Donor and Recipient Cells. Recipient PC3 or PC3-STEAP-1 cells were labeled with dextran-Texas Red. Donor PC3 or PC3-STEAP-1 cells were labeled with calcein-AM. The donor (green) and recipient (red) cells were co-cultured and analyzed by microscopy. Conclusion: The expression of STEAP-1 on both donor and recipient population is necessary for calcein transfer.
FIG. 24. Shows that the introduction of STEAP-1 specific RNAi reduces the expression of STEAP-1 in recombinant 3T3 and Rat-1 cells. Whole cell immunostaining revealed that the STEAP-1 RNAi reduced STEAP-1 expression in Rat-1 and 3T3 cells. This reduction was confirmed by Western blot analysis where STEAP-1 protein was substantially reduced in STEAP-1 RNAi treated cells relative to control and untreated cells.
FIG. 25. Shows that RNAi reduces the endogenous expression of STEAP-1 in the prostate cancer and LNCaP cell line.



DETAILED DESCRIPTION OF THE INVENTION
Outline of Sections


    
    
        I.) Definitions
        II.) STEAP-1 Polynucleotides
        II.A.) Uses of STEAP-1 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 STEAP-1-Encoding Nucleic Acid Molecules
        
        
        II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems
        III.) STEAP-1-related Proteins
        
            III.A.) Motif-bearing Protein Embodiments
            III.B.) Expression of STEAP-1-related Proteins
            III.C.) Modifications of STEAP-1-related Proteins
            III.D.) Uses of STEAP-1-related Proteins
        
        
        IV.) STEAP-1 Antibodies
        V.) STEAP-1 Cellular Immune Responses
        VI.) STEAP-1 Transgenic Animals
        VII.) Methods for the Detection of STEAP-1
        VIII.) Methods for Monitoring the Status of STEAP-1-related Genes and Their Products
        IX.) Identification of Molecules That Interact With STEAP-1
        X.) Therapeutic Methods and Compositions
        
            X.A.) Anti-Cancer Vaccines
        
        
        X.B.) STEAP-1 as a Target for Antibody-Based Therapy
        X.C.) STEAP-1 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 STEAP-1.
        XII.) Inhibition of STEAP-1 Protein Function
        
            XII.A.) Inhibition of STEAP-1 With Intracellular Antibodies
            XII.B.) Inhibition of STEAP-1 with Recombinant Proteins
            XII.C.) Inhibition of STEAP-1 Transcription or Translation
        
        
        XII.D.) General Considerations for Therapeutic Strategies
        XIII.) KITS/Articles of Manufacture
    
    


I.) Definitions
Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.
The terms “advanced prostate cancer”, “locally advanced prostate cancer”, “advanced disease” and “locally advanced disease” mean prostate cancers that have extended through the prostate capsule, and are meant to include stage C disease under the American Urological Association (AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, and stage T3-T4 and N+ disease under the TNM (tumor, node, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes compared to patients having clinically localized (organ-confined) prostate cancer. Locally advanced disease is clinically identified by palpable evidence of induration beyond the lateral border of the prostate, or asymmetry or induration above the prostate base. Locally advanced prostate cancer is presently diagnosed pathologically following radical prostatectomy if the tumor invades or penetrates the prostatic capsule, extends into the surgical margin, or invades the seminal vesicles.
“Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence STEAP-1 (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence STEAP-1. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.
The term “analog” refers to a molecule which is structurally similar or shares similar or corresponding attributes with another molecule (e.g. a STEAP-1-related protein). For example, an analog of a STEAP-1 protein can be specifically bound by an antibody or T cell that specifically binds to STEAP-1.
The term “antibody” is used in the broadest sense. Therefore, an “antibody” can be naturally occurring or man-made such as monoclonal antibodies produced by conventional hybridoma technology. Anti-STEAP-1 antibodies comprise monoclonal and polyclonal antibodies as well as fragments containing the antigen-binding domain and/or one or more complementarity determining regions of these antibodies.
An “antibody fragment” is defined as at least a portion of the variable region of the immunoglobulin molecule that binds to its target, i.e., the antigen-binding region. In one embodiment it specifically covers single anti-STEAP-1 antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-STEAP-1 antibody compositions with polyepitopic specificity.
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.”
The term “cytotoxic agent” refers to a substance that inhibits or prevents the expression activity of cells, function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Examples of cytotoxic agents include, but are not limited to auristatins, auromycins, maytansinoids, yttrium, bismuth, ricin, ricin A-chain, combrestatin, duocarmycins, dolostatins, doxorubicin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Sapaonaria officinalis inhibitor, and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes such as At211, I131, I125, Y90, R186, R188, 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 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 STEAP-1 genes or that encode polypeptides other than STEAP-1 gene product or fragments thereof. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated STEAP-1 polynucleotide. A protein is said to be “isolated,” for example, when physical, mechanical or chemical methods are employed to remove the STEAP-1 proteins from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated STEAP-1 protein. Alternatively, an isolated protein can be prepared by chemical means.
The term “mammal” refers to any organism classified as a mammal including mice, rats, rabbits, dogs, cats, cows, horses and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human.
The terms “metastatic prostate cancer” and “metastatic disease” mean prostate cancers that have spread to regional lymph nodes or to distant sites, and are meant to include stage D disease under the AUA system and stage T×N×M+ under the TNM system. As is the case with locally advanced prostate cancer, surgery is generally not indicated for patients with metastatic disease, and hormonal (androgen ablation) therapy is a preferred treatment modality. Patients with metastatic prostate cancer eventually develop an androgen-refractory state within 12 to 18 months of treatment initiation. Approximately half of these androgen-refractory patients die within 6 months after developing that status. The most common site for prostate cancer metastasis is bone. Prostate cancer bone metastases are often osteoblastic rather than osteolytic (i.e., resulting in net bone formation). Bone metastases are found most frequently in the spine, followed by the femur, pelvis, rib cage, skull and humerus. Other common sites for metastasis include lymph nodes, lung, liver and brain. Metastatic prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body radionuclide scans, skeletal radiography, and/or bone lesion biopsy.
The term “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 STEAP-1-related protein, refers to any pattern of amino acids forming part of the primary sequence of a protein, that is associated with a particular function (e.g. protein-protein interaction, protein-DNA interaction, etc) or modification (e.g. that is phosphorylated, glycosylated or amidated), or localization (e.g. secretory sequence, nuclear localization sequence, etc.) or a sequence that is correlated with being immunogenic, either humorally or cellularly. A motif can be either contiguous or capable of being aligned to certain positions that are generally correlated with a certain function or property. In the context of HLA motifs, “motif” refers to the pattern of residues in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino acids for a class II HLA motif, which is recognized by a particular HLA molecule. Peptide motifs for HLA binding are typically different for each protein encoded by each human HLA allele and differ in the pattern of the primary and secondary anchor residues.
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. In another embodiment, for example, the primary anchor residues of a peptide that will bind an HLA class II molecule are spaced relative to each other, rather than to the termini of a peptide, where the peptide is generally of at least 9 amino acids in length. The primary anchor positions for each motif and supermotif are set forth in Table IV. For example, analog peptides can be created by altering the presence or absence of particular residues in the primary and/or secondary anchor positions shown in Table IV. Such analogs are used to modulate the binding affinity and/or population coverage of a peptide comprising a particular HLA motif or supermotif.
“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 radio-


immunotherapy, 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 radio-


immunotherapy


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)









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 STEAP-1, ligands including hormones, neuropeptides, chemokines, odorants, phospholipids, and functional equivalents thereof that bind and preferably inhibit STEAP-1 protein function. Such non-limiting small molecules preferably have a molecular weight of less than about 10 kDa, more preferably below about 9, about 8, about 7, about 6, about 5 or about 4 kDa. In certain embodiments, small molecules physically associate with, or bind, STEAP-1 protein; are not found in naturally occurring metabolic pathways; and/or are more soluble in aqueous than non-aqueous solutions
“Stringency” of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured nucleic acid sequences to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).
“Stringent conditions” or “high stringency conditions”, as defined herein, are identified by, but not limited to, those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodium citrate) and 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C. “Moderately stringent conditions” are described by, but not limited to, those in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 37° C. in a solution comprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1×SSC at about 37-50° C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
An HLA “supermotif” is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles.
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 STEAP-1 protein shown in FIG. 2 or FIG. 3. An analog is an example of a variant protein. Splice isoform-s and single nucleotides polymorphisms (SNPs) are further examples of variants.
The “STEAP-1-related proteins” of the invention include those specifically identified herein, as well as allelic variants, conservative substitution variants, analogs and homologs that can be isolated/generated and characterized without undue experimentation following the methods outlined herein or readily available in the art. Fusion proteins that combine parts of different STEAP-1 proteins or fragments thereof, as well as fusion proteins of a STEAP-1 protein and a heterologous polypeptide are also included. Such STEAP-1 proteins are collectively referred to as the STEAP-1-related proteins, the proteins of the invention, or STEAP-1. The term “STEAP-1-related protein” refers to a polypeptide fragment or a STEAP-1 protein sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, or 664 or more amino acids.
II.) STEAP-1 Polynucleotides
One aspect of the invention provides polynucleotides corresponding or complementary to all or part of a STEAP-1 gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding a STEAP-1-related protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to a STEAP-1 gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides that hybridize to a STEAP-1 gene, mRNA, or to a STEAP-1 encoding polynucleotide (collectively, “STEAP-1 polynucleotides”). In all instances when referred to in this section, T can also be U in FIG. 2.
Embodiments of a STEAP-1 polynucleotide include: a STEAP-1 polynucleotide having the sequence shown in FIG. 2, the nucleotide sequence of STEAP-1 as shown in FIG. 2 wherein T is U; at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2; or, at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in FIG. 2 where T is U. For example, embodiments of STEAP-1 nucleotides comprise, without limitation:
(I) a polynucleotide comprising, consisting essentially of, or consisting of a sequence as shown in FIG. 2A, 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 66 through nucleotide residue number 1085, 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 96 through nucleotide residue number 872, 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 96 through nucleotide residue number 944, 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 96 through nucleotide residue number 872, 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 96 through nucleotide residue number 872, 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 96 through nucleotide residue number 872, 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 96 through nucleotide residue number 872, 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 96 through nucleotide residue number 872, 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. 2I, from nucleotide residue number 96 through nucleotide residue number 872, 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. 2J, from nucleotide residue number 96 through nucleotide residue number 872, 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. 2K, from nucleotide residue number 96 through nucleotide residue number 872, 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. 2L, from nucleotide residue number 96 through nucleotide residue number 872, including the stop codon, wherein T can also be U;
(XV) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2M, from nucleotide residue number 96 through nucleotide residue number 872, including the stop codon, wherein T can also be U;
(XVI) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2N, from nucleotide residue number 96 through nucleotide residue number 872, including the stop codon, wherein T can also be U;
(XVII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2O, from nucleotide residue number 96 through nucleotide residue number 872, including the stop codon, wherein T can also be U;
(XVIII) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2P, from nucleotide residue number 96 through nucleotide residue number 872, including the stop codon, wherein T can also be U;
(XIX) a polynucleotide comprising, consisting essentially of, or consisting of the sequence as shown in FIG. 2Q, from nucleotide residue number 96 through nucleotide residue number 872, including the stop codon, wherein T can also be U;
(XX) a polynucleotide that encodes a STEAP-1-related protein that is at least 90% homologous to an entire amino acid sequence shown in FIG. 2A-Q;
(XXI) a polynucleotide that encodes a STEAP-1-related protein that is at least 90% identical to an entire amino acid sequence shown in FIG. 2A-Q;
(XXII) a polynucleotide that encodes at least one peptide set forth in Tables V-XVIII and XXII-LI;
(XXIII) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of FIG. 3A in any whole number increment up to 339 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
(XXIV) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of FIG. 3A in any whole number increment up to 339 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
(XV) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of FIG. 3A in any whole number increment up to 339 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
(XVI) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of FIG. 3A in any whole number increment up to 339 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
(XXVII) a polynucleotide that encodes a peptide region of at least 5 amino acids of a peptide of FIG. 3A in any whole number increment up to 339 that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of FIG. 9;
(XXVIII) a polynucleotide that is fully complementary to a polynucleotide of any one of (I)-(XIX).
(XXIX) a peptide that is encoded by any of (I)-(XIX); and
(XXX) a polynucleotide of any of (I)-(XIX) or peptide of (XXIII-XXVII) together with a pharmaceutical excipient and/or in a human unit dose form
As used herein, a range is understood to disclose specifically all whole unit positions thereof.
Typical embodiments of the invention disclosed herein include STEAP-1 polynucleotides that encode specific portions of STEAP-1 mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example:
(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 330, 339 or more contiguous amino acids of STEAP-1 variant 1; the maximal lengths relevant for other variants are: variant 2, 258 amino acids; variant 3, 282 amino acids, and variant 4, 258 amino acids.
For example, representative embodiments of the invention disclosed herein include: polynucleotides and their encoded peptides themselves encoding about amino acid 1 to about amino acid 10 of the STEAP-1 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the STEAP-1 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the STEAP-1 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 30 to about amino acid 40 of the STEAP-1 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 40 to about amino acid 50 of the STEAP-1 protein shown in FIG. 2 or FIG. 3, polynucleotides-encoding about amino acid 50 to about amino acid 60 of the STEAP-1 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 60 to about amino acid 70 of the STEAP-1 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 70 to about amino acid 80 of the STEAP-1 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 80 to about amino acid 90 of the STEAP-1 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 90 to about amino acid 100 of the STEAP-1 protein shown in FIG. 2 or FIG. 3, in increments of about 10 amino acids, ending at the carboxyl terminal amino acid set forth in FIG. 2 or FIG. 3. Accordingly, polynucleotides encoding portions of the amino acid sequence (of about 10 amino acids), of amino acids, 100 through the carboxyl terminal amino acid of the STEAP-1 protein are embodiments of the invention. Wherein it is understood that each particular amino acid position discloses that position plus or minus five amino acid residues.
Polynucleotides encoding relatively long portions of a STEAP-1 protein are also within the scope of the invention. For example, polynucleotides encoding from about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 30, or 40 or 50 etc.) of the STEAP-1 protein “or variant” shown in FIG. 2 or FIG. 3 can be generated by a variety of techniques well known in the art. These polynucleotide fragments can include any portion of the STEAP-1 sequence as shown in FIG. 2.
Additional illustrative embodiments of the invention disclosed herein include STEAP-1 polynucleotide fragments encoding one or more of the biological motifs contained within a STEAP-1 protein “or variant” sequence, including one or more of the motif-bearing subsequences of a STEAP-1 protein “or variant” set forth in Tables V-XVIII and XXII-LI. In another embodiment, typical polynucleotide fragments of the invention encode one or more of the regions of STEAP-1 protein or variant that exhibit homology to a known molecule. In another embodiment of the invention, typical polynucleotide fragments can encode one or more of the STEAP-1 protein or variant N-glycosylation sites, cAMP and cGMP-dependent protein kinase phosphorylation sites, casein kinase II phosphorylation sites or N-myristoylation site and amidation sites.
Note that to determine the starting position of any peptide set forth in Tables V-XVIII and Tables XXII to LI (collectively HLA Peptide Tables) respective to its parental protein, e.g., variant 1, variant 2, etc., reference is made to three factors: the particular variant, the length of the peptide in an HLA Peptide Table, and the Search Peptides listed in Table LVII. Generally, a unique Search Peptide is used to obtain HLA peptides for a particular variant. The position of each Search Peptide relative to its respective parent molecule is listed in Table LXI. Accordingly, if a Search Peptide begins at position “X”, one must add the value “X minus 1” to each position in Tables V-XVIII and Tables XXII-IL to obtain the actual position of the HLA peptides in their parental molecule. For example if a particular Search Peptide begins at position 150 of its parental molecule, one must add 150-1, i.e., 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.
II.A.) Uses of STEAP-1 Polynucleotides
II.A.1.) Monitoring of Genetic Abnormalities
The polynucleotides of the preceding paragraphs have a number of different specific uses. The human STEAP-1 gene maps to the chromosomal location set forth in the Example entitled “Chromosomal Mapping of STEAP-1.” For example, because the STEAP-1 gene maps to this chromosome, polynucleotides that encode different regions of the STEAP-1 proteins are used to characterize cytogenetic abnormalities of this chromosomal locale, such as abnormalities that are identified as being associated with various cancers. In certain genes, a variety of chromosomal abnormalities including rearrangements have been identified as frequent cytogenetic abnormalities in a number of different cancers (see e.g. Krajinovic et al., Mutat. Res. 382(34): 81-83 (1998); Johansson et al., Blood 86(10): 3905-3914 (1995) and Finger et al., P.N.A.S. 85(23): 9158-9162 (1988)). Thus, polynucleotides encoding specific regions of the STEAP-1 proteins provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the chromosomal region that encodes STEAP-1 that may contribute to the malignant phenotype. In this context, these polynucleotides satisfy a need in the art for expanding the sensitivity of chromosomal screening in order to identify more subtle and less common chromosomal abnormalities (see e.g. Evans et al., Am. J. Obstet. Gynecol 171(4): 1055-1057 (1994)).
Furthermore, as STEAP-1 was shown to be highly expressed in bladder and other cancers, STEAP-1 polynucleotides are used in methods assessing the status of STEAP-1 gene products in normal versus cancerous tissues. Typically, polynucleotides that encode specific regions of the STEAP-1 proteins are used to assess the presence of perturbations (such as deletions, insertions, point mutations, or alterations resulting in a loss of an antigen etc.) in specific regions of the STEAP-1 gene, such as regions containing one or more motifs. Exemplary assays include both RT-PCR assays as well as single-strand conformation polymorphism (SSCP) analysis (see, e.g., Marrogi et al., J. Cutan. Pathol. 26(8): 369-378 (1999), both of which utilize polynucleotides encoding specific regions of a protein to examine these regions within the protein.
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 STEAP-1. For example, antisense molecules can be RNAs or other molecules, including peptide nucleic acids (PNAs) or non-nucleic acid molecules such as phosphorothioate derivatives that specifically bind DNA or RNA in a base pair-dependent manner. A skilled artisan can readily obtain these classes of nucleic acid molecules using the STEAP-1 polynucleotides and polynucleotide sequences disclosed herein.
Antisense technology entails the administration of exogenous oligonucleotides that bind to a target polynucleotide located within the cells. The term “antisense” refers to the fact that such oligonucleotides are complementary to their intracellular targets, e.g., STEAP-1. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The STEAP-1 antisense oligonucleotides of the present invention include derivatives such as S-oligonucleotides (phosphorothioate derivatives or S-oligos, see, Jack Cohen, supra), which exhibit enhanced cancer cell growth inhibitory action. S-oligos (nucleoside phosphorothioates) are isoelectronic analogs of an oligonucleotide (O-oligo) in which a nonbridging oxygen atom of the phosphate group is replaced by a sulfur atom. The S-oligos of the present invention can be prepared by treatment of the corresponding O-oligos with 3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transfer reagent. See, e.g., Iyer, R. P. et al., J. Org. Chem. 55:4693-4698 (1990); and Iyer, R. P. et al., J. Am. Chem. Soc. 112:1253-1254 (1990). Additional STEAP-1 antisense oligonucleotides of the present invention include morpholino antisense oligonucleotides known in the art (see, e.g., Partridge et al., 1996, Antisense & Nucleic Acid Drug Development 6: 169-175).
The STEAP-1 antisense oligonucleotides of the present invention typically can be RNA or DNA that is complementary to and stably hybridizes with the first 100 5′ codons or last 100 3′ codons of a STEAP-1 genomic sequence or the corresponding mRNA. Absolute complementarity is not required, although high degrees of complementarity are preferred. Use of an oligonucleotide complementary to this region allows for the selective hybridization to STEAP-1 mRNA and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, STEAP-1 antisense oligonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to STEAP-1 mRNA. Optionally, STEAP-1 antisense oligonucleotide is a 30-mer oligonucleotide that is complementary to a region in the first 10 5′ codons or last 10 3′ codons of STEAP-1. Alternatively, the antisense molecules are modified to employ ribozymes in the inhibition of STEAP-1 expression, see, e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).
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 STEAP-1 polynucleotide in a sample and as a means for detecting a cell expressing a STEAP-1 protein.
Examples of such probes include polypeptides comprising all or part of the human STEAP-1 cDNA sequence shown in FIG. 2. Examples of primer pairs capable of specifically amplifying STEAP-1 mRNAs are also described in the Examples. As will be understood by the skilled artisan, a great many different primers and probes can be prepared based on the sequences provided herein and used effectively to amplify and/or detect a STEAP-1 mRNA.
The STEAP-1 polynucleotides of the invention are useful for a variety of purposes, including but not limited to their use as probes and primers for the amplification and/or detection of the STEAP-1 gene(s), mRNA(s), or fragments thereof, as reagents for the diagnosis and/or prognosis of prostate cancer and other cancers; as coding sequences capable of directing the expression of STEAP-1 polypeptides; as tools for modulating or inhibiting the expression of the STEAP-1 gene(s) and/or translation of the STEAP-1 transcripts); and as therapeutic agents.
The present invention includes the use of any probe as described herein to identify and isolate a STEAP-1 or STEAP-1 related nucleic acid sequence from a naturally occurring source, such as humans or other mammals, as well as the isolated nucleic acid sequence per se, which would comprise all or most of the sequences found in the probe used.
II.A.4.) Isolation of STEAP-1-Encoding Nucleic Acid Molecules
The STEAP-1 cDNA sequences described herein enable the isolation of other polynucleotides encoding STEAP-1 gene product(s), as well as the isolation of polynucleotides encoding STEAP-1 gene product homologs, alternatively spliced isoforms, allelic variants, and mutant forms of a STEAP-1 gene product as well as polynucleotides that encode analogs of STEAP-1-related proteins. Various molecular cloning methods that can be employed to isolate full length cDNAs encoding a STEAP-1 gene are well known (see, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2d edition, Cold Spring Harbor Press, New York, 1989; Current Protocols in Molecular Biology. Ausubel et al, Eds., Wiley and Sons, 1995). For example, lambda phage cloning methodologies can be conveniently employed, using commercially available cloning systems (e.g., Lambda ZAP Express, Stratagene). Phage clones containing STEAP-1 gene cDNAs can be identified by probing with a labeled STEAP-1 cDNA or a fragment thereof. For example, in one embodiment, a STEAP-1 cDNA (e.g., FIG. 2) or a portion thereof can be synthesized and used as a probe to retrieve overlapping and full-length cDNAs corresponding to a STEAP-1 gene. A STEAP-1 gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with STEAP-1DNA 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 STEAP-1 polynucleotide, a fragment, analog or homologue thereof, including but not limited to phages, plasmids, phagemids, cosmids, YACs, BACs, as well as various viral and non-viral vectors well known in the art, and cells transformed or transfected with such recombinant DNA or RNA molecules. Methods for generating such molecules are well known (see, for example, Sambrook et al., 1989, supra).
The invention further provides a host-vector system comprising a recombinant DNA molecule containing a STEAP-1 polynucleotide, fragment, analog or homologue thereof within a suitable prokaryotic or eukaryotic host cell. Examples of suitable eukaryotic host cells include a yeast cell, a plant cell, or an animal cell, such as a mammalian cell or an insect cell (e.g., a baculovirus-infectible cell such as an Sf9 or HighFive cell). Examples of suitable mammalian cells include various prostate cancer cell lines such as DU145 and TsuPr1, other transfectable or transducible prostate cancer cell lines, primary cells (PrEC), as well as a number of mammalian cells routinely used for the expression of recombinant proteins (e.g., COS, CHO, 293, 293T cells). More particularly, a polynucleotide comprising the coding sequence of STEAP-1 or a fragment, analog or homolog thereof can be used to generate STEAP-1 proteins or fragments thereof using any number of host-vector systems routinely used and widely known in the art.
A wide range of host-vector systems suitable for the expression of STEAP-1 proteins or fragments thereof are available, see for example, Sambrook et al., 1989, supra; Current Protocols in Molecular Biology, 1995, supra). Preferred vectors for mammalian expression include but are not limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vector pSRαtkneo (Muller et al., 1991, MCB 11:1785). Using these expression vectors, STEAP-1 can be expressed in several prostate cancer and non-prostate cell lines, including for example 293, 293T, rat-1, NIH 3T3 and TsuPr1. The host-vector systems of the invention are useful for the production of a STEAP-1 protein or fragment thereof. Such host-vector systems can be employed to study the functional properties of STEAP-1 and STEAP-1 mutations or analogs.
Recombinant human STEAP-1 protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a STEAP-1-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding STEAP-1 or fragment, analog or homolog thereof, a STEAP-1-related protein is expressed in the 293T cells, and the recombinant STEAP-1 protein is isolated using standard purification methods (e.g., affinity purification using anti-STEAP-1 antibodies). In another embodiment, a STEAP-1 coding sequence is subcloned into the retroviral vector pSRαMSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293 and rat-1 in order to establish STEAP-1 expressing cell lines. Various other expression systems well known in the art can also be employed. Expression constructs encoding a leader peptide joined in frame to a STEAP-1 coding sequence can be used for the generation of a secreted form of recombinant STEAP-1 protein.
As discussed herein, redundancy in the genetic code permits variation in STEAP-1 gene sequences. In particular, it is known in the art that specific host species often have specific codon preferences, and thus one can adapt the disclosed sequence as preferred for a desired host. For example, preferred analog codon sequences typically have rare codons (i.e., codons having a usage frequency of less than about 20% in known sequences of the desired host) replaced with higher frequency codons. Codon preferences for a specific species are calculated, for example, by utilizing codon usage tables available on the INTERNET.
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.) STEAP-1-Related Proteins
Another aspect of the present invention provides STEAP-1-related proteins. Specific embodiments of STEAP-1 proteins comprise a polypeptide having all or part of the amino acid sequence of human STEAP-1 as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of STEAP-1 proteins comprise variant, homolog or analog polypeptides that have alterations in the amino acid sequence of STEAP-1 shown in FIG. 2 or FIG. 3.
Embodiments of a STEAP-1 polypeptide include: a STEAP-1 polypeptide having a sequence shown in FIG. 2, a peptide sequence of a STEAP-1 as shown in FIG. 2 wherein T is U; at least 10 contiguous nucleotides of a polypeptide having the sequence as shown in FIG. 2; or, at least 10 contiguous peptides of a polypeptide having the sequence as shown in FIG. 2 where T is U. For example, embodiments of STEAP-1 peptides comprise, without limitation:
(I) an protein comprising, consisting essentially of, or consisting of an amino acid sequence as shown in FIG. 2A-Q or FIG. 3A-D;
(II) a 101P3A11-related protein that is at least 90% homologous to an entire amino acid sequence shown in FIGS. 2A-Q;
(III) a 101P3A11-related protein that is at least 90% identical to an entire amino acid sequence shown in FIGS. 2A-Q or 3A-D;
(IV) a protein that comprises at least one peptide set forth in Tables V-XVIII or Tables XXII to LI, optionally with a proviso that it is not an entire protein of FIG. 2;
(V) a protein that comprises at least one peptide set forth in Tables V-XVIII, collectively, which peptide is also set forth in Tables XXII to LI, collectively, optionally with a proviso that it is not an entire protein of FIG. 2;
(VI) a protein that comprises at least two peptides selected from the peptides set forth in Tables V-XVIII and XXII to LI collectively, optionally with a proviso that it is not an entire protein of FIG. 2;
(VII) a protein that comprises at least two peptides selected from the peptides set forth in Tables V-XVII and XXII to LI 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 V-XVIII; and at least one peptide set forth in Tables XXII to LI, with a proviso that the protein is not a contiguous sequence from an amino acid sequence of FIG. 2;
(IX) a polypeptide comprising at least 5 amino acids of a protein of FIGS. 3A, 3B, 3C, or 3D in any whole number increment up to 339, 258, 282, or 258 respectively that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5;
(X) a polypeptide comprising at least 5 amino acids of a protein of FIGS. 3A, 3B, 3C, or 3D in any whole number increment up to 339, 258, 282, or 258, respectively, that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of FIG. 6;
(XI) a polypeptide comprising at least 5 amino acids of a protein of FIGS. 3A, 3B, 3C, or 3D in any whole number increment up to 339, 258, 282, or 258, respectively, that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7;
(XII) polypeptide comprising at least 5 amino acids of a protein of FIGS. 3A, 3B, 3C, or 3D in any whole number increment up to 339, 258, 282, or 258, respectively, that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile of FIG. 8;
(XIII) a polypeptide comprising at least 5 amino acids of a protein of FIGS. 3A, 3B, 3C, or 3D in any whole number increment up to 339, 258, 282, or 258, respectively, that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of FIG. 9;
(XIV) a peptide that occurs at least twice in Tables V-XVIII and XXII to LI, collectively;
(XV) a peptide that occurs at least three times in Tables V-XVIII and XXII to LI, collectively;
(XVI) a peptide that occurs at least four times in Tables V-XVIII and XXII to LI, collectively;
(XVII) a peptide that occurs at least five times in Tables V-XVIII and XXII to LI, collectively;
(XVIII) a peptide that occurs at least once in Tables V-XVIII, and at least once in tables XXII to LI;
(XIX) 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;
(XX) a peptide of (I)-(XIX) together with a pharmaceutical excipient and/or in a human unit dose form.
As used herein, a range is understood to specifically disclose all whole unit positions thereof.
Typical embodiments of the invention disclosed herein include 101P3A11 polynucleotides that encode specific portions of 101P3A11 mRNA sequences (and those which are complementary to such sequences) such as those that encode the proteins and/or fragments thereof, for example:
(a) 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 330, 339 or more contiguous amino acids of STEAP-1 variant 1; the maximal lengths relevant for other variants are: variant 2, 258 amino acids; variant 3, 282 amino acids, and variant 4, 258 amino acids.
In general, naturally occurring allelic variants of human STEAP-1 share a high degree of structural identity and homology (e.g., 90% or more homology). Typically, allelic variants of a STEAP-1 protein contain conservative amino acid substitutions within the STEAP-1 sequences described herein or contain a substitution of an amino acid from a corresponding position in a homologue of STEAP-1. One class of STEAP-1 allelic variants are proteins that share a high degree of homology with at least a small region of a particular STEAP-1 amino acid sequence, but further contain a radical departure from the sequence, such as a non-conservative substitution, truncation, insertion or frame shift. In comparisons of protein sequences, the terms, similarity, identity, and homology each have a distinct meaning as appreciated in the field of genetics. Moreover, orthology and paralogy can be important concepts describing the relationship of members of a given protein family in one organism to the members of the same family in other organisms.
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 1995 May 19; 270(20):11882-6).
Embodiments of the invention disclosed herein include a wide variety of art-accepted variants or analogs of STEAP-1 proteins such as polypeptides having amino acid insertions, deletions and substitutions. STEAP-1 variants can be made using methods known in the art such as site-directed mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)) or other known techniques can be performed on the cloned DNA to produce the STEAP-1 variant DNA.
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, STEAP-1 variants, analogs or homologs, have the distinguishing attribute of having at least one epitope that is “cross reactive” with a STEAP-1 protein having an amino acid sequence of FIG. 3. As used in this sentence, “cross reactive” means that an antibody or T cell that specifically binds, to a STEAP-1 variant also specifically binds to a STEAP-1 protein having an amino acid sequence set forth in FIG. 3. A polypeptide ceases to be a variant of a protein shown in FIG. 3, when it no longer contains any epitope capable of being recognized by an antibody or T cell that specifically binds to the starting STEAP-1 protein. Those skilled in the art understand that antibodies that recognize proteins bind to epitopes of varying size, and a grouping of the order of about four or five amino acids, contiguous or not, is regarded as a typical number of amino acids in a minimal epitope. See, e.g., Nair et al., J. Immunol. 2000 165(12): 6949-6955; Hebbes et al., Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Inmiunol (1985) 135(4):2598-608.
Other classes of STEAP-1-related protein variants share 70%, 75%, 80%, 85% or 90% or more similarity with an amino acid sequence of FIG. 3, or a fragment thereof. Another specific class of STEAP-1 protein variants or analogs comprises one or more of the STEAP-1 biological motifs described herein or presently known in the art. Thus, encompassed by the present invention are analogs of STEAP-1 fragments (nucleic or amino acid) that have altered functional (e.g. immunogenic) properties relative to the starting fragment. It is to be appreciated that motifs now or which become part of the art are to be applied to the nucleic or amino acid sequences of FIG. 2 or FIG. 3.
As discussed herein, embodiments of the claimed invention include polypeptides containing less than the full amino acid sequence of a STEAP-1 protein shown in FIG. 2 or FIG. 3. For example, representative embodiments of the invention comprise peptides/proteins having any 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of a STEAP-1 protein shown in FIG. 2 or FIG. 3.
Moreover, representative embodiments of the invention disclosed herein include polypeptides consisting of about amino acid 1 to about amino acid 10 of a STEAP-1 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 10 to about amino acid 20 of a STEAP-1 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 20 to about amino acid 30 of a STEAP-1 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 30 to about amino acid 40 of a STEAP-1 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 40 to about amino acid 50 of a STEAP-1 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 50 to about amino acid 60 of a STEAP-1 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 60 to about amino acid 70 of a STEAP-1 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 70 to about amino acid 80 of a STEAP-1 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 80 to about amino acid 90 of a STEAP-1 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 90 to about amino acid 100 of a STEAP-1 protein shown in FIG. 2 or FIG. 3, etc. throughout the entirety of a STEAP-1 amino acid sequence. Moreover, polypeptides consisting of about amino acid 1 (or 20 or 30 or 40 etc.) to about amino acid 20, (or 130, or 140 or 150 etc.) of a STEAP-1 protein shown in FIG. 2 or FIG. 3 are embodiments of the invention. It is to be appreciated that the starting and stopping positions in this paragraph refer to the specified position as well as that position plus or minus 5 residues.
STEAP-1-related proteins are generated using standard peptide synthesis technology or using chemical cleavage methods well known in the art. Alternatively, recombinant methods can be used to generate nucleic acid molecules that encode a STEAP-1-related protein. In one embodiment, nucleic acid molecules provide a means to generate defined fragments of a STEAP-1 protein (or variants, homologs or analogs thereof).
III.A.) Motif-Bearing Protein Embodiments
Additional illustrative embodiments of the invention disclosed herein include STEAP-1 polypeptides comprising the amino acid residues of one or more of the biological motifs contained within a STEAP-1 polypeptide sequence set forth in FIG. 2 or FIG. 3. Various motifs are known in the art, and a protein can be evaluated for the presence of such motifs by a number of publicly available sequence analysis tools (see, e.g., PFAM; BCM Search Launcher; PSORT; CBS; InterProScan; ScanProsite; EpimatrixTM and EpimerTM, Brown University; and BIMAS).
Motif bearing subsequences of all STEAP-1 variant proteins are set forth and identified in Tables V-XVIII and XXII-LI.
Table XIX sets forth several frequently occurring motifs based on pfam searches. The columns of Table XIX list (1) motif name abbreviation, (2) percent identity found amongst the different member of the motif family, (3) motif name or description and (4) most common function; location information is included if the motif is relevant for location.
Polypeptides comprising one or more of the STEAP-1 motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the STEAP-1 motifs discussed above are associated with growth dysregulation and because STEAP-1 is overexpressed in certain cancers (See, e.g., Table I). Casein kinase II, cAMP and camp-dependent protein kinase, and Protein Kinase C, for example, are enzymes known to be associated with the development of the malignant phenotype (see e.g. Chen et al., Lab Invest., 78(2): 165-174 (1998); Gaiddon et al., Endocrinology 136(10): 4331-4338 (1995); Hall et al., Nucleic Acids Research 24(6): 1119-1126 (1996); Peterziel et al., Oncogene 18(46): 6322-6329 (1999) and O'Brian, Oncol. Rep. 5(2): 305-309 (1998)). Moreover, both glycosylation and myristoylation are protein modifications also associated with cancer and cancer progression (see e.g. Dennis et al., Biochem. Biophys. Acta 1473(1):21-34 (1999); Raju et al., Exp. Cell Res. 235(1): 145-154 (1997)). Amidation is another protein modification also associated with cancer and cancer progression (see e.g. Treston et al., J. Natl. Cancer Inst. Monogr. (13): 169-175 (1992)).
In another embodiment, proteins of the invention comprise one or more of the immunoreactive epitopes identified in accordance with art-accepted methods, such as the peptides set forth in Tables V-XVII and XXII-LI. CTL epitopes can be determined using specific algorithms to identify peptides within a STEAP-1 protein that are capable of optimally binding to specified HLA alleles (e.g., Table IV; EpimatdrixTM and EpimerTM, Brown University; and BIMAS). Moreover, processes for identifying peptides that have sufficient binding affinity for HLA molecules and which are correlated with being immunogenic epitopes, are well known in the art, and are carried out without undue experimentation. In addition, processes for identifying peptides that are immunogenic epitopes, are well known in the art, and are carried out without undue experimentation either in vitro or in vivo.
Also known in the art are principles for creating analogs of such epitopes in order to modulate immunogenicity. For example, one begins with an epitope that bears a CTL or HTL motif (see, e.g., the HLA Class I and HLA Class II motifs/supermotifs of Table IV). The epitope is analoged by substituting out an amino acid at one of the specified positions, and replacing it with another amino acid specified for that position. For example, one can substitute out a deleterious residue in favor of any other residue, such as a preferred residue as defined in Table IV; substitute a less-preferred residue with a preferred residue as defined in Table IV; or substitute an originally-occurring preferred residue with another preferred residue as defined in Table IV. Substitutions can occur at primary anchor positions or at other positions in a peptide; see, e.g., Table IV.
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. 149:3580-1 (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 XX, and/or, one or more of the predicted CTL epitopes of Tables V-XVII and XXII-XLVII, and/or, one or more of the predicted HTL epitopes of Tables XLVIII-LI, and/or, one or more of the T cell binding motifs known in the art. Preferred embodiments contain no insertions, deletions or substitutions either within the motifs or within the intervening sequences of the polypeptides. In addition, embodiments which include a number of either N-terminal and/or C-terminal amino acid residues on either side of these motifs may be desirable (to, for example, include a greater portion of the polypeptide architecture in which the motif is located). Typically, the number of N-terminal and/or C-terminal amino acid residues on either side of a motif is between about 1 to about 100 amino acid residues, preferably 5 to about 50 amino acid residues.
STEAP-1-related proteins are embodied in many forms, preferably in isolated form. A purified STEAP-1 protein molecule will be substantially free of other proteins or molecules that impair the binding of STEAP-1 to antibody, T cell or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a STEAP-1-related proteins include purified STEAP-1-related proteins and functional, soluble STEAP-1-related proteins. In one embodiment, a functional, soluble STEAP-1 protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.
The invention also provided STEAP-1 proteins comprising biologically active fragments of a STEAP-1 amino acid sequence shown in FIG. 2 or FIG. 3. Such proteins exhibit properties of the starting STEAP-1 protein, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the starting STEAP-1 protein; to be bound by such antibodies; to elicit the activation of HTL or CTL; and/or, to be recognized by HTL or CTL that also specifically bind to the starting protein.
STEAP-1-related polypeptides that contain particularly interesting structures can be predicted and/or identified using various analytical techniques well known in the art, including, for example, the methods of Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis, or based on immunogenicity. Fragments that contain such structures are particularly useful in generating subunit-specific anti-STEAP-1 antibodies or T cells or in identifying cellular factors that bind to STEAP-1. For example, hydrophilicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can be generated, and immunogenic peptide fragments identified, using the method of Kyte, J. and Doolittle, R. F., 1982, J. Mol. Biol. 157:105-132. Percent (%) Accessible Residues profiles can be generated, and immunogenic peptide fragments identified, using the method of Janin J., 1979, Nature 277:491-492. Average Flexibility profiles can be generated, and immunogenic peptide fragments identified, using the method of Bhaskaran R., Ponnuswamy P. K., 1988, Int. J. Pept. Protein Res. 32:242-255. Beta-turn profiles can be generated, and immunogenic peptide fragments identified, using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294.
CTL epitopes can be determined using specific algorithms to identify peptides within a STEAP-1 protein that are capable of optimally binding to specified HLA alleles (e.g., by using the SYFPEITHI web site; the listings in Table IV(A)-E); EpimatrixTM and EpimerTM, Brown University; and BIMAS). Illustrating this, peptide epitopes from STEAP-1 that are presented in the context of human MHC Class I molecules, e.g., HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (see, e.g., Tables V-XVII, XXII-LI). Specifically, the complete amino acid sequence of the STEAP-1 protein and relevant portions of other variants, i.e., for HLA Class I predictions 9 flanking residues on either side of a point mutation, and for HLA Class II predictions 14 flanking residues on either side of a point mutation, were entered into the HLA Peptide Motif Search algorithm found in the Bioinformatics and Molecular Analysis Section (BIMAS) web site listed above; in addition to the site SYFPEITHI.
The HLA peptide motif search algorithm was developed by Dr. Ken Parker based on binding of specific peptide sequences in the groove of HLA Class I molecules, in particular HLA-A2 (see, e.g., Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75 (1994)). This algorithm allows location and ranking of 8-mer, 9-mer, and 10-mer peptides from a complete protein sequence for predicted binding to HLA-A2 as well as numerous other HLA Class I molecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-mers. For example, for Class I HLA-A2, the epitopes preferably contain a leucine (L) or methionine (M) at position 2 and a valine (V) or leucine (L) at the C-terminus (see, e.g., Parker et al., J. Immunol. 149:3580-7 (1992)). Selected results of STEAP-1 predicted binding peptides are shown in Tables V-XVIII and XXII-LI herein. In Tables V-XVIII and XXII-XLVII, selected candidates, 9-mers and 10-mers, for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score. In Tables XLVIII-LI, selected candidates, 15-mers, for each family member are shown along with their location, the amino acid sequence of each specific peptide, and an estimated binding score. The binding score corresponds to the estimated half time of dissociation of complexes containing the peptide at 37° 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 SYFPEITHI or BIMAS) are to be “applied” to a STEAP-1 protein in accordance with the invention. As used in this context “applied” means that a STEAP-1 protein is evaluated, e.g., visually or by computer-based patterns finding methods, as appreciated by those of skill in the relevant art. Every subsequence of a STEAP-1 protein of 8, 9, 10, or II amino acid residues that bears an HLA Class I motif, or a subsequence of 9 or more amino acid residues that bear an HLA Class II motif are within the scope of the invention.
III.B.) Expression STEAP-1-Related Proteins
In an embodiment described in the examples that follow, STEAP-1 can be conveniently expressed in cells (such as 293T cells) transfected with a commercially available expression vector such as a CMV-driven expression vector encoding STEAP-1 with a C-terminal 6× His and MYC tag (pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, Nashville Tenn.). The Tag5 vector provides an IgGK secretion signal that can be used to facilitate the production of a secreted STEAP-1 protein in transfected cells. The secreted HIS-tagged STEAP-1 in the culture media can be purified, e.g., using a nickel column using-standard techniques.
III.C.) Modifications of STEAP-1-Related Proteins
Modifications of STEAP-1-related proteins such as covalent modifications are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of a STEAP-1 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of a STEAP-1 protein. Another type of covalent modification of a STEAP-1 polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of a protein of the invention. Another type of covalent modification of STEAP-1 comprises linking a STEAP-1 polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
The STEAP-1-related proteins of the present invention can also be modified to form a chimeric molecule comprising STEAP-1 fused to another, heterologous polypeptide or amino acid sequence. Such a chimeric molecule can be synthesized chemically or recombinantly. A chimeric molecule can have a protein of the invention fused to another tumor-associated antigen or fragment thereof. Alternatively, a protein in accordance with the invention can comprise a fusion of fragments of a STEAP-1 sequence (amino or nucleic acid) such that a molecule is created that is not, through its length, directly homologous to the amino or nucleic acid sequences shown in FIG. 2 or FIG. 3. Such a chimeric molecule can comprise multiples of the same subsequence of STEAP-1. A chimeric molecule can comprise a fusion of a STEAP-1-related protein with a polyhistidine epitope tag, which provides an epitope to which immobilized nickel can selectively bind, with cytokines or with growth factors. The epitope tag is generally placed at the amino- or carboxyl-terminus of a STEAP-1 protein. In an alternative embodiment, the chimeric molecule can comprise a fusion of a STEAP-1-related protein with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an “immunoadhesin”), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a STEAP-1 polypeptide in place of at least one variable region within an Ig molecule. In a preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgGI molecule. For the production of immunoglobulin fusions see, e.g., U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
III.D.) Uses of STEAP-1-Related Proteins
The proteins of the invention have a number of different specific uses. As STEAP-1 is highly expressed in prostate and other cancers, STEAP-1-related proteins are used in methods that assess the status of STEAP-1 gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of a STEAP-1 protein are used to assess the presence of perturbations (such as deletions, insertions, point mutations etc.) in those regions (such as regions containing one or more motifs). Exemplary assays utilize antibodies or T cells targeting STEAP-1-related proteins comprising the amino acid residues of one or more of the biological motifs contained within a STEAP-1 polypeptide sequence in order to evaluate the characteristics of this region in normal versus cancerous tissues or to elicit an immune response to the epitope. Alternatively, STEAP-1-related proteins that contain the amino acid residues of one or more of the biological motifs in a STEAP-1 protein are used to screen for factors that interact with that region of STEAP-1.
STEAP-1 protein fragments/subsequences are particularly useful in generating and characterizing domain-specific antibodies (e.g., antibodies recognizing an extracellular or intracellular epitope of a STEAP-1 protein), for identifying agents or cellular factors that bind to STEAP-1 or a particular structural domain thereof, and in various therapeutic and diagnostic contexts, including but not limited to diagnostic assays, cancer vaccines and methods of preparing such vaccines.
Proteins encoded by the STEAP-1 genes, or by analogs, homologs or fragments thereof, have a variety of uses, including but not limited to generating antibodies and in methods for identifying ligands and other agents and cellular constituents that bind to a STEAP-1 gene product. Antibodies raised against a STEAP-1 protein or fragment thereof are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of STEAP-1 protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers. STEAP-1-related nucleic acids or proteins are also used in generating HTL or CTL responses.
Various immunological assays useful for the detection of STEAP-1 proteins are used, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), immunocytochemical methods, and the like. Antibodies can be labeled and used as immunological imaging reagents capable of detecting STEAP-1-expressing cells (e.g., in radioscintigraphic imaging methods). STEAP-1 proteins are also particularly useful in generating cancer vaccines, as further described herein.
IV.) STEAP-1 Antibodies
Another aspect of the invention provides antibodies that bind to STEAP-1-related proteins. Preferred antibodies specifically bind to a STEAP-1-related protein and do not bind (or bind weakly) to peptides or proteins that are not STEAP-1-related proteins. For example, antibodies that bind STEAP-1 can bind STEAP-1-related proteins such as the homologs or analogs thereof.
STEAP-1 antibodies of the invention are particularly useful in cancer (see, e.g., Table I) diagnostic and prognostic assays, and imaging methodologies. Similarly, such antibodies are useful in the treatment, diagnosis, and/or prognosis of other cancers, to the extent STEAP-1 is also expressed or overexpressed in these other cancers. Moreover, intracellularly expressed antibodies (e.g., single chain antibodies) are therapeutically useful in treating cancers in which the expression of STEAP-1 is involved, such as advanced or metastatic prostate cancers.
The invention also provides various immunological assays useful for the detection and quantification of STEAP-1 and mutant STEAP-1-related proteins. Such assays can comprise one or more STEAP-1 antibodies capable of recognizing and binding a STEAP-1-related protein, as appropriate. These assays are performed within various immunological assay formats well known in the art, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), and the like.
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 STEAP-1 are also provided by the invention, including but not limited to radioscintigraphic imaging methods using labeled STEAP-1 antibodies. Such assays are clinically useful in the detection, monitoring, and prognosis of STEAP-1 expressing cancers such as prostate cancer.
STEAP-1 antibodies are also used in methods for purifying a STEAP-1-related protein and for isolating STEAP-1 homologues and related molecules. For example, a method of purifying a STEAP-1-related protein comprises incubating a STEAP-1 antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a STEAP-1-related protein under conditions that permit the STEAP-1 antibody to bind to the STEAP-1-related protein; washing the solid matrix to eliminate impurities; and eluting the STEAP-1-related protein from the coupled antibody. Other uses of STEAP-1 antibodies in accordance with the invention include generating anti-idiotypic antibodies that mimic a STEAP-1 protein.
Various methods for the preparation of antibodies are well known in the art. For example, antibodies can be prepared by immunizing a suitable mammalian host using a STEAP-1-related protein, peptide, or fragment, in isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NY (1989)). In addition, fusion proteins of STEAP-1 can also be used, such as a STEAP-1 GST-fusion protein. In a particular embodiment, a GST fusion protein comprising all or most of the amino acid sequence of FIG. 2 or FIG. 3 is produced, then used as an immunogen to generate appropriate antibodies. In another embodiment, a STEAP-1-related protein is synthesized and used as an immunogen.
In addition, naked DNA immunization techniques known in the art are used (with or without purified STEAP-1-related protein or STEAP-1 expressing cells) to generate an immune response to the encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).
The amino acid sequence of a STEAP-1 protein as shown in FIG. 2 or FIG. 3 can be analyzed to select specific regions of the STEAP-1 protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of a STEAP-1 amino acid sequence are used to identify hydrophilic regions in the STEAP-1 structure. Regions of a STEAP-1 protein that show immunogenic structure, as well as other regions and domains, can readily be identified using various other methods known in the art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis. Hydrophilicity profiles can be generated using the method of Hopp, T. P. and Woods, K. R., 1981, Proc. Nail. 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 STEAP-1 antibodies are further illustrated by way of the examples provided herein. Methods for preparing a protein or polypeptide for use as an immunogen are well known in the art. Also well known in the art are methods for preparing immunogenic conjugates of a protein with a carrier, such as BSA, KLH or other carrier protein. In some circumstances, direct conjugation using, for example, carbodiimide reagents are used; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, Ill., are effective. Administration of a STEAP-1 immunogen is often conducted by injection over a suitable time period and with use of a suitable adjuvant, as is understood in the art. During the immunization schedule, titers of antibodies can be taken to determine adequacy of antibody formation.
STEAP-1 monoclonal antibodies can be produced by various means well known in the art. For example, immortalized cell lines that secrete a desired monoclonal antibody are prepared using the standard hybridoma technology of Kohler and Milstein or modifications that immortalize antibody-producing B cells, as is generally known. Immortalized cell lines that secrete the desired antibodies are screened by immunoassay in which the antigen is a STEAP-1-related protein. When the appropriate immortalized cell culture is identified, the cells can be expanded and antibodies produced either from in vitro cultures or from ascites fluid.
The antibodies or fragments of the invention can also be produced, by recombinant means. Regions that bind specifically to the desired regions of a STEAP-1 protein can also be produced in the context of chimeric or complementarity-determining region (CDR) grafted antibodies of multiple species origin. Humanized or human STEAP-1 antibodies can also be produced, and are preferred for use in therapeutic contexts. Methods for humanizing murine and other non-human antibodies, by substituting one or more of the non-human antibody CDRs for corresponding human antibody sequences, are well known (see for example, Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen et al., 1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151: 2296.
Methods for producing fully human monoclonal antibodies include phage display and transgenic methods (for review, see Vaughan et al., 1998, Nature Biotechnology 16: 535-539). Fully human STEAP-1 monoclonal antibodies can be generated using cloning technologies employing large human Ig gene combinatorial libraries (i.e., phage display) (Griffiths and Hoogenboom, Building an in vitro immune system: human antibodies from phage display libraries. In: Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man, Clark, M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatorial libraries. Id., pp 65-82). Fully human STEAP-1 monoclonal antibodies can also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application WO98/24893, Kucherlapati and Jakobovits et al., published Dec. 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614; U.S. Pat. No. 6,162,963 issued 19 Dec. 2000; U.S. Pat. No. 6,150,584 issued 12 Nov. 2000; and, U.S. Pat. No. 6,114,598 issued 5 Sep. 2000). This method avoids the in vitro manipulation required with phage display technology and efficiently produces high affinity authentic human antibodies.
Reactivity of STEAP-1 antibodies with a STEAP-1-related protein can be established by a number of well known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses using, as appropriate, STEAP-1-related proteins, STEAP-1-expressing cells or extracts thereof. A STEAP-1 antibody or fragment thereof can be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator or an enzyme. Further, bi-specific antibodies specific for two or more STEAP-1 epitopes are generated using methods generally known in the art. Homodimeric antibodies can also be generated by cross-linking techniques known in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).
V.) STEAP-1 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 cellar 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; Sette, A. and Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et al., Cell 74:929-937, 1993; Kondo et al., J. Immunol 155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490, 1996; Sidney et al., Human Immunol. 45:79-93, 1996; Sette, A. and Sidney, J. Immunogenetics 1999 November; 50(3-4):201-12, Review).
Furthermore, x-ray crystallographic analyses of HLA-peptide complexes have revealed pockets within the peptide binding cleft/groove of HLA molecules which accommodate, in an allele-specific mode, residues borne by peptide ligands; these residues in turn determine the HLA binding capacity of the peptides in which they are present. (See, e.g., Madden, D. R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203, 1996; Fremont et al., Immunity 8:305, 1998; Stern et al., Structure 2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H. et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. Sci USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M. L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927, 1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science 257:919, 1992; Saper, M. A., Bjorkman, P. J. and Wiley, D.C., J. Mol. Biol. 219:277, 1991.)
Accordingly, the definition of class I and class II allele-specific HLA binding motifs, or class I or class II supermotifs allows identification of regions within a protein that are correlated with binding to particular HLA antigen(s).
Thus, by a process of HLA motif identification, candidates for epitope-based vaccines have been identified; such candidates can be further evaluated by HLA-peptide binding assays to determine binding affinity and/or the time period of association of the epitope and its corresponding HLA molecule. Additional confirmatory work can be performed to select, amongst these vaccine candidates, epitopes with preferred characteristics in terms of population coverage, and/or immunogenicity.
Various strategies can be utilized to evaluate cellular immunogenicity, including:
1) Evaluation of primary T cell cultures from normal individuals (see, e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. et al., Proc. Natl. Acad. Sci. USA 91:2105, 1994; Tsai, V. et al., J. Immunol. 158:1796, 1997; Kawashima, I. et al., Human Immunol. 59:1, 1998). This procedure involves the stimulation of peripheral blood lymphocytes (PBL) from normal subjects with a test peptide in the presence of antigen presenting cells in vitro over a period of several weeks. T cells specific for the peptide become activated during this time and are detected using, e.g., a lymphokine- or 51Cr-release assay involving peptide sensitized target cells.
2) Immunization of HLA transgenic mice (see, e.g., Wentworth, P. A. et al., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol. 8:651, 1996; Alexander, J. et al., J. Immunol. 159:4753, 1997). For example, in such methods peptides in incomplete Freund's adjuvant are administered subcutaneously to HLA transgenic mice. Several weeks following immunization, splenocytes are removed and cultured in vitro in the presence of test peptide for approximately one week. Peptide-specific T cells are detected using, e.g., a 51Cr-release assay involving peptide sensitized target cells and target cells expressing endogenously generated antigen.
3) Demonstration of recall T cell responses from immune individuals who have been either effectively vaccinated and/or from chronically ill patients (see, e.g., Rehermann, B. et al., J. Exp. Med. 181:1047, 1995; Doolan, D. L. et al., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin. Invest. 100:503, 1997; Threlkeld, S. C. et al., J. Immunol. 159:1648, 1997; Diepolder, H. M. et al., J. Virol. 71:6011, 1997). Accordingly, recall responses are detected by culturing PBL from subjects that have been exposed to the antigen due to disease and thus have generated an immune response “naturally”, or from patients who were vaccinated against the antigen. PBL from subjects are cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APC) to allow activation of “memory” T cells, as compared to “naive” T cells. At the end of the culture period, T cell activity is detected using assays including 51Cr release involving peptide-sensitized targets, T cell proliferation, or lymphokine release.
VI.) STEAP-1 Transgenic Animals
Nucleic acids that encode a STEAP-1-related protein can also be used to generate either transgenic animals or “knock out” animals that, in turn, are useful in the development and screening of therapeutically useful reagents. In accordance with established techniques, cDNA encoding STEAP-1 can be used to clone genomic DNA that encodes STEAP-1. The cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode STEAP-1 Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Pat. No. 4,736,866 issued 12 Apr. 1988, and U.S. Pat. No. 4,870,009 issued 26 Sep. 1989. Typically, particular cells would be targeted for STEAP-1 transgene incorporation with tissue-specific enhancers.
Transgenic animals that include a copy of a transgene encoding STEAP-1 can be used to examine the effect of increased expression of DNA that encodes STEAP-1. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression. In accordance with this aspect of the invention, an animal is treated with a reagent and a reduced incidence of a pathological condition, compared to untreated animals that bear the transgene, would indicate a potential therapeutic intervention for the pathological condition.
Alternatively, non-human homologues of STEAP-1 can be used to construct a STEAP-1 “knock out” animal that has a defective or altered gene encoding STEAP-1 as a result of homologous recombination between the endogenous gene encoding STEAP-1 and altered genomic DNA encoding STEAP-1 introduced into an embryonic cell of the animal. For example, cDNA that encodes STEAP-1 can be used to clone genomic DNA encoding STEAP-1 in accordance with established techniques. A portion of the genomic DNA encoding STEAP-1 can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends) are included in the vector (see, e.g., Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected (see, e.g., Li et al., Cell, 69:915 (1992)). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras (see, e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal, and the embryo brought to term to create a “knock out” animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knock out animals can be characterized, for example, for their ability to defend against certain pathological conditions or for their development of pathological conditions due to absence of a STEAP-1 polypeptide.
VII.) Methods for the Detection of STEAP-1
Another aspect of the present invention relates to methods for detecting STEAP-1 polynucleotides and STEAP-1-related proteins, as well as methods for identifying a cell that expresses STEAP-1. The expression profile of STEAP-1 makes it a diagnostic marker for metastasized disease. Accordingly, the status of STEAP-1 gene products provides information useful for predicting a variety of factors including susceptibility to advanced stage disease, rate of progression, and/or tumor aggressiveness. As discussed in detail herein, the status of STEAP-1 gene products in patient samples can be analyzed by a variety protocols that are well known in the art including immunohistochemical analysis, the variety of Northern blotting techniques including in situ hybridization, RT-PCR analysis (for example on laser capture micro-dissected samples), Western blot analysis and tissue array analysis.
More particularly, the invention provides assays for the detection of STEAP-1 polynucleotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, semen, cell preparations, and the like. Detectable STEAP-1 polynucleotides include, for example, a STEAP-1 gene or fragment thereof, STEAP-mRNA, alternative splice variant STEAP-1 mRNAs, and recombinant DNA or RNA molecules that contain a STEAP-1 polynucleotide. A number of methods for amplifying and/or detecting the presence of STEAP-1 polynucleotides are well known in the art and can be employed in the practice of this aspect of the invention.
In one embodiment, a method for detecting a STEAP-1 mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; sampling the cDNA so produced using a STEAP-1 polynucleotides as sense and antisense primers to amplify STEAP-1 cDNAs therein; and detecting the presence of the amplified STEAP-1 cDNA. Optionally, the sequence of the amplified STEAP-1 cDNA can be determined.
In another embodiment, a method of detecting a STEAP-1 gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using STEAP-1 polynucleotides as sense and antisense primers; and detecting the presence of the amplified STEAP-1 gene. Any number of appropriate sense and antisense probe combinations can be designed from a STEAP-1 nucleotide sequence (see, e.g., FIG. 2) and used for this purpose.
The invention also provides assays for detecting the presence of a STEAP-1 protein in a tissue or other biological sample such as serum, semen, bone, prostate, urine, cell preparations, and the like. Methods for detecting a STEAP-1-related protein are also well known and include, for example, immunoprecipitation, immunohistochemical analysis, Western blot analysis, molecular binding assays, ELISA, ELIFA and the like. For example, a method of detecting the presence of a STEAP-1-related protein in a biological sample comprises first contacting the sample with a STEAP-1 antibody, a STEAP-1-reactive fragment thereof, or a recombinant protein containing an antigen-binding region of a STEAP-1 antibody; and then detecting the binding of STEAP-1-related protein in the sample.
Methods for identifying a cell that expresses STEAP-1 are also within the scope of the invention. In one embodiment, an assay for identifying a cell that expresses a STEAP-1 gene comprises detecting the presence of STEAP-1 mRNA in the cell. Methods for the detection of particular mRNAs in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled STEAP-1 riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for STEAP-1, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like). Alternatively, an assay for identifying a cell that expresses a STEAP-1 gene comprises detecting the presence of STEAP-1-related protein in the cell or secreted by the cell. Various methods for the detection of proteins are well known in the art and are employed for the detection of STEAP-L-related proteins and cells that express STEAP-1-related proteins.
STEAP-1 expression analysis is also useful as a tool for identifying and evaluating agents that modulate STEAP-1 gene expression. For example, STEAP-1 expression is significantly upregulated in prostate cancer, and is expressed in cancers of the tissues listed in Table I. Identification of a molecule or biological agent that inhibits STEAP-1 expression or over-expression in cancer cells is of therapeutic value. For example, such an agent can be identified by using a screen that quantifies STEAP-1 expression by RT-PCR, nucleic acid hybridization or antibody binding.
VIII.) Methods for Monitoring the Status of STEAP-1-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 STEAP-1 expression in cancers) allows for early detection of such aberrant physiology, before a pathologic state such as cancer has progressed to a stage that therapeutic options are more limited and or the prognosis is worse. In such examinations, the status of STEAP-1 in a biological sample of interest can be compared, for example, to the status of STEAP-1 in a corresponding normal sample (e.g. a sample from that individual or alternatively another individual that is not affected by a pathology). An alteration in the status of STEAP-1 in the biological sample (as compared to the normal sample) provides evidence of dysregulated cellular growth. In addition to using a biological sample that is not affected by a pathology as a normal sample, one can also use a predetermined normative value such as a predetermined normal level of mRNA expression (see, e.g., Grever et al., J. Comp. Neurol. 1996 Dec. 9; 376(2): 306-14 and U.S. Pat. No. 5,837,501) to compare STEAP-1 status in a sample.
The term “status” in this context is used according to its art accepted meaning and refers to the condition or state of a gene and its products. Typically, skilled artisans use a number of parameters to evaluate the condition or state of a gene and its products. These include, but are not limited to the location of expressed gene products (including the location of STEAP-1 expressing cells) as well as the level, and biological activity of expressed gene products (such as STEAP-1 mRNA, polynucleotides and polypeptides). Typically, an alteration in the status of STEAP-1 comprises a change in the location of STEAP-1 and/or STEAP-1 expressing cells and/or an increase in STEAP-1 mRNA and/or protein expression.
STEAP-1 status in a sample can be analyzed by a number of means well known in the art, including without limitation immunohistochemical analysis, in situ hybridization, RT-PCR analysis on laser capture micro-dissected samples, Western blot analysis, and tissue array analysis. Typical protocols for evaluating the status of a STEAP-1 gene and gene products are found, for example in Ausubel et al. eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Thus, the status of STEAP-1 in a biological sample is evaluated by various methods utilized by skilled artisans including, but not limited to genomic Southern analysis (to examine, for example perturbations in a STEAP-1 gene), Northern analysis and/or PCR analysis of STEAP-1 mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of STEAP-1 mRNAs), and, Western and/or immunohistochemical analysis (to examine, for example alterations in polypeptide sequences, alterations in polypeptide localization within a sample, alterations in expression levels of STEAP-1 proteins and/or associations of STEAP-1 proteins with polypeptide binding partners). Detectable STEAP-1 polynucleotides include, for example, a STEAP-1 gene or fragment thereof, STEAP-1 mRNA, alternative splice variants, STEAP-1 mRNAs, and recombinant DNA or RNA molecules containing a STEAP-1 polynucleotide.
The expression profile of STEAP-1 makes it a diagnostic marker for local and/or metastasized disease, and provides information on the growth or oncogenic potential of a biological sample. In particular, the status of STEAP-1 provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The invention provides methods and assays for determining STEAP-1 status and diagnosing cancers that express STEAP-1, such as cancers of the tissues listed in Table I. For example, because STEAP-1 mRNA is so highly expressed in prostate and other cancers relative to normal prostate tissue, assays that evaluate the levels of STEAP-1 mRNA transcripts or proteins in a biological sample can be used to diagnose a disease associated with STEAP-1 dysregulation, and can provide prognostic information useful in defining appropriate therapeutic options.
The expression status of STEAP-1 provides information including the presence, stage and location of dysplastic, precancerous and cancerous cells, predicting susceptibility to various stages of disease, and/or for gauging tumor aggressiveness. Moreover, the expression profile makes it useful as an imaging reagent for metastasized disease. Consequently, an aspect of the invention is directed to the various molecular prognostic and diagnostic methods for examining the status of STEAP-1 in biological samples such as those from individuals suffering from, or suspected of suffering from a pathology characterized by dysregulated cellular growth, such as cancer.
As described above, the status of STEAP-1 in a biological sample can be examined by a number of well-known procedures in the art. For example, the status of STEAP-1 in a biological sample taken from a specific location in the body can be examined by evaluating the sample for the presence or absence of STEAP-1 expressing cells (e.g. those that express STEAP-1 mRNAs or proteins). This examination can provide evidence of dysregulated cellular growth, for example, when STEAP-1-expressing cells are found in a biological sample that does not normally contain such cells (such as a lymph node), because such alterations in the status of STEAP-1 in a biological sample are often associated with dysregulated cellular growth. Specifically, one indicator of dysregulated cellular growth is the metastases of cancer cells from an organ of origin (such as the prostate) to a different area of the body (such as a lymph node). In this context, evidence of dysregulated cellular growth is important for example because occult lymph node metastases can be detected in a substantial proportion of patients with prostate cancer, and such metastases are associated with known predictors of disease progression (see, e.g., Murphy et al., Prostate 42(4): 315-317 (2000); Su et al., Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al., J Urol 1995 August 154(2 Pt 1):474-8).
In one aspect, the invention provides methods for monitoring STEAP-1 gene products by determining the status of STEAP-1 gene products expressed by cells from an individual suspected of having a disease associated with dysregulated cell growth (such as hyperplasia or cancer) and then comparing the status so determined to the status of STEAP-1 gene products in a corresponding normal sample. The presence of aberrant STEAP-1 gene products in the test sample relative to the normal sample provides an indication of the presence of dysregulated cell growth within the cells of the individual.
In another aspect, the invention provides assays useful in determining the presence of cancer in an individual, comprising detecting a significant increase in STEAP-1 mRNA or protein expression in a test cell or tissue sample relative to expression levels in the corresponding normal cell or tissue. The presence of STEAP-1 mRNA can, for example, be evaluated in tissues including but not limited to those listed in Table I. The presence of significant STEAP-1 expression in any of these tissues is useful to indicate the emergence, presence and/or severity of a cancer, since the corresponding normal tissues do not express STEAP-1 mRNA or express it at lower levels.
In a related embodiment, STEAP-1 status is determined at the protein level rather than at the nucleic acid level. For example, such a method comprises determining the level of STEAP-1 protein expressed by cells in a test tissue sample and comparing the level so determined to the level of STEAP-1 expressed in a corresponding normal sample. In one embodiment, the presence of STEAP-1 protein is evaluated, for example, using immunohistochemical methods. STEAP-1 antibodies or binding partners capable of detecting STEAP-1 protein expression are used in a variety of assay formats well known in the art for this purpose.
In a further embodiment, one can evaluate the status of STEAP-1 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules. These perturbations can include insertions, deletions, substitutions and the like. Such evaluations are useful because perturbations in the nucleotide and amino acid sequences are observed in a large number of proteins associated with a growth dysregulated phenotype (see, e.g., Marrogi et al., 1999, J. Cutan. Pathol. 26(8):369-378). For example, a mutation in the sequence of STEAP-1 may be indicative of the presence or promotion of a tumor. Such assays therefore have diagnostic and predictive value where a mutation in STEAP-1 indicates a potential loss of function or increase in tumor growth.
A wide variety of assays for observing perturbations in nucleotide and amino acid sequences are well known in the art. For example, the size and structure of nucleic acid or amino acid sequences of STEAP-1 gene products are observed by the Northern, Southern, Western, PCR and DNA sequencing protocols discussed herein. In addition, other methods for observing perturbations in nucleotide and amino acid sequences such as single strand conformation polymorphism analysis are well known in the art (see, e.g., U.S. Pat. No. 5,382,510 issued 7 Sep. 1999, and U.S. Pat No. 5,952,170 issued 17 Jan. 1995).
Additionally, one can examine the methylation status of a STEAP-1 gene in a biological sample. Aberrant demethylation and/or hypermethylation of CpG islands in gene 5′ regulatory regions frequently occurs in immortalized and transformed cells, and can result in altered expression of various genes. For example, promoter hypermethylation of the pi-class glutathione S-transferase (a protein expressed in normal prostate but not expressed in >90% of prostate carcinomas) appears to permanently silence transcription of this gene and is the most frequently detected genomic alteration in prostate carcinomas (De Marzo et al., Am. J. Pathol. 155(6): 1985-1992 (1999)). In addition, this alteration is present in at least 70% of cases of high-grade prostatic intraepithelial neoplasia (PIN) (Brooks et al., Cancer Epidemiol. Biomarkers Prev., 1998, 7:531-536). In another example, expression of the LAGE-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 to demethylation (Lethe et al., Int. J. Cancer 76(6): 903-908 (1998)). A variety of assays for examining methylation status of a gene are well known in the art. For example, one can utilize, in Southern hybridization approaches, methylation-sensitive restriction enzymes that cannot cleave sequences that contain methylated CpG sites to assess the methylation status of CpG islands. In addition, MSP (methylation specific PCR) can rapidly profile the methylation status of all the CpG sites present in a CpG island of a given gene. This procedure involves initial modification of DNA by sodium bisulfite (which will convert all unmethylated cytosines to uracil) followed by amplification using primers specific for methylated versus unmethylated DNA. Protocols involving methylation interference can also be found for example in Current Protocols In Molecular Biology, Unit 12, Frederick M. Ausubel et al. eds., 1995.
Gene amplification is an additional method for assessing the status of STEAP-1. Gene amplification is measured in a sample directly, for example, by conventional Southern blotting or Northern blotting to quantitate the in 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 STEAP-1 expression. The presence of RT-PCR amplifiable STEAP-1 mRNA provides an indication of the presence of cancer. RT-PCR assays are well known in the art. RT-PCR detection assays for tumor cells in peripheral blood are currently being evaluated for use in the diagnosis and management of a number of human solid tumors. In the prostate cancer field, these include RT-PCR assays for the detection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 13:1195-2000; Heston et al., 1995, Clin. Chem. 41:1687-1688).
A further aspect of the invention is an assessment of the susceptibility that an individual has for developing cancer. In one embodiment, a method for predicting susceptibility to cancer comprises detecting STEAP-1 mRNA or STEAP-1 protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of STEAP-1 mRNA expression correlates to the degree of susceptibility. In a specific embodiment, the presence of STEAP-1 in prostate or other tissue is examined, with the presence of STEAP-1 in the sample providing an indication of prostate cancer susceptibility (or the emergence or existence of a prostate tumor). Similarly, one can evaluate the integrity STEAP-1 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations in STEAP-1 gene products in the sample is an indication of cancer susceptibility (or the emergence or existence of a tumor).
The invention also comprises methods for gauging tumor aggressiveness. In one embodiment, a method for gauging aggressiveness of a tumor comprises determining the level of STEAP-1 mRNA or STEAP-1 protein expressed by tumor cells, comparing the level so determined to the level of STEAP-1 mRNA or STEAP-1 protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of STEAP-1 mRNA or STEAP-1 protein expression in the tumor sample relative to the normal sample indicates the degree of aggressiveness. In a specific embodiment, aggressiveness of a tumor is evaluated by determining the extent to which STEAP-1 is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors. Another embodiment is the evaluation of the integrity of STEAP-1 nucleotide and amino acid sequences in a biological sample, in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more perturbations indicates more aggressive tumors.
Another embodiment of the invention is directed to methods for observing the progression of a malignancy in an individual over time. In one embodiment, methods for observing the progression of a malignancy in an individual over time comprise determining the level of STEAP-1 mRNA or STEAP-1 protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of STEAP-1 mRNA or STEAP-1 protein expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of STEAP-1 mRNA or STEAP-1 protein expression in the tumor sample over time provides information on the progression of the cancer. In a specific embodiment, the progression of a cancer is evaluated by determining STEAP-1 expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer. Also, one can evaluate the integrity STEAP-1 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like, where the presence of one or more perturbations indicates a progression of the cancer.
The above diagnostic approaches can be combined with any one of a wide variety of prognostic and diagnostic protocols known in the art. For example, another embodiment of the invention is directed to methods for observing a coincidence between the expression of STEAP-1 gene and STEAP-1 gene products (or perturbations in STEAP-1 gene and STEAP-1 gene products) and a factor that is associated with malignancy, as a means for diagnosing and prognosticating the status of a tissue sample. A wide variety of factors associated with malignancy can be utilized, such as the expression of genes associated with malignancy (e.g. PSA, PSCA and PSM expression for prostate cancer etc.) as well as gross cytological observations (see, e.g., Bocking et al., 1984, Anal. Quant. Cytol. 6(2):74-88; Epstein, 1995, Hum. Pathol. 26(2):223-9; Thorson et al., 1998, Mod. Pathol. 11(6):543-51; Baisden et al., 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods for observing a coincidence between the expression of STEAP-1 gene and STEAP-1 gene products (or perturbations in STEAP-1 gene and STEAP-1 gene products) and another factor that is associated with malignancy are useful, for example, because the presence of a set of specific factors that coincide with disease provides information crucial for diagnosing and prognosticating the status of a tissue sample.
In one embodiment, methods for observing a coincidence between the expression of STEAP-1 gene and STEAP-1 gene products (or perturbations in STEAP-1 gene and STEAP-1 gene products) and another factor associated with malignancy entails detecting the overexpression of STEAP-1 mRNA or protein in a tissue sample, detecting the overexpression of PSA mRNA or protein in a tissue sample (or PSCA or PSM expression), and observing a coincidence of STEAP-1 mRNA or protein and PSA mRNA or protein overexpression (or PSCA or PSM expression). In a specific embodiment, the expression of STEAP-1 and PSA mRNA in prostate tissue is examined, where the coincidence of STEAP-1 and PSA mRNA overexpression in the sample indicates the existence of prostate cancer, prostate cancer susceptibility or the emergence or status of a prostate tumor.
Methods for detecting and quantifying the expression of STEAP-1 mRNA or protein are described herein, and standard nucleic acid and protein detection and quantification technologies are well known in the art. Standard methods for the detection and quantification of STEAP-1 mRNA include in situ hybridization using labeled STEAP-1 riboprobes, Northern blot and related techniques using STEAP-1 polynucleotide probes, RT-PCR analysis using primers specific for STEAP-1, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like. In a specific embodiment, semi-quantitative RT-PCR is used to detect and quantify STEAP-1 mRNA expression. Any number of primers capable of amplifying STEAP-1 can be used for this purpose, including but not limited to the various primer sets specifically described herein. In a specific embodiment, polyclonal or monoclonal antibodies specifically reactive with the wild-type STEAP-1 protein can be used in an immunohistochemical assay of biopsied tissue.
IX.) Identification of Molecules that Interact with STEAP-1
The STEAP-1 protein and nucleic acid sequences disclosed herein allow a skilled artisan to identify proteins, small molecules and other agents that interact with STEAP-1, as well as pathways activated by STEAP-1 via any one of a variety of art accepted protocols. For example, one can utilize one of the so-called interaction trap systems (also referred to as the “two-hybrid assay”). In such systems, molecules interact and reconstitute a transcription factor which directs expression of a reporter gene, whereupon the expression of the reporter gene is assayed. Other systems identify protein-protein interactions in vivo through reconstitution of a eukaryotic transcriptional activator, see, e.g., U.S. Pat. No. 5,955,280 issued 21 Sep. 1999, U.S. Pat. No. 5,925,523 issued 20 Jul. 1999, U.S. Pat. No. 5,846,722 issued 8 Dec. 1998 and U.S. Pat. No. 6,004,746 issued 21 Dec. 1999. Algorithms are also available in the art for genome-based predictions of protein function (see, e.g., Marcotte, et al., Nature 402: 4 Nov. 1999, 83-86).
Alternatively one can screen peptide libraries to identify molecules that interact with STEAP-1 protein sequences. In such methods, peptides that bind to STEAP-1 are identified by screening libraries that encode a random or controlled collection of amino acids. Peptides encoded by the libraries are expressed as fusion proteins of bacteriophage coat proteins, the bacteriophage particles are then screened against the STEAP-1 protein(s).
Accordingly, peptides having a wide variety of uses, such as therapeutic, prognostic or diagnostic reagents, are thus identified without any prior information on the structure of the expected ligand or receptor molecule. Typical peptide libraries and screening methods that can be used to identify molecules that interact with STEAP-1 protein sequences are disclosed for example in U.S. Pat. No. 5,723,286 issued 3 Mar. 1998 and U.S. Pat. No. 5,733,731 issued 31 Mar. 1998.
Alternatively, cell lines that express STEAP-1 are used to identify protein-protein interactions mediated by STEAP-1. Such interactions can be examined using immunoprecipitation techniques (see, e.g., Hamilton B. J., et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51). STEAP-1 protein can be immunoprecipitated from STEAP-1-expressing cell lines using anti-STEAP-1 antibodies. Alternatively, antibodies against His-tag can be used in a cell line engineered to express fusions of STEAP-1 and a His-tag (vectors mentioned above). The immunoprecipitated complex can be examined for protein association by procedures such as Western blotting, 35S-methionine labeling of proteins, protein microsequencing, silver staining and two-dimensional gel electrophoresis.
Small molecules and ligands that interact with STEAP-1 can be identified through related embodiments of such screening assays. For example, small molecules can be identified that interfere with protein function, including molecules that interfere with STEAP-1 's ability to mediate phosphorylation and de-phosphorylation, interaction with DNA or RNA molecules as an indication of regulation of cell cycles, second messenger signaling or tumorigenesis. Similarly, small molecules that modulate STEAP-1-related ion channel, protein pump, or cell communication functions are identified and used to treat patients that have a cancer that expresses STEAP-1 (see, e.g., Hille, B., Ionic Channels of Excitable Membranes 2nd Ed., Sinauer Assoc., Sunderland, Mass., 1992). Moreover, ligands that regulate STEAP-1 function can be identified based on their ability to bind STEAP-1 and activate a reporter construct. Typical methods are discussed for example in U.S. Pat. No. 5,928,868 issued 27 Jul. 1999, and include methods for forming hybrid ligands in which at least one ligand is a small molecule. In an illustrative embodiment, cells engineered to express a fusion protein of STEAP-1 and a DNA-binding protein are used to co-express a fusion protein of a hybrid ligand/small molecule and a cDNA library transcriptional activator protein. The cells further contain a reporter gene, the expression of which is conditioned on the proximity of the first and second fusion proteins to each other, an event that occurs only if the hybrid ligand binds to target sites on both hybrid proteins. Those cells that express the reporter gene are selected and the unknown small molecule or the unknown ligand is identified. This method provides a means of identifying modulators, which activate or inhibit STEAP-1.
An embodiment of this invention comprises a method of screening for a molecule that interacts with a STEAP-1 amino acid sequence shown in FIG. 2 or FIG. 3, comprising the steps of contacting a population of molecules with a STEAP-1 amino acid sequence, allowing the population of molecules and the STEAP-1 amino acid sequence to interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the STEAP-1 amino acid sequence, and then separating molecules that do not interact with the STEAP-1 amino acid sequence from molecules that do. In a specific embodiment, the method further comprises purifying, characterizing and identifying a molecule that interacts with the STEAP-1 amino acid sequence. The identified molecule can be used to modulate a function performed by STEAP-1. In a preferred embodiment, the STEAP-1 amino acid sequence is contacted with a library of peptides.
X.) Therapeutic Methods and Compositions
The identification of STEAP-1 as a protein that is normally expressed in a restricted set of tissues, but which is also expressed in prostate and other cancers, opens a number of therapeutic approaches to the treatment of such cancers. As contemplated herein, STEAP-1 functions as a transcription factor involved in activating tumor-promoting genes or repressing genes that block tumorigenesis.
Accordingly, therapeutic approaches that inhibit the activity of a STEAP-1 protein are useful for patients suffering from a cancer that expresses STEAP-1. These therapeutic approaches generally fall into two classes. One class comprises various methods for inhibiting the binding or association of a STEAP-1 protein with its binding partner or with other proteins. Another class comprises a variety of methods for inhibiting the transcription of a STEAP-1 gene or translation of STEAP-1 mRNA.
X.A.) Anti-Cancer Vaccines
The invention provides cancer vaccines comprising a STEAP-1-related protein or STEAP-1-related nucleic acid. In view of the expression of STEAP-1, cancer vaccines prevent and/or treat STEAP-1-expressing cancers with minimal or no effects on non-target tissues. The use of a tumor antigen in a vaccine that generates humoral and/or cell-mediated immune responses as anticancer 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 STEAP-1-related protein, or a STEAP-1-encoding nucleic acid molecule and recombinant vectors capable of expressing and presenting the STEAP-1 immunogen (which typically comprises a number of antibody or T cell epitopes). Skilled artisans understand that a wide variety of vaccine systems for delivery of immunoreactive epitopes are known in the art (see, e.g., Heryln et al., Ann Med 1999 February 31(1):66-78; Maruyama et al., Cancer Immunol Immunother 2000 June 49(3): 123-32) Briefly, such methods of generating an immune response (e.g. humoral and/or cell-mediated) in a mammal, comprise the steps of: exposing the mammal's immune system to an immunoreactive epitope (e.g. an epitope present in a STEAP-1 protein shown in FIG. 3 or analog or homolog thereof) so that the mammal generates an immune response that is specific for that epitope (e.g. generates antibodies that specifically recognize that epitope). In preferred method, a STEAP-1 immunogen contains a biological motif, see e.g., Tables V-XVIII and XXII-LI, or a peptide of a size range from STEAP-1 indicated in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and FIG. 9.
The entire STEAP-1 protein, immunogenic regions or epitopes thereof can be combined and delivered by various means. Such vaccine compositions can include, for example, lipopeptides (e.g., Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge, et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine 12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995), peptide compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin Exp Immunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs) (see e.g., Tarn, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tarn, 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 STEAP-1-associated cancer, the vaccine compositions of the invention can also be used in conjunction with other treatments used for cancer, e.g., surgery, chemotherapy, drug therapies, radiation therapies, etc. including use in combination with immune adjuvants such as IL-2, IL-12, GM-CSF, and the like.
Cellular Vaccines:
CTL epitopes can be determined using specific algorithms to identify peptides within STEAP-1 protein that bind corresponding HLA alleles (see e.g., Table IV; EpimerTM and EpimatrixTM, Brown University; BIMAS; and SYFPEITHI). In a preferred embodiment, a STEAP-1 immunogen contains one or more amino acid sequences identified using techniques well known in the art, such as the sequences shown in Tables V-XVIII and XXII-LI or a peptide of 8, 9, 10 or 11 amino acids specified by an HLA Class I motif/supermotif (e.g., Table IV (A), Table IV (D), or Table IV (E)) and/or a peptide of at least 9 amino acids that comprises an HLA Class II motif/supermotif (e.g., Table IV (B) or Table IV (C)). As is appreciated in the art, the HLA Class I binding groove is essentially closed ended so that peptides of only a particular size range can fit into the groove and be bound, generally HLA Class I epitopes are 8, 9, 10, or 11 amino acids long. In contrast, the HLA Class II binding groove is essentially open ended; therefore a peptide of about 9 or more amino acids can be bound by an HLA Class II molecule. Due to the binding groove differences between HLA Class I and II, HLA Class I motifs are length specific, i.e., position two of a Class I motif is the second amino acid in an amino to carboxyl direction of the peptide. The amino acid positions in a Class II motif are relative only to each other, not the overall peptide, i.e., additional amino acids can be attached to the amino and/or carboxyl termini of a motif-bearing sequence. HLA Class I 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 STEAP-1 protein) so that an immune response is generated. A typical embodiment consists of a method for generating an immune response to STEAP-1 in a host, by contacting the host with a sufficient amount of at least one STEAP-1 B cell or cytotoxic T-cell epitope or analog thereof; and at least one periodic interval thereafter re-contacting the host with the STEAP-1 B cell or cytotoxic T-cell epitope or analog thereof. A specific embodiment consists of a method of generating an immune response against a STEAP-1-related protein or a man-made multiepitopic peptide comprising: administering STEAP-1 immunogen (e.g. a STEAP-1 protein or a peptide fragment thereof, a STEAP-1 fusion protein or analog etc.) in a vaccine preparation to a human or another mammal. Typically, such vaccine preparations further contain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or a universal helper epitope such as a PADRET peptide (Epimmune Inc., San Diego, Calif.; see, e.g., Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., Immunity 1994 1(9): 751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92). An alternative method comprises generating an immune response in an individual against a STEAP-1 immunogen by: administering in vivo to muscle or skin of the individual's body a DNA molecule that comprises a DNA sequence that encodes a STEAP-1 immunogen, the DNA sequence operatively linked to regulatory sequences which control the expression of the DNA sequence; wherein the DNA molecule is taken up by cells, the DNA sequence is expressed in the cells and an immune response is generated against the immunogen (see, e.g., U.S. Pat. No. 5,962,428). Optionally a genetic vaccine facilitator such as anionic lipids; saponins; lectins; estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; and urea is also administered. In addition, an antiidiotypic antibody can be administered that mimics STEAP-1, in order to generate a response to the target antigen.
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 STEAP-1. Constructs comprising DNA encoding a STEAP-1-related protein/immunogen and appropriate regulatory sequences can be injected directly into muscle or skin of an individual, such that the cells of the muscle or skin take-up the construct and express the encoded STEAP-1 protein/immunogen. Alternatively, a vaccine comprises a STEAP-1-related protein. Expression of the STEAP-1-related protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells that bear a STEAP-1 protein. Various prophylactic and therapeutic genetic immunization techniques known in the art can be used (for review, see information and references published at Internet address genweb.com). Nucleic acid based delivery is described, for instance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720. Examples of DNA-base 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 STEAP-1 related protein into the patient (e.g., intramuscularly or intradermally) to induce an anti-tumor response.
Vaccinia virus is used, for example, as a vector to express nucleotide sequences that encode the peptides of the invention. Upon introduction into a host, the recombinant vaccinia virus expresses the protein immunogenic peptide, and thereby elicits a host immune response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al., Nature 351:456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g. adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled in the art from the description herein.
Thus, gene delivery systems are used to deliver a STEAP-1-related nucleic acid molecule. In one embodiment, the full-length human STEAP-1 cDNA is employed. In another embodiment, STEAP-1 nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopes are employed.
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 STEAP-1 antigen to a patient's immune system. Dendritic cells express MHC class I and II molecules, B7 co-stimulator, and IL-12, and are thus highly specialized antigen presenting cells. In prostate cancer, autologous dendritic cells pulsed with peptides of the prostate-specific membrane antigen (PSMA) are being used in a Phase I clinical trial to stimulate prostate cancer patients' immune systems (Tjoa et al., 1996, Prostate 28:65-69; Murphy et al., 1996, Prostate 29:371-380). Thus, dendritic cells can be used to present STEAP-1 peptides to T cells in the context of MHC class I or II molecules. In one embodiment, autologous dendritic cells are pulsed with STEAP-1 peptides capable of binding to MHC class I and/or class II molecules. In another embodiment, dendritic cells are pulsed with the complete STEAP-1 protein. Yet another embodiment involves engineering the overexpression of a STEAP-1 gene in dendritic cells using various implementing vectors known in the art, such as adenovirus (Arthur et al., 1997, Cancer Gene Ther. 4:17-25), retrovirus (Henderson et al., 1996, Cancer Res. 56:3763-3770), lentivirus, adeno-associated virus, DNA transfection (Ribas et al., 1997, Cancer Res. 57:2865-2869), or tumor-derived RNA transfection (Ashley et al., 1997, J. Exp. Med. 186:1177-1182). Cells that express STEAP-1 can also be engineered to express immune modulators, such as GM-CSF, and used as immunizing agents.
X.B.) STEAP-1 as a Target for Antibody-Based Therapy
STEAP-1 is an attractive target for antibody-based therapeutic strategies. A number of antibody strategies are known in the art for targeting both extracellular and intracellular molecules (see, e.g., complement and ADCC mediated killing as well as the use of intrabodies). Because STEAP-1 is expressed by cancer cells of various lineages relative to corresponding normal cells, systemic administration of STEAP-1-immunoreactive compositions are prepared that exhibit excellent sensitivity without toxic, non-specific and/or non-target effects caused by binding of the immunoreactive composition to non-target organs and tissues. Antibodies specifically reactive with domains of STEAP-1 are useful to treat STEAP-1-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.
STEAP-1 antibodies can be introduced into a patient such that the antibody binds to STEAP-1 and modulates a function, such as an interaction with a binding partner, and consequently mediates destruction of the tumor cells and/or inhibits the growth of the tumor cells. Mechanisms by which such antibodies exert a therapeutic effect can include complement-mediated cytolysis, antibody-dependent cellular cytotoxicity, modulation of the physiological function of STEAP-1, inhibition of ligand binding or signal transduction pathways, modulation of tumor cell differentiation, alteration of tumor angiogenesis factor profiles, and/or apoptosis.
Those skilled in the art understand that antibodies can be used to specifically target and bind immunogenic molecules such as an immunogenic region of a STEAP-1 sequence shown in FIG. 2 or FIG. 3. In addition, skilled artisans understand that it is routine to conjugate antibodies to cytotoxic agents (see, e.g., Slevers et al. Blood 93:11 3678-3684 (Jun. 1, 1999)). When cytotoxic and/or therapeutic agents are delivered directly to cells, such as by conjugating them to antibodies specific for a molecule expressed by that cell (e.g. STEAP-1), the cytotoxic agent will exert its known biological effect (i.e. cytotoxicity) on those cells.
A wide variety of compositions and methods for using antibody-cytotoxic agent conjugates to kill cells are known in the art. In the context of cancers, typical methods entail administering to an animal having a tumor a biologically effective amount of a conjugate comprising a selected cytotoxic and/or therapeutic agent linked to a targeting agent (e.g. an anti-STEAP-1 antibody) that binds to a marker (e.g. STEAP-1) expressed, accessible to binding or localized on the cell surfaces. A typical embodiment is a method of delivering a cytotoxic and/or therapeutic agent to a cell expressing STEAP-1, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a STEAP-1 epitope, and, exposing the cell to the antibody-agent conjugate. Another illustrative embodiment is a method of treating an individual suspected of suffering from metastasized cancer, comprising a step of administering parenterally to said individual a pharmaceutical composition comprising a therapeutically effective amount of an antibody conjugated to a cytotoxic and/or therapeutic agent.
Cancer immunotherapy using anti-STEAP-1 antibodies can be done in accordance with various approaches that have been successfully employed in the treatment of other types of cancer, including but not limited to colon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138), multiple myeloma (Ozali 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 (trastuzunab) with paclitaxel (Genentech, Inc.). The antibodies can be conjugated to a therapeutic agent. To treat prostate cancer, for example, STEAP-1 antibodies can be administered in conjunction with radiation, chemotherapy or hormone ablation. Also, antibodies can be conjugated to a toxin such as calicheamicin (e.g., 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 STEAP-1 antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well. Fan et al. (Cancer Res. 53:4637-4642, 1993), Prewett et al. (International J. of Onco. 9:217-224, 1996), and Hancock et al. (Cancer Res. 51:4575-4580, 1991) describe the use of various antibodies together with chemotherapeutic agents.
Although STEAP-1 antibody therapy is useful for all stages of cancer, antibody therapy can be particularly appropriate in advanced or metastatic cancers. Treatment with the antibody therapy of the invention is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy of the invention is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. Additionally, antibody therapy can enable the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate the toxicity of the chemotherapeutic agent very well.
Cancer patients can be evaluated for the presence and level of STEAP-1 expression, preferably using immunohistochemical assessments of tumor tissue, quantitative STEAP-1 imaging, or other techniques that reliably indicate the presence and degree of STEAP-1 expression. Immunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this purpose. Methods for immunohistochemical analysis of tumor tissues are well known in the art.
Anti-STEAP-1 monoclonal antibodies that treat prostate and other cancers include those that initiate a potent immune response against the tumor or those that are directly cytotoxic. In this regard, anti-STEAP-1 monoclonal antibodies (mAbs) can elicit tumor cell lysis by either complement-mediated or antibody-dependent cell cytotoxicity (ADCC) mechanisms, both of which require an intact Fc portion of the immunoglobulin molecule for interaction with effector cell Fc receptor sites on complement proteins. In addition, anti-STEAP-1 mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express STEAP-1. Mechanisms by which directly cytotoxic mAbs act include: inhibition of cell growth, modulation of cellular differentiation, modulation of tumor angiogenesis factor profiles, and the induction of apoptosis. The mechanism(s) by which a particular anti-STEAP-1 mAb exerts an anti-tumor effect is evaluated using any number of in vitro assays that evaluate cell death such as ADCC, ADMMC, complement-mediated cell lysis, and so forth, as is generally known in the art.
In some patients, the use of murine or other non-human monoclonal antibodies, or human/mouse chimeric mAbs can induce moderate to strong immune responses against the non-human antibody. This can result in clearance of the antibody from circulation and reduced efficacy. In the most severe cases, such an immune response can lead to the extensive formation of immune complexes which, potentially, can cause renal failure. Accordingly, preferred monoclonal antibodies used in the therapeutic methods of the invention are those that are either fully human or humanized and that bind specifically to the target STEAP-1 antigen with high affinity but exhibit low or no antigenicity in the patient.
Therapeutic methods of the invention contemplate the administration of single anti-STEAP-1 mAbs as well as combinations, or cocktails, of different mAbs. Such mAb cocktails can have certain advantages inasmuch as they contain mAbs that target different epitopes, exploit different effector mechanisms or combine directly cytotoxic mAbs with mAbs that rely on immune effector functionality. Such mAbs in combination can exhibit synergistic therapeutic effects. In addition, anti-STEAP-1 mAbs can be administered concomitantly with other therapeutic modalities, including but not limited to various chemotherapeutic agents, androgen-blockers, immune modulators (e.g., IL-2, GM-CSF), surgery or radiation. The anti-STEAP-1 mAbs are administered in their “naked” or unconjugated form, or can have a therapeutic agent(s) conjugated to them.
Anti-STEAP-1 antibody formulations are administered via any route capable of delivering the antibodies to a tumor cell. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumor, intradermal, and the like. Treatment generally involves repeated administration of the anti-STEAP-1 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg/kg body weight. In general, doses in the range of 10-1000 mg mAb per week are effective and well tolerated.
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-STEAP-1 mAb preparation represents an acceptable dosing regimen. Preferably, the initial loading dose is administered as a 90-minute or longer infusion. The periodic maintenance dose is administered as a 30 minute or longer infusion, provided the initial dose was well tolerated. As appreciated by those of skill in the art, various factors can influence the ideal dose regimen in a particular case. Such factors include, for example, the binding affinity and half life of the Ab or mAbs used, the degree of STEAP-1 expression in the patient, the extent of circulating shed STEAP-1 antigen, the desired steady-state antibody concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient.
Optionally, patients should be evaluated for the levels of STEAP-1 in a given sample (e.g. the levels of circulating STEAP-1 antigen and/or STEAP-1 expressing cells) in order to assist in the determination of the most effective dosing regimen, etc. Such evaluations are also used for monitoring purposes throughout therapy, and are useful to gauge therapeutic success in combination with the evaluation of other parameters (for example, urine cytology and/or ImmunoCyt levels in bladder cancer therapy, or by analogy, serum PSA levels in prostate cancer therapy).
Anti-idiotypic anti-STEAP-1 antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a STEAP-1-related protein. In particular, the generation of anti-idiotypic antibodies is well known in the art; this methodology can readily be adapted to generate anti-idiotypic anti-STEAP-1 antibodies that mimic an epitope on a STEAP-1-related protein (see, for example, Wagner et al., 1997, Hybridoma 16: 33-40; Foon et al., 1995, J. Clin. Invest. 96:334-342; Herlyn et al., 1996, Cancer Immunol. Immunother. 43:65-76). Such an anti-idiotypic antibody can be used in cancer vaccine strategies.
X.C.) STEAP-1 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 STEAP-1 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 PADRE® (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 collated with tumor clearance. For HLA Class I this includes 3-4 epitopes that come from at least one tumor associated antigen (TAA). For HLA Class II a similar rationale is employed; again 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 STEAP-1, the PADRE® universal helper T cell epitope or multiple HTL epitopes from STEAP-1 (see e.g., Tables V-XVIII and XXII to LI), and an endoplasmic reticulum-translocating signal sequence can be engineered. A vaccine may also comprise epitopes that are derived from other TAAs.
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 n pathway, thereby improving HTL induction. In contrast to HTL or CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-β) may be beneficial in certain diseases.
Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in E. coli, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and grown to saturation in shaker flasks or a bioreactor according to well-known techniques. Plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion-exchange resins supplied by QIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.
Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). This approach, known as “naked DNA,” is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of minigene DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. A variety of methods have been described, and new techniques may become available. Cationic lipids, glycolipids, and fusogenic liposomes can also be used in the formulation (see, e.g., as described by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987). In addition, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds (PINC) could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.
Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes. For example, the plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays. The transfection method used will be dependent on the final formulation. Electroporation can be used for “naked” DNA, whereas cationic lipids allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS). These cells are then chromium-51 (51Cr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by 51Cr release, indicates both production of, and HLA presentation of, minigene-encoded CTT epitopes. Expression of HTL epitopes may be evaluated in an analogous manner using assays to assess HTL activity.
In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations. Transgenic mice expressing appropriate human HLA proteins are immunized with the DNA product. The dose and route of administration are formulation dependent (e.g., IM for DNA in PBS, intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-one days after immunization, splenocytes are harvested and restimulated for one week in the presence of peptides encoding each epitope being tested. Thereafter, for CTL effector cells, assays are conducted for cytolysis of peptide-loaded, 51Cr-labeled target cells using standard techniques. Lysis of target cells that were sensitized by HLA loaded with peptide epitopes, corresponding to minigene-encoded epitopes, demonstrates DNA vaccine function for in vivo induction of CTLs. Immunogenicity of HTL epitopes is confirmed in transgenic mice in an analogous manner.
Alternatively, the nucleic acids can be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253. Using this technique, particles comprised solely of DNA are administered. In a further alternative embodiment, DNA can be adhered to particles, such as gold particles.
Minigenes can also be delivered using other bacterial or viral delivery systems well known in the art, e.g., an expression construct encoding epitopes of the invention can be incorporated into a viral vector such as vaccinia.
X.C.2. Combinations of CTL Peptides with Helper Peptides
Vaccine compositions comprising CTL peptides of the invention can be modified, e.g., analoged, to provide desired attributes, such as improved serum half life, broadened population coverage or enhanced immunogenicity.
For instance, the ability of a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence which contains at least one epitope that is capable of inducing a T helper cell response. Although a CTL peptide can be directly linked to a T helper peptide, often CTL epitope/HTL epitope conjugates are linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues and sometimes 10 or more residues. The CTL peptide epitope can be linked to the T helper peptide epitope either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide may be acylated.
In certain embodiments, the T helper peptide is one that is recognized by T helper cells present in a majority of a genetically diverse population. This can be accomplished by selecting peptides that bind to many, most, or all of the HLA class II molecules. Examples of such amino acid bind many HLA Class II molecules include sequences from antigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO: 44), Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 45), and Streptococcus 18 kD protein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO: 46). Other examples include peptides bearing a DR 1-4-7 supermotif, or either of the DR3 motifs.
Alternatively, it is possible to prepare synthetic peptides capable of stimulating T helper lymphocytes, in a loosely HLA-restricted fashion, using amino acid sequences not found in nature (see, e.g., PCT publication WO 95/07707). These synthetic compounds called Pan-DR-binding epitopes (e.g., PADRESTM, 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: aKXVAAMMLKAAa (SEQ ID NO: 47), where “X” is either-cyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanine or L-alanine, has been found to bind to most HLA-DR alleles, and to stimulate the response of T helper lymphocytes from most individuals, regardless of their HLA type. An alternative of a pan-DR binding epitope comprises all “L” natural amino acids and can be provided in the form of nucleic acids that encode the epitope.
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 STEAP-1. Optionally, a helper T cell (HTL) peptide, such as a natural or artificial loosely restricted HLA Class II peptide, can be included to facilitate the CTL response. Thus, a vaccine in accordance with the invention is used to treat a cancer which expresses or overexpresses STEAP-1.
X.D. Adoptive Immunotherapy
Antigenic STEAP-1-related peptides are used to elicit a CTL and/or HTL response ex vivo, as well. The resulting CTL or HTL cells, can be used to treat tumors in patients that do not respond to other conventional forms of therapy, or will not respond to a therapeutic vaccine peptide or nucleic acid in accordance with the invention. Ex vivo CTL or HTL responses to a particular antigen are induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of antigen-presenting cells (APC), such as dendritic cells, and the appropriate immunogenic peptide. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cell (e.g., a tumor cell). Transfected dendritic cells may also be used as antigen presenting cells.
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 STEAP-1. In therapeutic applications, peptide and/or nucleic acid compositions are administered to a patient in an amount sufficient to elicit an effective B cell, CTL and/or HTL response to the antigen and to cure or at least partially arrest or slow symptoms and/or complications. An amount adequate to accomplish this is defined as “therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the particular composition administered, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.
For pharmaceutical compositions, the immunogenic peptides of the invention, or DNA encoding them, are generally administered to an individual already bearing a tumor that expresses STEAP-1. The peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences. Patients can be treated with the immunogenic peptides separately or in conjunction with other treatments, such as surgery, as appropriate.
For therapeutic use, administration should generally begin at the first diagnosis of STEAP-1-associated cancer. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. The embodiment of the vaccine composition (i.e., including, but not limited to embodiments such as peptide cocktails, polyepitopic polypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells) delivered to the patient may vary according to the stage of the disease or the patient's health status. For example, in a patient with a tumor that expresses STEAP-1, a vaccine comprising STEAP-1-specific CTL may be more efficacious in killing tumor cells in patient with advanced disease than alternative embodiments.
It is generally important to provide an amount of the peptide epitope delivered by a mode of administration sufficient to stimulate effectively a cytotoxic T cell response; compositions which stimulate helper T cell responses can also be given in accordance with this embodiment of the invention.
The dosage for an initial therapeutic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1,000 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0 μg to about 50,000 μg of peptide pursuant to a boosting regimen over weeks to months may be administered depending upon the patient's response and condition as determined by measuring the specific activity of CTL and HTL obtained from the patient's blood. Administration should continue until at least clinical symptoms or laboratory tests indicate that the neoplasia, has been eliminated or reduced and for a period thereafter. The dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.
In certain embodiments, the peptides and compositions of the present invention are employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, as a result of the minimal amounts of extraneous substances and the relative nontoxic nature of the peptides in preferred compositions of the invention, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions relative to these stated dosage amounts.
The vaccine compositions of the invention can also be used purely as prophylactic agents. Generally the dosage for an initial prophylactic immunization generally occurs in a unit dosage range where the lower value is about 1, 5, 50, 500, or 1000 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. This is followed by boosting dosages of between about 1.0 μg to about 50,000 μg of peptide administered at defined intervals from about four weeks to six months after the initial administration of vaccine. The immunogenicity of the vaccine can be assessed by measuring the specific activity of CTL and HTL obtained from a sample of the patient's blood.
The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral, nasal, intrathecal, or local (e.g. as a cream or topical ointment) administration. Preferably, the pharmaceutical compositions are administered parentally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
A variety of aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
The concentration of peptides of the invention in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
A human unit dose form of a composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable carrier, in one embodiment an aqueous carrier, and is administered in a volume/quantity that is known by those of skill in the art to be used for administration of such compositions to humans (see, e.g., Remington's Pharmaceutical Sciences, 17th Edition, A. Gennaro, Editor, Mack Publishing Co., Easton, Pa., 1985). For example a peptide dose for initial immunization can be from about 1 to about 50,000 μg, generally 100-5,000 μg, for a 70 kg patient. For example, for nucleic acids an initial immunization may be performed using an expression vector in the form of naked nucleic acid administered IM (or SC or ID) in the amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 μg) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-107 to 5×109 pfu.
For antibodies, a treatment generally involves repeated administration of the anti-STEAP-1 antibody preparation, via an acceptable route of administration such as intravenous injection (IV), typically at a dose in the range of about 0.1 to about 10 mg/kg body weight. In general, doses in the range of 10-500 mg mAb per week are effective and well tolerated. Moreover, an initial loading dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of about 2 mg/kg IV of the anti-STEAP-1 mAb preparation represents an acceptable dosing regimen. As appreciated by those of skill in the art, various factors can influence the ideal dose in a particular case. Such factors include, for example, half life of a composition, the binding affinity of an Ab, the immunogenicity of a substance, the degree of STEAP-1 expression in the patient, the extent of circulating shed STEAP-1 antigen, the desired steady-state concentration level, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the treatment method of the invention, as well as the health status of a particular patient. Non-limiting preferred human unit doses are, for example, 500 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; 225400 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 stability 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 STEAP-1.
As disclosed herein, STEAP-1 polynucleotides, polypeptides, reactive cytotoxic T cells (CTL), reactive helper T cells (HTL) and anti-polypeptide antibodies are used in well known diagnostic, prognostic and therapeutic assays that examine conditions associated with dysregulated cell growth such as cancer, in particular the cancers listed in Table I (see, e.g., both its specific pattern of tissue expression as well as its overexpression in certain cancers as described for example in the Example entitled “Expression analysis of STEAP-1 in normal tissues, and patient specimens”).
STEAP-1 can be analogized to a prostate associated antigen PSA, the archetypal marker that has been used by medical practitioners for years to identify and monitor the presence of prostate cancer (see, e.g., Merrill et al., J. Urol. 163(2): 503-5120 (2000); Polascik et al., J. Urol. August; 162(2):293-306 (1999) and Fortier et al., J. Nat. Cancer Inst. 91(19): 1635-1640 (1999)). A variety of other diagnostic markers are also used in similar contexts including p53 and K-ras (see, e.g., Tulchinsky et al., Int J Mol Med 1999 July 4(1):99-102 and Minimoto et al., Cancer Detect Prev 2000; 24(1):1-12). Therefore, this disclosure of STEAP-1 polynucleotides and polypeptides (as well as STEAP-1 polynucleotide probes and anti-STEAP-1 antibodies used to identify the presence of these molecules) and their properties allows skilled artisans to utilize these molecules in methods that are analogous to those used, for example, in a variety of diagnostic assays directed to examining conditions associated with cancer.
Typical embodiments of diagnostic methods which utilize the STEAP-1 polynucleotides, polypeptides, reactive T cells and antibodies are analogous to those methods from well-established diagnostic assays, which employ, e.g., PSA polynucleotides, polypeptides, reactive T cells and antibodies. For example, just as PSA polynucleotides are used as probes (for example in Northern analysis, see, e.g., Sharief et al., Biochem. Mol. Biol. Int. 33(3):567-74 (1994)) and primers (for example in PCR analysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189-1190 (2000)) to observe the presence and/or the level of PSA mRNAs in methods of monitoring PSA overexpression or the metastasis of prostate cancers, the STEAP-1 polynucleotides described herein can be utilized in the same way to detect STEAP-1 overexpression or the metastasis of prostate and other cancers expressing this gene. Alternatively, just as PSA polypeptides are used to generate antibodies specific for PSA which can then be used to observe the presence and/or the level of PSA proteins in methods to monitor PSA protein overexpression (see, e.g., Stephan et al., Urology 55(4):560-3 (2000)) or the metastasis of prostate cells (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3):233-7 (1996)), the STEAP-1 polypeptides described herein can be utilized to generate antibodies for use in detecting STEAP-1 overexpression or the metastasis of prostate cells and cells of other cancers expressing this gene.
Specifically, because metastases involves the movement of cancer cells from an organ of origin (such as the lung or prostate gland etc.) to a different area of the body (such as a lymph node), assays which examine a biological sample for the presence of cells expressing STEAP-1 polynucleotides and/or polypeptides can be used to provide evidence of metastasis. For example, when a biological sample from tissue that does not normally contain STEAP-1-expressing cells (lymph node) is found to contain STEAP-1-expressing cells such as the STEAP-1 expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis.
Alternatively STEAP-1 polynucleotides and/or polypeptides can be used to provide evidence of cancer, for example, when cells in a biological sample that do not normally express STEAP-1 or express STEAP-1 at a different level are found to express STEAP-1 or have an increased expression of STEAP-1 (see, e.g., the STEAP-1 expression in the cancers listed in Table I and in patient samples etc. shown in the accompanying Figures). In such assays, artisans may further wish to generate supplementary evidence of metastasis by testing the biological sample for the presence of a second tissue restricted marker (in addition to STEAP-1) such as PSA, PSCA etc. (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)).
Just as PSA polynucleotide fragments and polynucleotide variants are employed by skilled artisans for use in methods of monitoring PSA, STEAP-1 polynucleotide fragments and polynucleotide variants are used in an analogous manner. In particular, typical PSA polynucleotides used in methods of monitoring PSA are probes or primers which consist of fragments of the PSA cDNA sequence. Illustrating this, primers used to PCR amplify a PSA polynucleotide must include less than the whole PSA sequence to function in the polymerase chain reaction. In the context of such PCR reactions, skilled artisans generally create a variety of different polynucleotide fragments that can be used as primers in order to amplify different portions of a polynucleotide of interest or to optimize amplification reactions (see, e.g., Caetano-Anolles, G. Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., Methods Mol. Biol. 98:121-154 (1998)). An additional illustration of the use of such fragments is provided in the Example entitled “Expression analysis of STEAP-1 in normal tissues, and patient specimens,” where a STEAP-1 polynucleotide fragment is used as a probe to show the expression of STEAP-1 RNAs in cancer cells. In addition, variant polynucleotide sequences are typically used as primers and probes for the corresponding mRNAs in PCR and Northern analyses (see, e.g., Sawai et al, Fetal Diagn. Ther. 1996 November-December 11 (6):407-13 and Current Protocols In Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubel et al. eds., 1995)). Polynucleotide fragments and variants are useful in this context where they are capable of binding to a target polynucleotide sequence (e.g., a STEAP-1 polynucleotide shown in FIG. 2 or variant thereof) under conditions of high stringency.
Furthermore, PSA polypeptides which contain an epitope that can be recognized by an antibody or T cell that specifically binds to that epitope are used in methods of monitoring PSA. STEAP-1 polypeptide fragments and polypeptide analogs or variants can also be used in an analogous manner. This practice of using polypeptide-fragments or polypeptide variants to generate antibodies (such as anti-PSA antibodies or T cells) is typical in the art with a wide variety of systems such as fusion proteins being used by practitioners (see, e.g., Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubel et al. eds., 1995). In this context, each epitope(s) functions to provide the architecture with which an antibody or T cell is reactive. Typically, skilled artisans create a variety of different polypeptide fragments that can be used in order to generate immune responses specific for different portions of a polypeptide of interest (see, e.g., U.S. Pat. Nos. 5,840,501 and 5,939,533). For example it may be preferable to utilize a polypeptide comprising one of the STEAP-1 biological motifs discussed herein or a motif-bearing subsequence which is readily identified by one of skill in the art based on motifs available in the art. Polypeptide fragments, variants or analogs are typically useful in this context as long as they comprise an epitope capable of generating an antibody or T cell specific for a target polypeptide sequence (e.g. a STEAP-1 polypeptide shown in FIG. 3).
As shown herein, the STEAP-1 polynucleotides and polypeptides (as well as the STEAP-1 polynucleotide probes and anti-STEAP-1 antibodies or T cells used to identify the presence of these molecules) exhibit specific properties that make them useful in diagnosing cancers such as those listed in Table I. Diagnostic assays that measure the presence of STEAP-1 gene products, in order to evaluate the presence or onset of a disease condition described herein, such as prostate cancer, are used to identify patients for preventive measures or further monitoring, as has been done so successfully with PSA. Moreover, these materials satisfy a need in the art for molecules having similar or complementary characteristics to PSA in situations where, for example, a definite diagnosis of metastasis of prostatic origin cannot be made on the basis of a test for PSA alone (see, e.g., Alanen et al., Pathol. Res. Pract. 192(3): 233-237 (1996)), and consequently, materials such as STEAP-1 polynucleotides and polypeptides (as well as the STEAP-1 polynucleotide probes and anti-STEAP-1 antibodies used to identify the presence of these molecules) need to be employed to confirm a metastases of prostatic origin.
Finally, in addition to their use in diagnostic assays, the STEAP-1 polynucleotides disclosed herein have a number of other utilities such as their use in the identification of oncogenetic associated chromosomal abnormalities in the chromosomal region to which the STEAP-1 gene maps (see the Example entitled “Chromosomal Mapping of STEAP-1” below). Moreover, in addition to their use in diagnostic assays, the STEAP-1-related proteins and polynucleotides disclosed herein have other utilities such as their use in the forensic analysis of tissues of unknown origin (see, e.g., Takahama K Forensic Sci Int 1996 Jun. 28; 80(1-2): 63-9).
Additionally, STEAP-1-related proteins or polynucleotides of the invention can be used to treat a pathologic condition characterized by the over-expression of STEAP-1. For example, the amino acid or nucleic acid sequence of FIG. 2 or FIG. 3, or fragments of either, can be used to generate an immune response to a STEAP-1 antigen. Antibodies or other molecules that react with STEAP-1 can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.
XII.) Inhibition of STEAP-1 Protein Function
The invention includes various methods and compositions for inhibiting the binding of STEAP-1 to its binding partner or its association with other protein(s) as well as methods for inhibiting STEAP-1 function.
XII.A.) Inhibition of STEAP-1 With Intracellular Antibodies
In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to STEAP-1 are introduced into STEAP-1 expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti-STEAP-1 antibody is expressed intracellularly, binds to STEAP-1 protein, and thereby inhibits its function. Methods for engineering such intracellular single chain antibodies are well known. Such intracellular antibodies, also known as “intrabodies”, are specifically targeted to a particular compartment within the cell, providing control-over where the inhibitory activity of the treatment is focused. This technology has been successfully applied in the art (for review, see Richardson and Marasco, 1995, TIBTECH vol. 13). Intrabodies have been shown to virtually eliminate the expression of otherwise abundant cell surface receptors (see, e.g., Richardson et al.; 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol. Chem. 289: 23931-23936; Deshane et al., 1994, Gene Ther. 1: 332-337).
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 STEAP-1 in the nucleus, thereby preventing its activity within the nucleus. Nuclear targeting signals are engineered into such STEAP-1 intrabodies in order to achieve the desired targeting. Such STEAP-1 intrabodies are designed to bind specifically to a particular STEAP-1 domain. In another embodiment, cytosolic intrabodies that specifically bind to a STEAP-1 protein are used to prevent STEAP-1 from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus (e.g., preventing STEAP-1 from forming transcription complexes with other factors).
In order to specifically direct the expression of such intrabodies to particular cells, the transcription of the intrabody is placed under the regulatory control of an appropriate tumor-specific promoter and/or enhancer. In order to target intrabody expression specifically to prostate, for example, the PSA promoter and/or promoter/enhancer can be utilized (See, for example, U.S. Pat. No. 5,919,652 issued 6 Jul. 1999).
XII.B.) Inhibition of STEAP-1 with Recombinant Proteins
In another approach, recombinant molecules bind to STEAP-1 and thereby inhibit STEAP-1 function. For example, these recombinant molecules prevent or inhibit STEAP-1 from accessing/binding to its binding partner(s) or associating with other protein(s). Such recombinant molecules can, for example, contain the reactive part(s) of a STEAP-1 specific antibody molecule. In a particular embodiment, the STEAP-1 binding domain of a STEAP-1 binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two STEAP-1 ligand binding domains linked to the Fc portion of a human IgG, such as human IgG1. Such IgG portion can contain, for example, the 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 STEAP-1, whereby the dimeric fusion protein specifically binds to STEAP-1 and blocks STEAP-1 interaction with a binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking technologies.
XII.C.) Inhibition of STEAP-1 Transcription or Translation
The present invention also comprises various methods and compositions for inhibiting the transcription of the STEAP-1 gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of STEAP-1 mRNA into protein.
In one approach, a method of inhibiting the transcription of the STEAP-11 gene comprises contacting the STEAP-1 gene with a STEAP-1 antisense polynucleotide. In another approach, a method of inhibiting STEAP-1 mRNA translation comprises contacting a STEAP-1 mRNA with an antisense polynucleotide. In another approach, a STEAP-1 specific ribozyme is used to cleave a STEAP-1 message, thereby inhibiting translation. Such antisense and ribozyme based methods can also be directed to the regulatory regions of the STEAP-1 gene, such as STEAP-1 promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a STEAP-1 gene transcription factor are used to inhibit STEAP-1 mRNA transcription. The various polynucleotides and compositions useful in the aforementioned methods have been described above. The use of antisense and ribozyme molecules to inhibit transcription and translation is well known in the art.
Other factors that inhibit the transcription of STEAP-1 by interfering with STEAP-1 transcriptional activation are also useful to treat cancers expressing STEAP-1. Similarly, factors that interfere with STEAP-1 processing are useful to treat cancers that express STEAP-1. Cancer treatment methods utilizing such factors are also within the scope of the invention.
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 STEAP-1 (i.e., antisense, ribozyme, polynucleotides encoding intrabodies and other STEAP-1 inhibitory molecules). A number of gene therapy approaches are known in the art. Recombinant vectors encoding STEAP-1 antisense polynucleotides, ribozymes, factors capable of interfering with STEAP-1 transcription, and so forth, can be delivered to target tumor cells using such gene therapy approaches.
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 STEAP-1 to a binding partner, etc.
In vivo the effect of a STEAP-1 therapeutic composition can be evaluated in a suitable animal model. For example, xenogeneic prostate cancer models can be used, wherein human prostate cancer explants or passaged xenograft tissue 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 apoptic 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.
XII.) 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 vitro use, such as those described herein. Directions and or other information can also be included on an insert(s) or label(s) which is included with or on the kit.
The 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 STEAP-1 and modulating the function of STEAP-1.
The label can be on or associated with the container. A label a can be on a container when letters, numbers or other characters forming the label are molded or etched into the container itself; a label can be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. The label can indicate that the composition is used for diagnosing, treating, prophylaxing or prognosing a condition, such as a neoplasia of a tissue set forth in Table I. The article of manufacture can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution and/ordextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, stirrers, needles, syringes, and/or package inserts with indications and/or instructions for use.
EXAMPLES
Various aspects of the invention are further described and illustrated by way of the several examples that follow, none of which are intended to limit the scope of the invention.
Example 1
SSH-Generated Isolation of cDNA Fragment of the STEAP Gene
Materials and Methods
LAPC Xenografts:
LAPG xenografts were obtained from Dr. Charles Sawyers (UCLA) and generated as described (Klein et al, 1997, Nature Med. 3: 402-408; Craft et al., 1999, Cancer Res. 59: 5030-5036). Androgen dependent and independent LAPC-4 xenografts (LAPC4 AD and AI, respectively) and LAPC-9 xenografts (LAPC-9 AD and AI, respectively) were grown in intact male SCID mice or in castrated males, respectively, and were passaged as small tissue chunks in recipient males. LAPC4 AI xenografts were derived from LAPC4 AD tumors and LAPC-9 AI-xenografts were derived from LAPC-9 AD tumors. To generate the AI xenografts, male mice bearing LAPC AD-tumors were castrated and maintained for 2-3 months. After the LAPC tumors re-grew, the tumors were harvested and passaged in castrated males or in female SCID mice.
LAPC-4AD xenografts were grown intratibially as follows. LAPC-4 AD xenograft tumor tissue grown subcutaneously was minced into 1-2 mm3 sections while the tissue was bathed in 1× Iscoves medium, minced tissue was then centrifuged at 1.3K rpm for 4 minutes, the supernatant was resuspended in 10 ml ice cold 1× Iscoves medium and centrifuged at 1.3K rpm for 4 minutes. The pellet was then resuspended in 1× Iscoves with 1% pronase E and incubated for 20 minutes at room temperature with mild rocking agitation followed by incubation on ice for 2-4 minutes. Filtrate was centrifuged at 1.3K rpm for 4 minutes, and the pronase was removed from the aspirated pellet by resuspending in 10 ml Iscoves and re-centrifuging. Clumps of cells were then plated in PrEGM medium and grown overnight. The cells were then harvested, filtered, washed 2× RPMI, and counted. Approximately 50,000 cells were mixed with and equal volume of ice-cold Matrigel on ice, and surgically injected into the proximal tibial metaphyses of SCID mice via a 27 gauge needle. After 10-12 weeks, LAPC4 tumors growing in bone marrow were recovered.
Cell Lines and Tissues:
Human cell lines (e.g., HeLa) were obtained from the ATCC and were maintained in DMEM with 5% fetal calf serum. Human tissues for RNA and protein analyses were obtained from the Human Tissue Resource Center (HTRC) at the UCLA (Los Angeles, Calif.) and from QualTek, Inc. (Santa Barbara, Calif.).
RNA Isolation:
Tumor tissue and cell lines were homogenized in Trizol reagent (Life Technologies, Gibco BRL) using 10 ml/g tissue or 10 mV 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):









(SEQ ID NO: 48)









5′TTTTGATCAAGCTT303′



 


Adaptor 1:








(SEQ ID NO: 49)









5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′









(SEQ ID NO: 50)









3′GGCCCGTCCTAG5′



 


Adaptor 2:







(SEQ ID NO: 51)








5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′









(SEQ ID NO: 52)









3′CGGCTCCTAG5′



 


PCR primer 1:








(SEQ ID NO: 53)









5′CTAATACGACTCACTATAGGGC3′



 


Nested primer (NP)1:








(SEQ ID NO: 54)









5′TCGAGCGGCCGCCCGGGCAGGA3′



 


Nested primer (NP)2:








(SEQ ID NO: 55)









5′AGCGTGGTCGCGGCCGAGGA3′







Suppression Subtractive Hybridization:

Suppression Subtractive Hybridization (SSH) was used to identify cDNAs corresponding to genes, which may be up-regulated in androgen dependent prostate cancer compared to benign prostatic hyperplasia (BPH).
Double stranded cDNAs corresponding to the LAPC-4 AD xenograft (tester) and the BPH tissue (driver) were synthesized from 2 μg of poly(A)+ RNA isolated from xenograft and BPH tissue, as described above, using CLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide RSACDN as primer. First- and second-strand synthesis were carried out as described in the Kit's user manual protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1). The resulting cDNA was digested with Rsa I for 3 hrs. at 37° C. Digested cDNA was extracted with phenol/chloroform (1:1) and ethanol precipitated.
Driver cDNA (BPH) was generated by combining in a 4 to 1 ratio Rsa I digested BPH cDNA with digested cDNA from mouse liver, in order to ensure that murine genes were subtracted from the tester cDNA (LAPC-4 AD).
Tester cDNA (LAPC-4 AD) was generated by diluting 1 μl of Rsa I digested LAPC4 AD cDNA (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 overlayed with mineral oil, denatured in an MJ Research thermal cycler at 98° C. for 1.5 minutes, and then were allowed to hybridize for 8 hrs at 68° C. The two hybridizations were then mixed together with an additional 1 μl of fresh denatured driver cDNA and were allowed to hybridize overnight at 68° C. The second hybridization was then diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA, heated at 70° C. for 7 min. and stored at −20° C.
PCR Amplification, Cloning and Sequencing of Gene Fragments Generated from SSH:
To amplify gene fragments resulting from SSH reactions, two PCR amplifications were performed. In the primary PCR reaction 1 μl of the diluted final hybridization mix was added to 1 μl of PCR primer 1 (10 μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and 0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5 min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C. for 30 sec, 72° C. for 1.5 min. Five separate-primary PCR reactions were performed for each experiment. The products were pooled and diluted 1:10 with water. For the secondary PCR reaction, 11 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, 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 were generated from 1 μg of mRNA with oligo (dT) 12-18 priming using the Gibco-BRL Superscript-Preamplification system-L The manufacturer's protocol was used and 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 was increased to 200 μl with water prior to normalization. First strand cDNAs from 16 different normal human tissues were obtained from Clontech.
Normalization of the first strand cDNAs from multiple tissues was performed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID NO: 56) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 57) to amplify β-actin. First strand cDNA (5 μl) was 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 was removed at 18, 20, and 22 cycles and used for agarose gel electrophoresis. PCR was performed using an MJ Research thermal cycler under the following conditions: initial denaturation was at 94° 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 were required to achieve equal band intensities in all tissues after 22 cycles of PCR.
To determine expression levels of the 8P1D4 gene, 5 μl of normalized first strand cDNA was analyzed by PCR using 25, 30, and 35 cycles of amplification using the following primer pairs:








(SEQ ID NO: 58)











5′ ACT TTG TTG ATG ACC AGG ATT GGA 3′




 








(SEQ ID NO: 59)











5′ CAG AAC TTC AGC ACA CAC AGG AAC 3′







Semi quantitative expression analysis was achieved by comparing the PCR products at cycle numbers that give light band intensities.

Results

Several SSH experiments were conduced as described in the Materials and Methods, supra, and led to the isolation of numerous candidate gene fragment clones. All candidate clones were sequenced and subjected to homology analysis against all sequences in the major public gene and EST databases in order to provide information on the identity of the corresponding gene and to help guide the decision to analyze a particular gene for differential expression. In general, gene fragments which had no homology to any known sequence in any of the searched databases, and thus considered to represent novel genes, as well as gene fragments showing homology to previously sequenced expressed sequence tags (ESTs), were subjected to differential expression analysis by RT-PCR and/or Northern analysis.
One of the cDNA clones, designated 8P1D4, was 436 bp in length and showed homology to an EST sequence in the NCI-CGAP tumor gene database. The full length cDNA encoding the 8P1D4 gene was subsequently isolated using this cDNA and re-named STEAP-1. The 8P1D4 cDNA nucleotide sequence corresponds to nucleotide residues 150 through 585 in the STEAP-1 cDNA sequence as shown in FIG. 1A-B. Another clone, designated 28P3E1, 561 bp in length showed homology to a number of EST sequences in the NCI-CGAP tumor gene database or in other databases. Part of the 28P3E1 sequence (356 bp) is identical to an EST derived from human fetal tissue. After the full-length STEAP-1 cDNA was obtained and sequenced, it became apparent that this clone also corresponds to STEAP-1 (more specifically, to residues 622 through the 3′ end of the STEAP-1 nucleotide sequence as shown in FIG. 1).
Differential expression analysis by RT-PCR using primers derived from the 8P1D4 cDNA clone showed that the 8P1D4 (STEAP-1) gene is expressed at approximately equal levels in normal prostate and the LAPC4 and LAPC-9 xenografts. Further RT-PCR expression analysis of first strand cDNAs from 16 normal tissues showed greatest levels of 8P1D4 expression in prostate. Substantially lower level expression in several other normal tissues (i.e., colon, ovary, small intestine, spleen and testis) was detectable only at 30 cycles of amplification in brain, pancreas, colon and small intestine.
Example 2
Isolation of Full Length STEAP-1 Encoding cDNA
The 436 bp 8P1D4 gene fragment (Example 1) was used to isolate additional cDNAs encoding the 8P 1D4/STEAP-1 gene. Briefly, a normal human prostate cDNA library (Clontech) was screened with a labeled probe generated from the 436 bp 8P1D4 cDNA. One of the positive clones, clone 10, is 1195 bp in length and encodes a 339 amino acid protein having nucleotide and encoded amino acid sequences bearing no significant homology to any known human genes or proteins (homology to a rat Kidney Injury Protein recently described in International Application WO98/53071). The encoded protein contains at least 6 predicted transmembrane motifs implying a cell surface orientation (see FIG. 1A-B, predicted transmembrane motifs underlined). These structural features led to the designation “STEAP”, for “Six Transmembrane Epithelial Antigen of the Prostate”.
Subsequent identification of additional “STEAP” proteins led to the re-designation of the 8P1D4 gene product as “STEAP-1”. The STEAP-1 cDNA and encoded amino acid sequences are shown in FIG. 2A-Q. STEAP-1 cDNA clone 10 was deposited with the American Type Culture Collection (“ATCC”) (10801 University Blvd., Manassas, Va. 20110-2209 USA) as plasmid 8P1D4 clone 10.1 on Aug. 26, 1998 as ATCC Accession Number 98849. The STEAP-1 cDNA clone can be excised therefrom using EcoRI/XbaI double digest (EcoRI at the 5′ end, XbaI at the 3′ end).
This deposit was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations there under (Budapest Treaty). This assures maintenance of a viable culture of the deposit for 30 years from the date of deposit and for at least five (5) years after the most recent request for the furnishing of a sample of the deposit received by the depository. The deposits will be made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc. and ATCC, which assures that all restrictions imposed by the depositor on the availability to the public of the deposited material will be irrevocably removed upon the granting of the pertinent U.S. patent, assures permanent and unrestricted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 U.S.C. §122 and the Commissioner's rules pursuant thereto (including 37 C.F.R. §1.14 with particular reference to 886 OG 638).
The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.
Example 3
Chromosomal Mapping of STEAP-1
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.).
STEAP-1 maps to chromosome 7q21 using STEAP-1 sequence and the NCBI BLAST tool:
Example 4
Expression Analysis of STEAP-1
Expression of STEAP-1 in stomach cancer patient specimens is shown in FIG. 14. RNA was extracted from normal stomach (N) and from 10 different stomach cancer patient specimens (T). Northern blot with 10 μg of total RNA/lane was probed with STEAP-1 sequence. Results show strong expression of an approximately 1.6 kb STEAP-1 in the stomach tumor tissues. The lower panel represents ethidium bromide staining of the blot showing quality of the RNA samples.
FIG. 15 shows that STEAP-1 was expressed in rectum cancer patient tissues. RNA was extracted from normal rectum (N), rectum cancer patient tumors (T), and rectum cancer metastasis (M). Northern blots with 10 μg of total RNA were probed with the STEAP-1 sequence. Results show strong expression of STEAP-1 in the rectum cancer patient tissues. The lower panel represents ethidium bromide staining of the blot showing quality of the RNA samples.
Expression of STEAP-1 by RT-PCR demonstrated that STEAP-1 is strongly expressed in human umbilical vein endothelial cells (HUVEC) (FIG. 16). First strand cDNA was prepared from HUVEC cells, LAPC-4AD and LAPC-9AD prostate cancer xenografts, as well as from human brain tissues. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to STEAP-1, was performed at 27 and 30 cycles of amplification (FIG. 16A). As a control, PCR using primers to actin is shown in FIG. 16B. Results show strong expression of STEAP-1 in HUVEC cells similar to the expression detected in prostate cancer xenograft tissues. Expression of STEAP-1 in HUVEC cells indicates that targeting STEAP-1 may also target endothelial cells of the neovasculature of the tumors.
Example 5
Transcript Variants of 8P1D4
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 (see, e.g., Kan, Z., et al., Gene structure prediction and alternative splicing analysis using genomically aligned ESTs, Genome Research, 2001 May, 11(5):889-900.) Even when a variant is identified that is not a full-length clone, that portion of the variant is very useful for antigen generation and for further cloning of the full-length splice variant, using techniques known in the art.
Moreover, computer programs are available in the art that identify transcript variants based on genomic sequences. Genomic-based transcript variant identification programs include FgenesH (A. Salamov and V. Solovyev, “Ab initio gene finding in Drosophila genomic DNA,” Genome Research 2000 April; 10(4):516-22); Grail and GenScan. For a general discussion of splice variant identification protocols see., e.g., Southan, C., A genomic perspective on human proteases, FEBS Lett. 2001 Jun. 8; 498(2-3):214-8; de Souza, S. J., et al., Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags, Proc. Natl. Acad Sci USA. 2000 Nov. 7; 97(23):12690-3.
To further confirm the parameters of a transcript variant, a variety of techniques are available in the art, such as full-length cloning, proteomic validation, PCR-based validation, and 5′ RACE validation, etc. (see e.g., Proteomic Validation: Brennan, S. O., et al., Albumin banks peninsula: a new termination variant characterized by electrospray mass spectrometry, Biochem Biophys Acta. 1999 Aug. 17; 1433(1-2):321-6; Ferranti P, et al., Differential splicing of pre-messenger RNA produces multiple forms of mature caprine alpha(s1)-casein, Eur J Biochem. 1997 October 1; 249(1):1-7. For PCR-based Validation: Wellaann S, et al., Specific reverse transcription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCycler technology, Clin Chem. 2001 April; 47(4):654-60; Jia, H. P., et al., Discovery of new human beta-defensins using a genomics-based approach, Gene. 2001 Jan. 24; 263(1-2):211-8. For PCR-based and 5′ RACE Validation: Brigle, K. E., et al., Organization of the murine reduced-folate carrier gene and identification of variant splice forms, Biochem Biophys Acta. 1997 Aug. 7; 1353(2): 191-8).
It is known in the art that genomic regions are modulated in cancers. When the genomic region to which a gene maps is modulated in a particular cancer, the alternative transcripts or splice variants of the gene are modulated as well. Disclosed herein is that 8P1D4 has a particular expression profile related to cancer. Alternative transcripts and splice variants of 8P1D4 may also be involved in cancers in the same or different tissues, thus serving as tumor-associated markers/antigens.
The exon composition of the original transcript, designated as 8P1D4 v.1, is shown in Table LXI. Using the full-length gene and EST sequences, two transcript variants were identified, designated as 8P1D4 v.2 and v.3. Compared with 8P1D4 v.1, transcript variant 8P1D4 v.2 did not splice out intron 4 of 8P1D4 v.1 and variant 8P1D4 v.3 spliced out one additional exon from intron 4 of 8P1D4 v.1, as shown in FIG. 11. Theoretically, each different combination of exons in spatial order, e.g. exons 2 and 3, is a potential splice variant. FIG. 11 shows the schematic alignment of exons of the transcript variants.
Tables LIII through LX are set forth on a variant by variant bases. Tables LIII and LVII show the nucleotide sequence of the transcript variants. Table LIV shows the alignment of the transcript variant with the nucleic acid sequence of 8P 1D4 v.1. Table LVIII shows the alignment of the transcript variant with the nucleic acid sequence of 8P1D4 v.2. Tables LV and LIX lays out amino acid translation of the transcript variant for the identified reading frame orientation. Table LVI displays the alignment of the amino acid sequence encoded by the splice variant with that of 8P1D4 v.1. Table LX displays the alignment of the amino acid sequence encoded by the splice variant with that of 8P1D4 v.2.
Example 6
Single Nucleotide Polymorphisms of 8P1D4
A Single Nucleotide Polymorphism (SNP) is a single base pair variation in a nucleotide sequence at a specific location. At any given point of the genome, there are four possible nucleotide base pairs: A/T, C/G, G/C and T/A. Genotype refers to the specific base pair sequence of one or more locations in the genome of an individual. Haplotype refers to the base pair sequence of more than one location on the same DNA molecule (or the same chromosome in higher organisms), often in the context of one gene or in the context of several tightly linked genes. SNPs that occur on a cDNA are called cSNPs. These cSNPs may change amino acids of the protein encoded by the gene and thus change the functions of the protein. Some SNPs cause inherited diseases; others contribute to quantitative variations in phenotype and reactions to environmental factors including diet and drugs among individuals. Therefore, SNPs and/or combinations of alleles (called haplotypes) have many applications, including diagnosis of inherited diseases, determination of drug reactions and dosage, identification of genes responsible for diseases, and analysis of the genetic relationship between individuals (P. Nowotny, J. M. Kwon and A. M. Goate, “SNP analysis to dissect human traits,” Curr. Opin Neurobiol. 2001 October; 11(5):637-641; M. Pirmohamed and B. K. Park, “Genetic susceptibility to adverse drug reactions,” Trends Pharmacol. Sci. 2001 June; 22(6):298-305; J. H. Riley, C. J. Allan, E. Lai and A. Roses, “The use of single nucleotide polymorphisms in the isolation of common disease genes,” Pharmacogenomics. 2000 February; 1(1):39-47; R. Judson, J. C. Stephens and A. Windemuth, “The predictive power of haplotypes in clinical response,” Pharmacogenomics. 2000 February; 1(1):15-26).
SNPs are identified by a variety of art-accepted methods (P. Bean, “The promising voyage of SNP target discovery,” Am. Clin. Lab. 2001 October-November; 20(9):18-20; K. M. Weiss, “In search of human variation,” Genome Res. 1998 July; 8(7):691-697; M. M. She, “Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies,” Clin. Chem. 2001 February; 47(2):164-172). For example, SNPs are identified by sequencing DNA fragments that show polymorphism by gel-based methods such as restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). They can also be discovered by direct sequencing of DNA samples pooled from different individuals or by comparing sequences from different DNA samples. With the rapid accumulation of sequence data in public and private databases, one can discover SNPs by comparing sequences using computer programs (Z. Gu, L. Hillier and P. Y. Kwok, “Single nucleotide polymorphism hunting in cyberspace,” Hum. Mutat. 1998; 12(4):221-225). SNPs can be verified and genotype or haplotype of an individual can be determined by a variety of methods including direct sequencing and high throughput microarrays (P. Y. Kwok, “Methods for genotyping single nucleotide polymorphisms,” Annu. Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K. Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft, “High-throughput SNP genotyping with the Masscode system,” Mol Diagn. 2000 December; 5(4):329-340).
Using the methods described above, fourteen SNPs were identified in the transcript from clone GTH9, designated as 8P1D4v.2, at positions 602 (C/G), 386 (C/T), 1087 (T/G), 1447 (T/C), 1621 (A/T), 1625 (G/T), 1716 (C/A), 2358 (C/T), 2646 (T/G), 2859 (T/G), 2908 (A/T), 3006 (G/C), 3107 (C/T), and 3180 (A/T). The transcripts or proteins with alternative alleles were designated as variants 8P1D4 v.4, v.5, v.6, v.7, v.8, v.9, v.10, v.11, v.12, v.13, v. 14, v. 15, v. 16 and v.17, respectively. FIG. 10 shows the schematic alignment of the SNP variants. FIG. 12 shows the schematic alignment of protein variants, corresponding to nucleotide variants. These alleles of the NPs, though shown separately here, can occur in different combinations (haplotypes) and in any one of the transcript variants (such as 8P1D4 v.1 and v.3) that contains the sequence context of the SNPs. E.g., the first two SNP were also on 8P1D4 v.3 at the same positions, but at 572 and 356, respectively, on 8P1D4 v.1.
Example 7
Production of Recombinant 8P1D4 in Prokaryotic Systems
To express recombinant 8P1D4 and 8P1D4 variants in prokaryotic cells, the full or partial length 8P1D4 and 8P1D4 variant cDNA sequences are cloned into any one of a variety of expression vectors known in the art. The full length cDNA, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 8P1D4, variants, or analogs thereof are used.
A. In Vitro Transcription and Translation Constructs:
pCRII: To generate 8P1D4 sense and anti-sense RNA probes for RNA in situ investigations, pCRII constructs (Invitrogen, Carlsbad Calif.) are generated encoding either all or fragments of the 8P1D4 cDNA. The pCRII vector has Sp6 and T7 promoters flanking the insert to drive the transcription of 8P1D4 RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze the cell and tissue expression of 8P1D4 at the RNA level. Transcribed 8P1D4 RNA representing the cDNA amino acid coding region of the 8P1D4 gene is used in in vitro translation systems such as the TnTTM Coupled Reticulolysate System (Promega, Corp., Madison, Wis.) to synthesize 8P1D4 protein.
B. Bacterial Constructs:
pGEX Constructs: To generate recombinant 8P1D4 proteins in bacteria that are fused to the Glutathione S-transferase (GST) protein, all or parts of the 8P1D4 cDNA or variants are cloned into the GST-fusion vector of the pGEX family (Amersham Pharmacia Biotech, Piscataway, N.J.). These constructs allow controlled expression of recombinant 8P1D4 protein sequences with GST fused at the amino-terminus and a six histidine epitope (6× 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 8P1D4-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 8P1D4 proteins that are fused to maltose-binding protein (MBP), all or parts of the 8P1D4 cDNA protein coding sequence are fused to the MBP gene by cloning into the pMAL-c2X and pMAL-p2X vectors (New England Biolabs, Beverly, Mass.). These constructs allow controlled expression of recombinant 8P1D4 protein sequences with MBP fused at the amino-terminus and a 6× 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 8P1D4. The pMAL-c2X and pMAL-p2X vectors are optimized to express the recombinant protein in the cytoplasm or periplasm respectively. Periplasm expression enhances folding of proteins with disulfide bonds.
pET Constructs: To express 8P1D4 in bacterial cells, all or parts of the 8P1D4 cDNA protein coding sequence are cloned into the pET family of vectors (Novagen, Madison, Wis.). These vectors allow tightly controlled expression of recombinant 8P1D4 protein in bacteria with and without fusion to proteins that enhance solubility, such as NusA and thioredoxin (Trx), and epitope tags, such as 6× His and S-TagTM that aid purification and detection of the recombinant protein. For example, constructs are made utilizing pET NusA fusion system 43.1 such that regions of the 8P1D4 protein are expressed as amino-terminal fusions to NusA.
C. Yeast Constructs:
pESC Constructs: To express 8P1D4 in the yeast species Saccharomyces cerevisiae for generation of recombinant protein and functional studies, all or parts of the 8P1D4 cDNA protein coding sequence are cloned into the pESC family of vectors each of which contain 1 of 4 selectable markers, HIS3, TRP1, LEU2, and URA3 (Stratagene, La Jolla, Calif.). These vectors allow controlled expression from the same plasmid of up to 2 different genes or cloned sequences containing either FlagTM or Myc epitope tags in the same yeast cell. This system is useful to confirm protein-protein interactions of 8P1D4. In addition, expression in yeast yields similar post-translational modifications, such as glycosylations and phosphorylations, that are found when expressed in eukaryotic cells.
pESP Constructs: To express 8P1D4 in the yeast species Saccharomyces pombe, all or parts of the 8P1D4 cDNA protein coding sequence are cloned into the pESP family of vectors. These vectors allow controlled high level of expression of a 8P1D4 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 STEAP-1 in Eukaryotic Systems
A. Mammalian Constructs:
To express recombinant STEAP-1 in eukaryotic cells, the full or partial length STEAP-1 cDNA sequences can be cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of STEAP-1 are expressed in these constructs, amino acids 1 to 339 of STEAP-1 v.1, v.4, amino acids 1 to 258 of v.2, amino acids 1 to 282 of v.3, or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more contiguous amino acids from STEAP-1, variants, or analogs thereof. In certain embodiments a region of a specific variant of STEAP-1 is expressed that encodes an amino acid at a specific position which differs from the amino acid of any other variant found at that position. In other embodiments, a region of a variant of STEAP-1 is expressed that lies partly or entirely within a sequence that is unique to that variant.
The constructs can be transfected into any one of a wide variety of mammalian cells such as 293T cells. Transfected 293T cell lysates can be probed with the anti-STEAP-1 polyclonal serum, described herein.
pcDNA4/HisMax Constructs: To express STEAP-1 in mammalian cells, a STEAP-1 ORF, or portions thereof, of STEAP-1 are cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter and the SP 16 translational enhancer. The recombinant protein has XpressTM and six histidine (6× His) epitopes fused to the amino-terminus. The pcDNA4/HisMax vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Zeocin resistance gene allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli. 
pcDNA3.1/MycHis Constructs: To express STEAP-1 in mammalian cells, a STEAP-1 ORF, or portions thereof, of STEAP-1 with a consensus Kozak translation initiation site was cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the myc epitope and 6× His epitope fused to the carboxyl-terminus. The pcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability, along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene was used, as it allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli. 
pcDNA3.1/CT-GFP-TOPO Construct: To express STEAP-1 in mammalian cells and to allow detection of the recombinant proteins using fluorescence, a STEAP-1 ORF, or portions thereof, with a consensus Kozak translation initiation site are cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the Green Fluorescent Protein (GFP) fused to the carboxyl-terminus facilitating non-invasive, in vivo detection and cell biology studies. The pcDNA3.1CT-GFP-TOPO vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli. Additional constructs with an amino-terminal GFP fusion are made in pcDNA3.1/NT-GFP-TOPO spanning the entire length of a STEAP-1 protein.
PAPtag: A STEAP-1 ORF, or portions thereof, is cloned into pAPtag-5 (GenHunter Corp. Nashville, Tenn.). This construct generates an alkaline phosphatase fusion at the carboxyl-terminus of a STEAP-1 protein while fusing the IgGκ signal sequence to the amino-terminus. Constructs are also generated in which alkaline phosphatase with an amino-terminal IgGκ signal sequence is fused to the amino-terminus of a STEAP-1 protein. The resulting recombinant STEAP-1 proteins are optimized for secretion into the media of transfected mammalian cells and can be used to identify proteins such as ligands or receptors that interact with STEAP-1 proteins. Protein expression is driven from the CMV promoter and the recombinant proteins also contain myc and 6× His epitopes fused at the carboxyl-terminus that facilitates detection and purification. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the recombinant protein and the ampicillin resistance gene permits selection of the plasmid in E. coli. 
ptag5: A STEAP-1 ORF, or portions thereof, was cloned into pTag-5. This vector is similar to pAPtag but without the alkaline phosphatase fusion. This construct generated STEAP-1 protein with an amino-terminal IgGκ signal sequence and myc and 6× His epitope tags at the carboxyl-terminus that facilitate detection and affinity purification. The resulting recombinant STEAP-1 protein was optimized for secretion into the media of transfected mammalian cells, and is used as immunogen or ligand to identify proteins such as ligands or receptors that interact with the STEAP-1 proteins. Protein expression was driven from the CMV promoter. The Zeocin resistance gene present in the vector allowed for selection of mammalian cells expressing the protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli. 
PsecFc: A STEAP-1 ORF, or portions thereof, was also cloned into psecFc. The psecFc vector was assembled by cloning the human immunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen, California). This construct generated an IgG1 Fc fusion at the carboxyl-terminus of the STEAP-1 proteins, while fusing the IgGK signal sequence to N-terminus. STEAP-1 fusions utilizing the murine IgG1 Fc region are also used. The resulting recombinant STEAP-1 proteins were optimized for secretion into the media of transfected mammalian cells, and can were used as immunogens or to identify proteins such as ligands or receptors that interact with STEAP-1 protein. Protein expression is driven from the CMV promoter. The hygromycin resistance gene present in the vector allowed for selection of mammalian cells that express the recombinant protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli. 
pSRα Constructs: To generate mammalian cell lines that express STEAP-1 constitutively, STEAP-1 ORF, or portions thereof, of STEAP-1 were cloned into pSRα constructs. Amphotropic and ecotropic retroviruses were generated by transfection of pSRα constructs into the 293T-10A1 packaging line or co-transfection of pSRα and a helper plasmid (containing deleted packaging sequences) into the 293 cells, respectively. The retrovirus was used to infect a variety of mammalian cell lines, resulting in the integration of the cloned gene, STEAP-1, into the host cell-lines. Protein expression was driven from a long terminal repeat (LTR). The Neomycin resistance gene present in the vector allowed for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permit selection and maintenance of the plasmid in E. coli. The retroviral vectors were thereafter be used for infection and generation of various cell lines using, for example, PC3, NIH 3T3, TsuPr1, 293 or rat-1 cells.
Additional pSRα constructs are made that fuse an epitope tag such as the FLAGTM tag to the carboxyl-terminus of STEAP-1 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: 60) is added to cloning primer at the 3′ end of the ORF. Additional pSRα constructs were made to produce both amino-terminal and carboxyl-terminal GFP and myc/6× His fusion proteins of the full-length STEAP-1 proteins.
Additional Viral Vectors: Additional constructs are made for viral-mediated delivery and expression of STEAP-1. High virus titer leading to high level expression of STEAP-1 is achieved in viral delivery systems such as adenoviral vectors and herpes amplicon vectors. A STEAP-1 coding sequences or fragments thereof are amplified by PCR and subcloned into the AdEasy shuttle vector (Stratagene). Recombination and virus packaging are performed according to the manufacturer's instructions to generate adenoviral vectors. Alternatively, STEAP-1 coding sequences or fragments thereof are cloned into the HSV-1 vector (Imgenex) to generate herpes viral vectors. The viral vectors are thereafter used for infection of various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
Regulated Expression Systems: To control expression of STEAP-1 in mammalian cells, coding sequences of STEAP-1, or portions thereof, are cloned into regulated mammalian expression systems such as the T-Rex System (Invitrogen), the GeneSwitch System (Invitrogen) and the tightly-regulated Ecdysone System (Sratagene). These systems allow the study of the temporal and concentration dependent effects of recombinant STEAP-1. These vectors are thereafter used to control expression of STEAP-1 in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
B. Baculovirus Expression Systems
To generate recombinant STEAP-1 proteins in a baculovirus expression system, STEAP-1 ORF, or portions thereof, are cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag at the N-terminus. Specifically, pBlueBac-STEAP-1 is co-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda) insect cells to generate recombinant baculovirus (see Invitrogen instruction manual for details). Baculovirus is then collected from cell supernatant and purified by plaque assay.
Recombinant STEAP-1 protein is then generated by infection of HighFive insect cells (Invitrogen) with purified baculovirus. Recombinant STEAP-1 protein can be detected using anti-STEAP-1 or anti-His-tag antibody. STEAP-1 protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for STEAP-1.
Example 9
Antigenicity Profiles and Secondary Structure
FIGS. 5-9 and FIGS. 5a-9a depict graphically five amino acid profiles of the 8P1D4 variants 1 and 3 respectively, each assessment available by accessing the ProtScale website 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 the 8P1D4 protein. Each of the above amino acid profiles of 8P1D4 were generated using the following ProtScale parameters for analysis: 1) A window size of 9; 2) 100% weight of the window edges compared to the window center; and, 3) amino acid profile values normalized to lie between 0 and 1.
Hydrophilicity (FIG. 5), Hydropathicity (FIG. 6) and Percentage Accessible Residues (FIG. 7) profiles were used to determine stretches of hydrophilic amino acids (i.e., values greater than 0.5 on the Hydrophilicity and Percentage Accessible Residues profile, and values less than 0.5 on the Hydropathicity profile). Such regions are likely to be exposed to the aqueous environment, be present on the surface of the protein, and thus available for immune recognition, such as by antibodies.
Average Flexibility (FIG. 8) and Beta-turn (FIG. 9) profiles determine stretches of amino acids (i.e., values greater than 0.5 on the Beta-turn profile and the Average Flexibility profile) that are not constrained in secondary structures such as beta sheets and alpha helices. Such regions are also more likely to be exposed on the protein and thus accessible to immune recognition, such as by antibodies.
Antigenic sequences of the 8P1D4 protein and of the variant proteins indicated, e.g., by the profiles set forth in FIG. 5, FIG. 6, FIG. 7, FIG. 8, and/or FIG. 9 are used to prepare immunogens, either peptides or nucleic acids that encode them, to generate therapeutic and diagnostic anti-8P1D4 antibodies. The immunogen can be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50 contiguous amino acids, or the corresponding nucleic acids that encode them, from the 8P1D4 protein variants listed in FIGS. 2 and 3. In particular, peptide immunogens of the invention can comprise, a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of FIG. 5; a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of FIG. 6; a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 7; a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile on FIG. 8; and, a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of FIG. 9. Peptide immunogens of the invention can also comprise nucleic acids that encode any of the forgoing.
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 structures of 8P1D4 variant 1 and variant 3, namely the predicted presence and location of alpha helices, extended strands, and random coils, are predicted from the respective primary amino acid sequences using the HNN—Hierarchical Neural Network method, accessed from the ExPasy molecular biology server. The analysis indicates that 8P1D4 variant 1 is composed of 64.60% alpha helix, 4.72% extended strand, and 30.68% random coil (FIG. 13a). 8P1D4 variant 2 is composed of 62.79% alpha helix, 3.10% extended strand, and 34.11% random coil (FIG. 13b). 8P1D4 variant 3 is composed of 58.87% alpha helix, 5.32% extended strand, and 35.82% random coil (FIG. 13c).
Analysis for the potential presence of transmembrane domains in 8P1D4 variants were carried out using a variety of transmembrane prediction algorithms accessed from the ExPasy molecular biology server. Shown graphically are the results of analysis of variant 1 depicting the presence and location of 6 transmembrane domains using the TMpred program (FIG. 13d) and TMHMM program (FIG. 13e). Also shown are the results of analysis of variant 2 depicting the presence and location of 4 transmembrane domains using TMpred (FIG. 13f) and 3 transmembrane domains using TMHMM (FIG. 13g). Analysis of variant 3 predicts the presence of 4 transmembrane domains using the TMpred (Figure h) and 3 transmembrane domains with TMHMM (FIG. 13i). The results of each program, namely the amino acids encoding the transmembrane domains are summarized in Table XX.
Example 10
Generation of 8P1D4 Polyclonal Antibodies
Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. In addition to immunizing with the full length 8P1D4 protein, computer algorithms are employed in design of immunogens that, based on amino acid sequence analysis contain characteristics of being antigenic and available for recognition by the immune system of the immunized host (see the Example entitled “Antigenicity Profiles”). Such regions would be predicted to be hydrophilic, flexible, in beta-turn conformations, and be exposed on the surface of the protein (see, e.g., FIG. 5, FIG. 6, FIG. 7, FIG. 8, or FIG. 9 for amino acid profiles that indicate such regions of 8P1D4 and variants).
For example, 8P1D4 recombinant bacterial fusion proteins or peptides containing hydrophilic, flexible, beta-turn regions of 8P1D4 variant proteins are used as antigens to generate polyclonal antibodies in New Zealand White rabbits. For example, such regions include, but are not limited to, amino acids 1-40, amino acids 143-165, amino acids 180-220, of 8P1D4 variants 1, 2, and 3, amino acids 312-339 of 8P1D4 variant 1, and amino acids 250-282 of 8P1D4 variant 3. It is useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. In one embodiment, a peptide encoding amino acids 250-282 of 8P1D4 variant 3 is conjugated to KLH and used to immunize a rabbit. Alternatively the immunizing agent may include all or portions of the 8P1D4 variant proteins, analogs or fusion proteins thereof. For example, the 8P1D4 variant 1 amino acid sequence can be fused using recombinant DNA techniques to any one of a variety of fusion protein partners that are well known in the art, such as glutathione-S-transferase (GST) and HIS tagged fusion proteins. Such fusion proteins are purified from induced bacteria using the appropriate affinity matrix.
In one embodiment, a GST-fusion protein encoding amino acids 250-282, encompassing the second predicted extracellular loop, is produced, purified, and used as immunogen. Other recombinant bacterial fusion proteins that may be employed include maltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see the section entitled “Production of 8P1D4 in Prokaryotic Systems” and Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, P. S., Brady, W., Urnes, M., Grosmaire, L., Damle, N., and Ledbetter, L. (1991) J. Exp. Med. 174, 561-566).
In addition to bacterial derived fusion proteins, mammalian expressed protein antigens are also used. These antigens are expressed from mammalian expression vectors such as the Tag5 and Fc-fusion vectors (see the Example entitled “Production of Recombinant 8P1D4 in Eukaryotic Systems”), and retain post-translational modifications such as glycosylations found in native protein. In one embodiment, the predicted extracellular loop of variant 1, amino acids 185-218, is cloned into the Tag5 mammalian secretion vector. The recombinant protein is purified by metal chelate chromatography from tissue culture supernatants of 293T cells stably expressing the recombinant vector. The purified Tag5 8P1D4 protein is then used as immunogen.
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 a KLH-conjugated peptide encoding amino acids 250-282 of variant 3, the full-length 8P1D4 variant 1 cDNA is cloned into pCDNA 3.1 myc-his expression vector (Invitrogen, see the Example entitled “Production of Recombinant 8P 1D4 in Eukaryotic Systems”). After transfection of the constructs into 293T cells, cell lysates are probed with the anti-8P 1D4 serum and with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, Calif.) to determine specific reactivity to denatured 8P1D4 protein using the Western blot technique. The immune serum is then tested by the Western blot technique against 293T-8P 1D4 cells. In addition, the immune serum is tested by fluorescence microscopy, flow cytometry and immunoprecipitation against 293T and other recombinant 8P1D4-expressing cells to determine specific recognition of native protein. Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometric techniques using cells that endogenously express 8P1D4 are also carried out to test reactivity and specificity.
Anti-serum from rabbits immunized with 8P1D4 variant fusion proteins, such as GST and MBP fusion proteins, are purified by depletion of antibodies reactive to the fusion partner sequence by passage over an affinity column containing the fusion partner either alone or in the context of an irrelevant fusion protein. For example, antiserum derived from a GST-8P1D4 fusion protein encoding amino acids 250-282 of variant 3 is first purified by passage over a column of GST protein covalently coupled to AffiGel matrix (BioRad, Hercules, Calif.). The antiserum is then affinity purified by passage over a column composed of a MBP-fusion protein also encoding amino acids 250-282 covalently coupled to AffiGel matrix. The serum is then further purified by protein G affinity chromatography to isolate the IgG fraction. Sera from other His-tagged antigens and peptide immunized rabbits as well as fusion partner depleted sera are affinity purified by passage over a column matrix composed of the original protein immunogen or free peptide.
Example 11
Generation of 8P1D4 Monoclonal Antibodies (mAbs)
In one embodiment, therapeutic mAbs to 8P1D4 variants comprise those that react with epitopes specific for each variant protein or specific to sequences in common between the variants that would disrupt or modulate the biological function of the 8P1D4 variants, for example those that would disrupt the interaction with ligands and substrates or disrupt its biological activity. Immunogens for generation of such mAbs include those designed to encode or contain the entire 8P1D4 protein variant sequence, regions of the 8P1D4 protein variants predicted to be antigenic from computer analysis of the amino acid sequence (see, e.g., FIG. 5, FIG. 6, FIG. 7, FIG. 8, or FIG. 9, and the Example entitled “Antigenicity Profiles”). Immunogens include peptides, recombinant bacterial proteins, and mammalian expressed Tag 5 proteins and human and murine IgG FC fusion proteins. In addition, cells engineered to express high levels of a respective 8P1D4 variant, such as 293T-8P1D4 variant 1 or 300.19-8P1D4 variant 1murine Pre-B cells, are used to immunize mice.
To generate mAbs to an 8P1D4 variant, mice are first immunized intraperitoneally (IP) with, typically, 10-50 μg of protein immunogen or 107 8P1D4-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 an 8P1D4 variant sequence is used to immunize mice by direct injection of the plasmid DNA. For example amino acids 185-218, is cloned into the Tag5 mammalian secretion vector and the recombinant vector is used as immunogen. In another example the same amino acids are cloned into an Fc-fusion secretion vector in which the 8P1D4 variant 1 sequence is fused at the amino-terminus to an IgK leader sequence and at the carboxyl-terminus to the coding sequence of the human or murine IgG Fc region. This recombinant vector is then used as immunogen. The plasmid immunization protocols are used in combination with purified proteins expressed from the same vector and with cells expressing the respective 8P1D4 variant.
During the immunization protocol, test bleeds are taken 7-10 days following an injection to monitor titer and specificity of the immune response. Once appropriate reactivity and specificity is obtained as determined by ELISA, Western blotting, immunoprecipitation, fluorescence microscopy, and flow cytometric analyses, fusion and hybridoma generation is then carried out with established procedures well known in the art (see, e.g., Harlow and Lane, 1988).
In one embodiment for generating 8P1D4 variant 3 specific monoclonal antibodies, a GST-8P1D4 variant 3 antigen encoding amino acids 250-282, is expressed and purified from bacteria. Balb C mice are initially immunized intraperitoneally with 25 μg of the GST-8P1D4 variant 3 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 an MBP-fusion protein encoding amino acids 250-282 determines the titer of serum from immunized mice. Reactivity and specificity of serum to full length 8P1D4 variant 3 protein is monitored by Western blotting, immunoprecipitation and flow cytometry using 293T cells transfected with an expression vector encoding the 8P1D4 variant 3 cDNA compared to cells transfected with variant 1 and 2 cDNA (see e.g., the Example entitled “Production of Recombinant 8P1D4 in Eukaryotic Systems”). Other recombinant 8P1D4 variant 3-expressing cells or cells endogenously expressing 8P1D4 variant 3 are also used. Mice showing the strongest specific reactivity to 8P1D4 variant 3 are rested and given a final injection of GST-antigen in PBS and then sacrificed four days later. The spleens of the sacrificed mice are harvested and fused to SPO/2 myeloma cells using standard procedures (Harlow and Lane, 1988). Supernatants from HAT selected growth wells are screened by ELISA, Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometry to identify 8P1D4 specific antibody-producing clones.
The binding affinity of a 8P1D4 monoclonal antibody is determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which 8P1D4 monoclonal antibodies preferred for diagnostic or therapeutic use, as appreciated by one of skill in the art. The BIAcore system (Uppsala, Sweden) is a preferred method for determining binding affinity. The BIAcore system uses surface plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23: 1; Morton and Myszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecular interactions in real time. BIAcore analysis conveniently generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants.
Example 12
HLA Class I and Class H 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 V-XVIII and XXII-LI employ the protein sequence data from the gene product of STEAP-1 set forth in FIGS. 2 and 3, the specific peptides used to generate the tables are listed in Table LXI.
Computer searches for epitopes bearing HLA Class I or Class II supermotifs or motifs are performed as follows. All translated STEAP-1 protein sequences are analyzed using a text string search software program to identify potential peptide sequences containing appropriate HLA binding motifs; such programs are readily produced in accordance with information in the art in view of known motif/supermotif disclosures. Furthermore, such calculations can be made mentally.
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 n molecules. These polynomial algorithms account for the impact of different amino acids at different positions, and are essentially based on the premise that the overall affinity (or ΔG) of peptide-HLA molecule interactions can be approximated as a linear polynomial function of the type:

“ΔG”=a1i×a2i×a3i . . . ×ani 

where aji is a coefficient which represents the effect of the presence of a given amino acid (j) at a given position (i) along the sequence of a peptide of n amino acids. The crucial assumption of this method is that the effects at each position are essentially independent of each other (i.e., independent binding of individual side-chains). When residue j occurs at position i in the peptide, it is assumed to contribute a constant amount ji to the free energy of binding of the peptide irrespective of the sequence of the rest of the peptide.
The method of derivation of specific algorithm coefficients has been described in Gulukota et al., J. Mol. Biol. 267:1258-126, 1997; (see also Sidney et al., Human Immunol. 45:79-93, 1996; and Southwood et al., J. Immunol. 160:3363-3373, 1998). Briefly, for all i positions, anchor and non-anchor alike, the geometric mean of the average relative binding (ARB) of all peptides carrying j is calculated relative to the remainder of the group, and used as the estimate of ji. For Class II peptides, if multiple alignments are possible, only the highest scoring alignment is utilized, following an iterative procedure. To calculate an algorithm score of a given peptide in a test set, the ARB values corresponding to the sequence of the peptide are multiplied. If this product exceeds a chosen threshold, the peptide is predicted to bind. Appropriate thresholds are chosen as a function of the degree of stringency of prediction desired.
Selection of HLA-A2 Supertype Cross-Reactive Peptides
Protein sequences from STEAP-1 are scanned utilizing motif identification software, to identify 8-, 9-10- and 11-mer sequences containing the HLA-A2-supermotif main anchor specificity. Typically, these sequences are then scored using the protocol described above and the peptides corresponding to the positive-scoring sequences are synthesized and tested for their capacity to bind purified HLA-A*0201 molecules in vitro (HLA-A*0201 is considered a prototype A2 supertype molecule).
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 STEAP-1 protein sequence(s) scanned above is also examined for the presence of peptides with the HLA-A3-supermotif primary anchors. Peptides corresponding to the HLA A3 supermotif-bearing sequences are then synthesized and tested for binding to HLA-A*0301 and HLA-A*1101 molecules, the molecules encoded by the two most prevalent A3-supertype alleles. The peptides that bind at least one of the two alleles with binding affinities of ≦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 STEAP-1 protein(s) scanned above is also analyzed for the presence of 8-, 9-10-, or 11-mer peptides with the HLA-B7-supermotif. Corresponding peptides are synthesized and tested for binding to HLA-B*0702, the molecule encoded by the most common B7-supertype allele (i.e., the prototype B7 supertype allele). Peptides binding B*0702 with 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 STEAP-1 protein can also be performed to identify HLA-A 1- 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® M-450) and the detacha-bead® reagent. Typically about 200-250×106 PBMC are processed to obtain 24×106 CD8+ T-cells (enough for a 48-well plate culture). Briefly, the PBMCs are thawed in RPMI with 30 μg/ml DNAse, washed once with PBS containing 1% human AB serum and resuspended in PBS/1% AB serum at a concentration of 20×106 cells/ml. The magnetic beads are washed 3 times with PBS/AB serum, added to the cells (140 μl beads/20×106 cells) and incubated for 1 hour at 4° C. with continuous mixing. The beads and cells are washed 4× with PBS/AB serum to remove the nonadherent cells and resuspended at 100×106 cells/ml (based on the original cell number) in PBS/AB serum containing 100 μl/ml detacha-bead® reagent and 30 μg/ml DNAse. The mixture is incubated for 1 hour at room temperature with continuous mixing. The beads are washed again with PBS/AB/DNAse to collect the CD8+ T-cells. The DC are collected and centrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1% BSA, counted and pulsed with 40 μg/ml of peptide at a cell concentration of 1-2×106/ml in the presence of 3 μg/ml β2− microglobulin for 4 hours at 20° C. The DC are then irradiated (4,200 rads), washed 1 time with medium and counted again.
Setting up induction cultures: 0.25 ml cytokine-generated DC (at 1×105 cells/ml) are co-cultured with 0.25 ml of CD8+ T-cells (at 2×106 cell/ml) in each well of a 48-well plate in the presence of 10 ng/ml of IL-7. Recombinant human IL-10 is added the next day at a final concentration of 10 ng/ml and rhuman IL-2 is added 48 hours later at 10 IU/ml.
Restimulation of the induction cultures with peptide-pulsed adherent cells: Seven and fourteen days after the primary induction, the cells are restimulated with peptide-pulsed adherent cells. The PBMCs are thawed and washed twice with RPMI and DNAse. The cells are resuspended at 5×106 cells/ml and irradiated at  ̃4200 rads. The PBMCs are plated at 2×106 in 0.5 ml complete medium per well and incubated for 2 hours at 37° C. The plates are washed twice with RPMI by tapping the plate gently to remove the nonadherent cells and the adherent cells pulsed with 10 μg/ml of peptide in the presence of 3 μg/ml β2 microglobulin in 0.25 ml RPMI/5% AB per well for 2 hours at 37° C. Peptide solution from each well is aspirated and the wells are washed once with RPMI. Most of the media is aspirated from the induction cultures (CD8+ cells) and brought to 0.5 ml with fresh media. The cells are then transferred to the wells containing the peptide-pulsed adherent cells. Twenty four hours later recombinant human IL-10 is added at a final concentration of 10 ng/ml and recombinant human IL2 is added the next day and again 2-3 days later at 50IU/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 both assays for a side-by-side comparison.
Measurement of CTL Lytic Activity by 51Cr Release.
Seven days after the second restimulation, cytotoxicity is determined in a standard (5 hr) 51Cr release assay by assaying individual wells at a single E:T. Peptide-pulsed targets are prepared by incubating the cells with 10 μg/ml peptide overnight at 37° C.
Adherent target cells are removed from culture flasks with trypsin-EDTA. Target cells are labeled with 200 μCi of 51Cr sodium chromate (Dupont, Wilmington, Del.) for 1 hour at 37° C. Labeled target cells are resuspended at 106 per ml and diluted 1:10 with K562 cells at a concentration of 3.3×106/ml (an NK-sensitive erythroblastoma cell line used to reduce non-specific lysis). Target cells (100 μl) and effectors (100 μl) are plated in 96 well round-bottom plates and incubated for 5 hours at 37° C. At that time, 100 μl of supernatant are collected from each well and percent lysis is determined according to the formula:

[(cpm of the test sample−cpm of the spontaneous 51Cr release sample)/(cpm of the maximal 51Cr release sample−cpm of the spontaneous 51Cr release sample)]×100.

Maximum and spontaneous release are determined by incubating the labeled targets with 1% Triton X-100 and media alone, respectively. A positive culture is defined as one in which the specific lysis (sample-background) is 10% or higher in the case of individual wells and is 15% or more at the two highest E:T ratios when expanded cultures are assayed.
In Situ Measurement of Human IFNγ Production as an Indicator of Peptide-Specific and Endogenous Recognition
Immulon 2 plates are coated with mouse anti-human IFNγ monoclonal antibody (4 μg/ml 0.1M NaHCO3, pH8.2) overnight at 4° C. The plates are washed with Ca2+, Mg2+-free PBS/0.05% Tween 20 and blocked with PBS/10% FCS for two hours, after which the CTLs (100 μl/well) and targets (100 μl/well) are added to each well, leaving empty wells for the standards and blanks (which received media only). The target cells, either peptide-pulsed or endogenous targets, are used at a concentration of 1×106 cells/ml. The plates are incubated for 48 hours at 37° C. with 5% CO2.
Recombinant human IFN-gamma is added to the standard wells starting at 400 pg or 1200 pg/100 microliter/well and the plate incubated for two hours at 37° C. The plates are washed and 100 μl of biotinylated mouse anti-human IFN-gamma monoclonal antibody (2 microgram/ml in PBS/3% FCS/0.05% Tween 20) are added and incubated for 2 hours at room temperature. After washing again, 100 microliter HRP-streptavidin (1:4000) are added and the plates incubated for one hour at room temperature. The plates are then washed 6× with wash buffer, 100 microliter/well developing solution (TMB 1:1) are added, and the plates allowed to develop for 5-15 minutes. The reaction is stopped with 50 microliter/well 1M H3PO4 and read at OD450. A culture is considered positive if it measured at least 50 pg of IFN-gamma/well above background and is twice the background level of expression.
CTL Expansion.
Those cultures that demonstrate specific lytic activity against peptide-pulsed targets and/or tumor targets are expanded over a two week period with anti-CD3. Briefly, 5×104 CD8+ cells are added to a T25 flask containing the following: 1×106 irradiated (4,200 rad) PBMC (autologous or allogeneic) per ml, 2×105 irradiated (8,000 rad) EBV-transformed cells per ml, and OKT3 (anti-CD3) at 30 ng per ml in RPMI-1640 containing 10% (v/v) human AB serum, non-essential amino acids, sodium pyruvate, 25 μM 2-mercaptoethanol, L-glutamine and penicillin/streptomycin. Recombinant human IL2 is added 24 hours later at a final concentration of 200 IU/ml and every three days thereafter with fresh media at 50 IU/ml. The cells are split if the cell concentration exceeds 1×106/ml and the cultures are assayed between days 13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the 51Cr release assay or at 1×106/ml in the in situ IFNγ assay using the same targets as before the expansion.
Cultures are expanded in the absence of anti-CD3+ as follows. Those cultures that demonstrate specific lytic activity against peptide and endogenous targets are selected and 5×104 CD8+ cells are added to a T25 flask containing the following: 1×106 autologous PBMC per ml which have been peptide-pulsed with 10 μg/ml peptide for two hours at 37° C. and irradiated (4,200 rad); 2×105 irradiated (8,000 rad) EBV-transformed cells per ml RPMI-1640 containing 10% (v/v) human AB serum, non-essential AA, sodium pyruvate, 25 mM 2-ME, L-glutamine and gentamicin.
Immunogenicity of A2 Supermotif-Bearing Peptides
A2-supermotif cross-reactive binding peptides are tested in the cellular assay for the ability to induce peptide-specific CTL in normal individuals. In this analysis, a peptide is typically considered to be an epitope if it induces peptide-specific CTLs in at least individuals, and preferably, also recognizes the endogenously expressed peptide.
Immunogenicity can also be confirmed using PBMCs isolated from patients bearing a tumor that expresses STEAP-1. Briefly, PBMCs are isolated from patients, re-stimulated with peptide-pulsed monocytes and assayed for the ability to recognize peptide-pulsed target cells as well as transfected cells endogenously expressing the antigen.
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 Creatine 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 HLA-B7-transgenic mice, following for example, IFA immunization or lipopeptide immunization. Analoged peptides are additionally tested for the ability to stimulate a recall response using PBMC from patients with STEAP-1-expressing tumors.
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 I. 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 STEAP-1-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 STEAP-1-derived, HLA class II HTL epitopes, a STEAP-1 antigen is analyzed for the presence of sequences bearing an HLA-DR-motif or supermotif. Specifically, 15-mer sequences are selected comprising a DR-supermotif, comprising a 9-mer core, and three-residue N- and C-terminal flanking regions (15 amino acids total).
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 STEAP-1-derived peptides identified above are tested for their binding capacity for various common HLA-DR molecules. All peptides are initially tested for binding to the DR molecules in the primary panel: DR1, DR4w4, and DR7. Peptides binding at least two of these three DR molecules are then tested for binding to DR2w2 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. STEAP-1-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 STEAP-1 antigens are analyzed for sequences carrying one of the two DR3-specific binding motifs reported by Geluk et al. (J. Immunol. 152:5742-5748, 1994). The corresponding peptides are then synthesized and confirmed as having the ability to bind DR3 with an affinity of 1 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 STEAP-1-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 STEAP-1-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 include 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%. An analogous approach can be used to estimate population coverage achieved with combinations of class II motif-bearing epitopes.
Immunogenicity studies in humans (e.g., Bertoni et al., J. Clin. Invest. 100:503, 1997; Doolan et al., Immunity 7:97, 1997; and Threlkeld et al., J. Immunol. 159:1648, 1997) have shown that highly cross-reactive binding peptides are almost always recognized as epitopes. The use of highly cross-reactive binding peptides is an important selection criterion in identifying candidate epitopes for inclusion in a vaccine that is immunogenic in a diverse population.
With a sufficient number of epitopes (as disclosed herein and from the art), an average population coverage is predicted to be greater than 95% in each of five major ethnic populations. The game theory Monte Carlo simulation analysis, which is known in the art (see e.g., Osborne, M. J. and Rubinstein, A. “A course in game theory” MIT Press, 1994), can be used to estimate what percentage of the individuals in a population comprised of the Caucasian, North American Black, Japanese, Chinese, and Hispanic ethnic groups would recognize the vaccine epitopes described herein. A preferred percentage is 90%. A more preferred percentage is 95%.
Example 19
CTL Recognition of Endogenously Processed Antigens After Priming
This example confirms that CTL induced by native or analoged peptide epitopes identified and selected as described herein recognize endogenously synthesized, i.e., native antigens.
Effector cells isolated from transgenic mice that are immunized with peptide epitopes, for example HLA-A2 supermotif-bearing epitopes, are re-stimulated in vitro using peptide-coated stimulator cells. Six days later, effector cells are assayed for cytotoxicity and the cell lines that contain peptide-specific cytotoxic activity are further re-stimulated. An additional six days later, these cell lines are tested for cytotoxic activity on 51Cr labeled Jurkat-A2.1/Kb target cells in the absence or presence of peptide, and also tested on 51Cr labeled target cells bearing the endogenously synthesized antigen, i.e. cells that are stably transfected with STEAP-1 expression vectors.
The results demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized STEAP-1 antigen. The choice of transgenic mouse model to be used for such an analysis depends upon the epitope(s) that are being evaluated. In addition to HLA-A*0201/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 STEAP-1-derived CTL and HTL peptide vaccine compositions. The vaccine composition used herein comprise peptides to be administered to a patient with a STEAP-1-expressing tumor. The peptide composition can comprise multiple CTL and/or HTL epitopes. The epitopes are identified using methodology as described herein. This example also illustrates that enhanced immunogenicity can be achieved by inclusion of one or more HTL epitopes in a CTL vaccine composition; such a peptide composition can comprise an HTL epitope conjugated to a CTL epitope. The CTL epitope can be one that binds to multiple HLA family members at an affinity of 500 nM or less, or analogs of that epitope. The peptides may be lipidated, if desired.
Immunization procedures: Immunization of transgenic mice is performed as described (Alexander et al., J. Immunol. 159:4753-4761, 1997). For example, A2/Kb mice, which are transgenic for the human HLA A2.1 allele and are used to confirm the immunogenicity of HLA-A*0201 motif- or HLA-A2 supermotif-bearing epitopes, and are primed subcutaneously (base of the tail) with a 0.1 ml of peptide in Incomplete Freund's Adjuvant, or if the peptide composition is a lipidated CTL/HTL conjugate, in DMSO/saline, or if the peptide composition is a polypeptide, in PBS or Incomplete Freund's Adjuvant. Seven days after priming, splenocytes obtained from these animals are restimulated with syngenic irradiated LPS-activated lymphoblasts coated with peptide.
Cell lines: Target cells for peptide-specific cytotoxicity assays are Jurkat cells transfected with the HLA-A2.1/Kb chimeric gene (e.g., Vitiello et al., J. Exp. Med. 173:1007, 1991)
In vitro CTL activation: One week after priming, spleen cells (30×106 cells/flask) are co-cultured at 37° C. with syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (10×106 cells/flask) in 10 ml of culture medium/T25 flask. After six days, effector cells are harvested and assayed for cytotoxic activity.
Assay for cytotoxic activity: Target cells (1.0 to 1.5×106) are incubated at 37° C. in the presence of 200 μl of 51Cr. After 60 minutes, cells are washed three times and resuspended in R10 medium. Peptide is added where required at a concentration of 1 μg/ml. For the assay, 104 51Cr-labeled target cells are added to different concentrations of effector cells (final volume of 200 μl) in U-bottom 96-well plates. After a six hour incubation period at 37° C., a 0.1 ml aliquot of supernatant is removed from each well and radioactivity is determined in a Micromedic automatic gamma counter. The percent specific lysis is determined by the formula:

percent specific release=100×(experimental release−spontaneous release)/(maximum release−spontaneous release).

To facilitate comparison between separate CTL assays run under the same conditions, % 51Cr release data is expressed as lytic units/106 cells. One lytic unit is arbitrarily defined as the number of effector cells required to achieve 30% lysis of 10,000 target cells in a six hour 51Cr release assay. To obtain specific lytic units/106, the lytic units/106 obtained in the absence of peptide is subtracted from the lytic units/106 obtained in the presence of peptide. For example, if 30% 51Cr release is obtained at the effector (E):target (T) ratio of 50:1 (i.e., 5×105 effector cells for 10,000 targets) in the absence of peptide and 5:1 (i.e., 5×104 effector cells for 10,000 targets) in the presence of peptide, the specific lytic units would be: [(1/50,000)−(1/500,000)]×106=18 LU.

The results are analyzed to assess the magnitude of the CTL responses of animals injected with the immunogenic CTL/HTL conjugate vaccine preparation and are compared to the magnitude of the CTL response achieved using, for example, CTL epitopes as outlined above in the Example entitled “Confirmation of Immunogenicity.” Analyses similar to this may be performed to confirm the immunogenicity of peptide conjugates containing multiple CTL epitopes and/or multiple HTL epitopes. In accordance with these procedures, it is found that a CTL response is induced, and concomitantly that an HTL response is induced upon administration of such compositions.
Example 21
Selection of CTL and HTL Epitopes for Inclusion in a STEAP-1-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 STEAP-1 clearance. The number of epitopes used depends on observations of patients who spontaneously clear STEAP-1. For example, if it has been observed that patients who spontaneously clear STEAP-1-expressing cells generate an immune response to at least three (3) epitopes from STEAP-1 antigen, then at least three epitopes should be included for HLA class I. A similar rationale is used to determine HLA class II epitopes.
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 STEAP-1, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing nucleic acid vaccine compositions. Related to this embodiment, computer programs can be derived in accordance with principles in the art, which identify in a target sequence, the greatest number of epitopes per sequence length.
A vaccine composition comprised of selected peptides, when administered, is safe, efficacious, and elicits an immune response similar in magnitude to an immune response that controls or clears cells that bear or overexpress STEAP-1.
Example 22
Construction of “Minigene” Multi-Epitope DNA Plasmids
This example discusses the construction of a minigene expression plasmid. Minigene plasmids may, of course, contain various configurations of B cell, CTL and/or HTL epitopes or epitope analogs as described herein.
A minigene expression plasmid typically includes multiple CTL and HTL peptide epitopes. In the present example, HLA-A2, -A3, -B7 supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearing peptide epitopes are used in conjunction with DR supermotif-bearing epitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearing peptide epitopes derived STEAP-1, are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from STEAP-1 to provide broad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct. The selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.
Such a construct may additionally include sequences that direct the HTL epitopes to the endoplasmic reticulum. For example, the Ii protein may be fused to one or more HTL epitopes as described in the art, wherein the CLIP sequence of the Ii protein is removed and replaced with an HLA class II epitope sequence so that HLA class II epitope is directed to the endoplasmic reticulum, where the epitope binds to an HLA class II molecules.
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., Barnett et al., Aids Res. and Human Retroviruses 14, Supplement 3:S299-S309, 1998) or recombinant vaccinia, for example, expressing a minigene or DNA encoding the complete protein of interest (see, e.g., Hanke et al., Vaccine 16:439-445, 1998; Sedegah et al., Proc. Natl. Acad. Sci. USA 95:7648-53, 1998; Hanke and McMichael, Immunol. Letters 66:177-181, 1999; and Robinson et al., Nature Med. 5:526-34, 1999).
For example, the efficacy of the DNA minigene used in a prime boost protocol is initially evaluated in transgenic mice. In this example, A2.1/Kb transgenic mice are immunized IM with 100 μg of a DNA minigene encoding the immunogenic peptides including at least one HLA-A2 supermotif-bearing peptide. After an incubation period (ranging from 3-9 weeks), the mice are boosted IP with 107 pfu/mouse of a recombinant vaccinia virus expressing the same sequence encoded by the DNA minigene. Control mice are immunized with 100 μg of DNA or recombinant vaccinia without the minigene sequence, or with DNA encoding the minigene, but without the vaccinia boost. After an additional incubation period of two weeks, splenocytes from the mice are immediately assayed for peptide-specific activity in an ELISPOT assay. Additionally, splenocytes are stimulated in vitro with the A2-restricted peptide epitopes encoded in the minigene and recombinant vaccinia, then assayed for peptide-specific activity in an alpha, beta and/or gamma IFN ELISA.
It is found that the minigene utilized in a prime-boost protocol elicits greater immune responses toward the HLA-A2 supermotif peptides than with DNA alone. Such an analysis can also be performed using HLA-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 STEAP-1 expression in persons who are at risk for tumors that bear this antigen. For example, a polyepitopic peptide epitope composition (or a nucleic acid comprising the same) containing multiple CTL and HTL epitopes such as those selected in the above Examples, which are also selected to target greater than 80% of the population, is administered to individuals at risk for a STEAP-1-associated tumor.
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 STEAP-1-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 STEAP-1 Sequences
A native STEAP-1 polyprotein sequence is analyzed, preferably using computer algorithms defined for each class I and/or class II supermotif or motif, to identify “relatively short” regions of the polyprotein that comprise multiple epitopes. The “relatively short” regions are preferably less in length than an entire native antigen. This relatively short sequence that contains multiple distinct or overlapping, “nested” epitopes can be used to generate a minigene construct. The construct is engineered to express the peptide, which corresponds to the native protein sequence. The “relatively short” peptide is generally less than 250 amino acids in length, often less than 100 amino acids in length, preferably less than 75 amino acids in length, and more preferably less than 50 amino acids in length. The protein sequence of the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, i.e., it has a high concentration of epitopes. As noted herein, epitope motifs may be nested or overlapping (i.e., frame shifted relative to one another). For example, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes.
The vaccine composition will include, for example, multiple CTL epitopes from STEAP-1 antigen and at least one HTL epitope. This polyepitopic native sequence is administered either as a peptide or as a nucleic acid sequence which encodes the peptide. Alternatively, an analog can be made of this native sequence, whereby one or more of the epitopes comprise substitutions that alter the cross-reactivity and/or binding affinity properties of the polyepitopic peptide.
The embodiment of this example provides for the possibility that an as yet undiscovered aspect of immune system processing will apply to the native nested sequence and thereby facilitate the production of therapeutic or prophylactic immune response-inducing vaccine compositions. Additionally, such an embodiment provides for the possibility of motif-bearing epitopes for an HLA makeup(s) that is presently unknown. Furthermore, this embodiment (excluding an analoged embodiment) directs the immune response to multiple peptide sequences that are actually present in native STEAP-1, thus avoiding the need to evaluate any junctional epitopes. Lastly, the embodiment provides an economy of scale when producing peptide or nucleic acid vaccine compositions.
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 STEAP-1 peptide epitopes of the present invention are used in conjunction with epitopes from other target tumor-associated antigens, to create a vaccine composition that is useful for the prevention or treatment of cancer that expresses STEAP-1 and such other antigens. For example, a vaccine composition can be provided as a single polypeptide that incorporates multiple epitopes from STEAP-1 as well as tumor-associated antigens that are often expressed with a target cancer associated with STEAP-1 expression, or can be administered as a composition comprising a cocktail of one or more discrete epitopes. Alternatively, the vaccine can be administered as a minigene construct or as dendritic cells which have been loaded with the peptide epitopes in vitro.
Example 27
Use of Peptides to Evaluate an Immune Response
Peptides of the invention may be used to analyze an immune response for the presence of specific antibodies, CTL or HTL directed to STEAP-1. Such an analysis can be performed in a manner described by Ogg et al., Science 279:2103-2106, 1998. In this Example, peptides in accordance with the invention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen.
In this example highly sensitive human leukocyte antigen tetrameric complexes (“tetramers”) are used for a cross-sectional analysis of, for example, STEAP-1 HLA-A*0201-specific CTL frequencies from HLA A*0201-positive individuals at different stages of disease or following immunization comprising a STEAP-1 peptide containing an A*0201 motif. Tetrameric complexes are synthesized as described (Musey et al., N. Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201 in this example) and β2-microglobulin are synthesized by means of a prokaryotic expression system. The heavy chain is modified by deletion of the transmembrane-cytosolic tail and COOH-terminal addition of a sequence containing a BirA enzymatic biotinylation site. The heavy chain, β2-microglobulin, and peptide are refolded by dilution. The 45-kD refolded product is isolated by fast protein liquid chromatography and then biotinylated by BirA in the presence of biotin (Sigma, St. Louis, Mo.), adenosine 5′ triphosphate and magnesium. Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio, and the tetrameric product is concentrated to 1 mg/ml. The resulting product is referred to as tetramer-phycoerythrin.
For the analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300 g for 5 minutes and resuspended in 50 μl of cold phosphate-buffered saline. Tri-color analysis is performed with the tetramer-phycoerythrin, along with anti-CD8-Tricolor, and anti-CD38. The PBMCs are incubated with tetramer and antibodies on ice for 30 to 60 min and then washed twice before formaldehyde fixation. Gates are applied to contain >99.98% of control samples. Controls for the tetramers include both A*0201-negative individuals and A*0201-positive non-diseased donors. The percentage of cells stained with the tetramer is then determined by flow cytometry. The results indicate the number of cells in the PBMC sample that contain epitope-restricted CTLs, thereby readily indicating the extent of immune response to the STEAP-1 epitope, and thus the status of exposure to STEAP-1, or exposure to a vaccine that elicits a protective or therapeutic response.
Example 28
Use of Peptide Epitopes to Evaluate Recall Responses
The peptide epitopes of the invention are used as reagents to evaluate T cell responses, such as acute or recall responses, in patients. Such an analysis may be performed on patients who have recovered from STEAP-1-associated disease or who have been vaccinated with a STEAP-1 vaccine.
For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any STEAP-1 vaccine. PBMC are collected from vaccinated individuals and HLA typed. Appropriate peptide epitopes of the invention that, optimally, bear supermotifs to provide cross-reactivity with multiple HLA supertype family members, are then used for analysis of samples derived from individuals who bear that HLA type.
PBMC from vaccinated individuals are separated on Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis, Mo.), washed three times in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 μg/ml), and Hepes (10 mM) containing 10% heat-inactivated human AB serum (complete RPMI) and plated using microculture formats. A synthetic peptide comprising an epitope of the invention is added at 10 μg/ml to each well and HBV core 128-140 epitope is added at 1 μg/ml to each well as a source of T cell help during the first week of stimulation.
In the microculture format, 4×105 PBMC are stimulated with peptide in 8 replicate cultures in 96-well round bottom plate in 100 μl/well of complete RPMI. On days 3 and 10, 100 μl of complete RPMI and 20 U/ml final concentration of 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 STEAP-1 or a STEAP-1 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 STEAP-1 antigen, or PHA. Cells are routinely plated in replicates of 4-6 wells for each condition. After seven days of culture, the medium is removed and replaced with fresh medium containing 10 U/ml IL-2. Two days later, 1 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 STEAP-1
Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients having cancer that expresses STEAP-1. The main objectives of the trial are to determine an effective dose and regimen for inducing CTLs in cancer patients that express STEAP-1, to establish the safety of inducing a CTL and HTL response in these patients, and to see to what extent activation of CTLs improves the clinical picture of these patients, as manifested, e.g., by the reduction and/or shrinking of lesions. Such a study is designed, for example, as follows:
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 STEAP-1.
Clinical manifestations or antigen-specific T-cell responses are monitored to assess the effects of administering the peptide compositions. The vaccine composition is found to be both safe and efficacious in the treatment of STEAP-1-associated disease.
Example 31
Induction of CTL Responses Using a Prime Boost Protocol
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 cell 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 STEAP-1 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 STEAP-1 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 STEAP-1 antigens can be induced by incubating, in tissue culture, the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of APC, such as DC, and immunogenic peptides. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cells, i.e., tumor cells.
Example 33
An Alternative Method of Identifying and Confirming Motif-Bearing Peptides
Another method of identifying and confirming motif-bearing peptides is to elute them from cells bearing defined MHC molecules. For example, EBV transformed B cell lines used for tissue typing have been extensively characterized to determine which HLA molecules they express. In certain cases these cells express only a single type of HLA molecule. These cells can be transfected with nucleic acids that express the antigen of interest, e.g. STEAP-1. Peptides produced by endogenous antigen processing of peptides produced as a result of transfection will then bind to HLA molecules within the cell and be transported and displayed on the cell's surface. Peptides are then eluted from the HLA molecules by exposure to mild acid conditions and their amino acid sequence determined, e.g., by mass spectral analysis (e.g., Kubo et al., J. Immunol. 152:3913, 1994). Because the majority of peptides that bind a particular HLA molecule are motif-bearing, this is an alternative modality for obtaining the motif-bearing peptides correlated with the particular HLA molecule expressed on the cell.
Alternatively, cell lines that do not express endogenous HLA molecules can be transfected with an expression construct encoding a single HLA allele. These cells can then be used as described, i.e., they can then be transfected with nucleic acids that encode STEAP-1 to isolate peptides corresponding to STEAP-1 that have been presented on the cell surface. Peptides obtained from such an analysis will bear motif(s) that correspond to binding to the single HLA allele that is expressed in the cell.
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 STEAP-1-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring STEAP-1. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06 software (National Biosciences) and the coding sequence of STEAP-1. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5′ sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to a STEAP-1-encoding transcript.
Example 35
Purification of Naturally-Occurring or Recombinant STEAP-1 Using STEAP-1-Specific Antibodies
Naturally occurring or recombinant STEAP-1 is substantially purified by immunoaffinity chromatography using antibodies specific for STEAP-1. An immunoaffinity column is constructed by covalently coupling anti-STEAP-1 antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
Media containing STEAP-1 are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of STEAP-1 (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/STEAP-1 binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and GCR.P is collected.
Example 36
Identification of Molecules Which Interact with STEAP-1
STEAP-1, 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 STEAP-1, washed, and any wells with labeled STEAP-1 complex are assayed. Data obtained using different concentrations of STEAP-1 are used to calculate values for the number, affinity, and association of STEAP-1 with the candidate molecules.
Example 37
In Vivo Assay for STEAP-1 Tumor Growth Promotion
The effect of the STEAP-1 protein on tumor cell growth is evaluated in vivo by evaluating tumor development and growth of cells expressing or lacking STEAP-1. For example, SCID mice are injected subcutaneously on each flank with 1×106 of either 3T3, or prostate cancer cell lines (e.g. PC3 cells) containing tkNeo empty vector or STEAP-1. At least two strategies may be used: (1) Constitutive STEAP-1 expression under regulation of a promoter such as a constitutive promoter obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or from heterologous mammalian promoters, e.g. the actin promoter or an immunoglobulin promoter, provided such promoters are compatible with the host cell systems, and (2) Regulated expression under control of an inducible vector system, such as ecdysone, tetracycline, etc., provided such promoters are compatible with the host cell systems. Tumor volume is then monitored by caliper measurement at the appearance of palpable tumors and followed over time to determine if STEAP-1-expressing cells grow at a faster rate and whether tumors produced by STEAP-1-expressing cells demonstrate characteristics of altered aggressiveness (e.g. enhanced metastasis, vascularization, reduced responsiveness to chemotherapeutic drugs).
Additionally, mice can be implanted with 1×105 of the same cells orthotopically to determine if STEAP-1 has an effect on local growth in the prostate, and whether STEAP-1 affects the ability of the cells to metastasize, specifically to lymph nodes, and bone (Miki T et al, Oncol Res. 2001; 12:209; Fu X et al, Int J Cancer. 1991, 49:938). The effect of STEAP on bone tumor formation and growth may be assessed by injecting prostate tumor cells intratibially.
The assay is also useful to determine the STEAP-1 inhibitory effect of candidate therapeutic compositions, such as for example, STEAP-1 intrabodies, STEAP-1 antisense molecules and ribozymes.
Example 38
STEAP-1 Monoclonal Antibody-Mediated Inhibition of Prostate Tumors In Vivo
The significant expression of STEAP-1 in cancer tissues and surface localization, together with its restrictive expression in normal tissues makes STEAP-1 a good target for antibody therapy. Similarly, STEAP-1 is a target for T cell-based immunotherapy. Thus, the therapeutic efficacy of anti-STEAP-1 mAbs in human prostate cancer xenograft mouse models is evaluated by using recombinant cell lines such as PC3-STEAP-1, and 3T3-STEAP-1 (see, e.g., Kaighn, M. E., et al., Invest Urol, 1979. 17(1): 16-23), as well as human prostate xenograft models such as LAPC 9AD (Saffran et al PNAS 1999, 10:1073-1078).
Antibody efficacy on tumor growth and metastasis formation is studied, e.g., in a mouse orthotopic prostate cancer xenograft models. The antibodies can be unconjugated, as discussed in this Example, or can be conjugated to a therapeutic modality, as appreciated in the art. Anti-STEAP-1 mAbs inhibit formation of both lung and prostate xenografts. Anti-STEAP-1 mAbs also retard the growth of established orthotopic tumors and prolonged survival of tumor-bearing mice. These results indicate the utility of anti-STEAP-1 mAbs in the treatment of local and advanced stages prostate cancer. (See, e.g., Saffran, D., et al., PNAS 10:1073-1078).
Administration of the anti-STEAP-1 mAbs led to retardation of established orthotopic tumor growth and inhibition of metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies indicate that STEAP-1 as an attractive target for immunotherapy and demonstrate the therapeutic potential of anti-STEAP-1 mAbs for the treatment of local and metastatic prostate cancer. This example demonstrates that unconjugated STEAP-1 monoclonal antibodies are effective to inhibit the growth of human prostate tumor xenografts grown in SCID mice; accordingly a combination of such efficacious monoclonal antibodies is also effective.
Tumor Inhibition Using Multiple Unconjugated STEAP-1 mAbs
Materials and Methods
STEAP-1 Monoclonal Antibodies:
Monoclonal antibodies are raised against STEAP-1 as described in the Example entitled “Generation of STEAP-1 Monoclonal Antibodies (mAbs).” The antibodies are characterized by ELISA, Western blot, FACS, and immunoprecipitation for their capacity to bind STEAP-1. Epitope mapping data for the anti-STEAP-1 mAbs, as determined by ELISA and Western analysis, recognize epitopes on the STEAP-1 protein. Immunohistochemical analysis of prostate cancer tissues and cells with these antibodies is performed.
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 UM-UC3 and CaLu1 tumor xenografts.
Cell Lines and Xenografts
The prostate cancer cell lines, PC3 and LNCaP cell line as well as the fibroblast line NIH 3T3 (American Type Culture Collection) are maintained in RPMI and DMEM respectively, supplemented with L-glutamine and 10% FBS.
PC3-STEAP-1 and 3T3-STEAP-1 cell populations are generated by retroviral gene transfer as described in Hubert, R. S., et al., Proc Natl Acad Sci USA, 1999. 96(25): 14523.
The LAPC-9 xenograft, which expresses a wild-type androgen receptor and produces prostate-specific antigen (PSA), is passaged in 6- to 8-week-old male ICR-severe combined immunodeficient (SCID) mice (Taconic Farms) by s.c. trocar implant (Craft, N., et al., Nat. Med. 1999, 5:280). Single-cell suspensions of LAPC-9 tumor cells are prepared as described in Craft, et al.
Xenograft Mouse Models.
Subcutaneous (s.c.) tumors are generated by injection of 1×106 cancer cells mixed at a 1:1 dilution with Matrigel (Collaborative Research) in the right flank of male SCID mice. To test antibody efficacy on tumor formation, i.e. antibody injections are started on the same day as tumor-cell injections. As a control, mice are injected with either purified mouse IgG (ICN) or PBS; or a purified monoclonal antibody that recognizes an irrelevant antigen not expressed in human cells. In preliminary studies, no difference is found between mouse IgG or PBS on tumor growth. Tumor sizes are determined by caliper measurements, and the tumor volume is calculated as length×width×height. Mice with Subcutaneous tumors greater than 1.5 cm in diameter are sacrificed.
Orthotopic injections are performed under anesthesia by using ketamine/xylazine. For prostate orthotopic studies, an incision is made through the abdomen to expose the prostate and LAPC or PC3 tumor cells (5×105) mixed with Matrigel are injected into the prostate capsule in a 10-μl volume. To monitor tumor growth, mice are palpated and blood is collected on a weekly basis to measure PSA levels. The mice are segregated into groups for the appropriate treatments, with anti-STEAP-1 or control mAbs being injected i.p.
Anti-STEAP-1 mAbs Inhibit Growth of STEAP-1-Expressing Xenograft-Cancer Tumors
The effect of anti-STEAP-1 mAbs on tumor formation is tested by using LNCaP and LAPC9 orthotopic models. As compared with the s.c. tumor model, the orthotopic model, which requires injection of tumor cells directly in the mouse prostate, respectively, results in a local tumor growth, development of metastasis in distal sites, deterioration of mouse health, and subsequent death (Saffran, D., et al., PNAS supra). The features make the orthotopic model more representative of human disease progression and allowed us to follow the therapeutic effect of mAbs on clinically relevant end points.
Accordingly, tumor cells are injected into the mouse prostate, and 2 days later, the mice are segregated into two groups and treated with either: a) 200-500 μg, of anti-STEAP-1 Ab, or b) PBS three times per week for two to five weeks.
A major advantage of the orthotopic cancer models is the ability to study the development of metastases. Formation of metastasis in mice bearing established orthotopic tumors is studies by IHC analysis on lung sections using an antibody against a tumor-specific cell-surface protein such as anti-CK20 for prostate cancer (Lin S et al, Cancer Detect Prev. 2001; 25:202).
Another advantage of xenograft cancer models is the ability to study neovascularization and angiogenesis. Tumor growth is partly dependent on new blood vessel development. Although the capillary system and developing blood network is of host origin, the initiation and architecture of the neovasculature is regulated by the xenograft tumor (Davidoff A M et al, Clin Cancer Res. 2001; 7:2870; Solesvik O et al., Eur J Cancer Clin Oncol. 1984, 20:1295). The effect of antibody and small molecule on neovascularization is studied in accordance with procedures known in the art, such as by IHC analysis of tumor tissues and their surrounding microenvironment.
Mice bearing established orthotopic tumors are administered 1000 μg injections of either anti-STEAP-1 mAb or PBS over a 4-week period. Mice in both groups are allowed to establish a high tumor burden, to ensure a high frequency of metastasis formation in mouse lungs. Mice then are killed and their bladders, livers, bone and lungs are analyzed for the presence of tumor cells by IHC analysis. These studies demonstrate a broad anti-tumor efficacy of anti-STEAP-1 antibodies on initiation and progression of prostate cancer in xenograft mouse models. Anti-STEAP-1 antibodies inhibit tumor formation of tumors as well as retarding the growth of already established tumors and prolong the survival of treated mice. Moreover, anti-STEAP-1 mAbs demonstrate a dramatic inhibitory effect on the spread of local prostate tumor to distal sites, even in the presence of a large tumor burden. Thus, anti-STEAP-1 mAbs are efficacious on major clinically relevant end points (tumor growth), prolongation of survival, and health.
Example 39
Therapeutic and Diagnostic use of Anti-STEAP-1 Antibodies in Humans
Anti-STEAP-1 monoclonal antibodies are safely and effectively used for diagnostic, prophylactic, prognostic and/or therapeutic purposes in humans. Western blot and immunohistochemical analysis of cancer tissues and cancer xenografts with anti-STEAP-1 mAb show strong extensive staining in carcinoma but significantly lower or undetectable levels in normal tissues. Detection of STEAP-1 in carcinoma and in metastatic disease demonstrates the usefulness of the mAb as a diagnostic and/or prognostic indicator. Anti-STEAP-1 antibodies are therefore used in diagnostic applications such as immunohistochemistry of kidney biopsy specimens to detect cancer from suspect patients.
As determined by flow cytometry, anti-STEAP-1 mAb specifically binds to carcinoma cells. Thus, anti-STEAP-1 antibodies are used in diagnostic whole body imaging applications, such as radioimmunoscintigraphy and radioimnuunotherapy, (see, e.g., Potamianos S., et. al. Anticancer Res 20(2A):925-948 (2000)) for the detection of localized and metastatic cancers that exhibit expression of STEAP-1. Shedding or release of an extracellular domain of STEAP-1 into the extracellular milieu, such as that seen for alkaline phosphodiesterase BIO (Meerson, N. R., Hepatology 27:563-568 (1998)), allows diagnostic detection of STEAP-1 by anti-STEAP-1 antibodies in serum and/or urine samples from suspect patients.
Anti-STEAP-1 antibodies that specifically bind STEAP-1 are used in therapeutic applications for the treatment of cancers that express STEAP-1. Anti-STEAP-1 antibodies are used as an unconjugated modality and as conjugated form in which the antibodies are attached to one of various therapeutic or imaging modalities well known in the art, such as a prodrugs, enzymes or radioisotopes. In preclinical studies, unconjugated and conjugated anti-STEAP-1 antibodies are tested for efficacy of tumor prevention and growth inhibition in the SCID mouse cancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6, (see, e.g., the Example entitled “STEAP-1 Monoclonal Antibody-mediated Inhibition of Bladder and Lung Tumors In Vivo”). Either conjugated and unconjugated anti-STEAP-1 antibodies are used as a therapeutic modality in human clinical trials either alone or in combination with other treatments as described in following Examples.
Example 40
Human Clinical Trials for the Treatment and Diagnosis of Human Carcinomas Through Use of Human Anti-STEAP-1 Antibodies In Vivo
Antibodies are used in accordance with the present invention which recognize an epitope on STEAP-1, and are used in the treatment of certain tumors such as those listed in Table I. Based upon a number of factors, including STEAP-1 expression levels, tumors such as those listed in Table I are presently preferred indications. In connection with each of these indications, three clinical approaches are successfully pursued.
I.) Adjunctive therapy: In adjunctive therapy, patients are treated with anti-STEAP-1 antibodies in combination with a chemotherapeutic or antineoplastic agent and/or radiation therapy. Primary cancer targets, such as those listed in Table I, are treated under standard protocols by the addition anti-STEAP-1 antibodies to standard first and second line therapy. Protocol designs address effectiveness as assessed by reduction in tumor mass as well as the ability to reduce usual doses of standard chemotherapy. These dosage reductions allow additional and/or prolonged therapy by reducing dose-related toxicity of the chemotherapeutic agent. Anti-STEAP-1 antibodies are utilized in several adjunctive clinical trials in combination with the chemotherapeutic or antineoplastic agents adriamycin (advanced prostrate carcinoma), cisplatin (advanced head and neck and lung carcinomas), taxol (breast cancer), and doxorubicin (preclinical).
II.) Monotherapy: In connection with the use of the anti-STEAP-1 antibodies in monotherapy of tumors, the antibodies are administered to patients without a chemotherapeutic or antineoplastic agent. In one embodiment, monotherapy is conducted clinically in end stage cancer patients with extensive metastatic disease. Patients show some disease stabilization. Trials demonstrate an effect in refractory patients with cancerous tumors.
III.) Imaging Agent: Through binding a radionuclide (e.g., iodine or yttrium (I131, Y90) to anti-STEAP-1 antibodies, the radiolabeled antibodies are utilized as a diagnostic and/or imaging agent. In such a role, the labeled antibodies localize to both solid tumors, as well as, metastatic lesions of cells expressing STEAP-1. In connection with the use of the anti-STEAP-1 antibodies as imaging agents, the antibodies are used as an adjunct to surgical treatment of solid tumors, as both a pre-surgical screen as well as a post-operative follow-up to determine what tumor remains and/or returns. In one embodiment, a (111In)-STEAP-1 antibody is used as an imaging agent in a Phase I human clinical trial in patients having a carcinoma that expresses STEAP-1 (by analogy see, e.g., Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991)). Patients are followed with standard anterior and posterior gamma camera. The results indicate that primary lesions and metastatic lesions are identified
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-STEAP-1 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-STEAP-1 antibodies relative to the affinity of a known antibody for its target is one parameter used by those of skill in the art for determining analogous dose regimens. Further, anti-STEAP-1 antibodies that are fully human antibodies, as compared to the chimeric antibody, have slower clearance; accordingly, dosing in patients with such fully human anti-STEAP-1 antibodies can be lower, perhaps in the range of 50 to 300 mg/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-STEAP-1 antibodies. Conventional intravenous delivery is one standard delivery technique for many tumors. However, in connection with tumors in the peritoneal cavity, such as tumors of the ovaries, biliary duct, other ducts, and the like, intraperitoneal administration may prove favorable for obtaining high dose of antibody at the tumor and to also minimize antibody clearance. In a similar manner, certain solid tumors possess vasculature that is appropriate for regional perfusion. Regional perfusion allows for a high dose of antibody at the site of a tumor and minimizes short term clearance of the antibody.
Clinical Development Plan (CDP)
Overview: The CDP follows and develops treatments of anti-STEAP-1 antibodies in connection with adjunctive therapy, monotherapy, and as an imaging agent. Trials initially demonstrate safety and thereafter confirm efficacy in repeat doses. Trials are open label comparing standard chemotherapy with standard therapy plus anti-STEAP-1 antibodies. As will be appreciated, one criteria that can be utilized in connection with enrollment of patients is STEAP-1 expression levels in their tumors as determined by biopsy.
As with any protein or antibody infusion-based therapeutic, safety concerns are related primarily to (i) cytokine release syndrome, i.e., hypotension, fever, shaking, chills; (ii) the development of an immunogenic response to the material (i.e., development of human antibodies by the patient to the antibody therapeutic, or HAHA response); and, (iii) toxicity to normal cells that express STEAP-1. Standard tests and follow-up are utilized to monitor each of these safety concerns. Anti-STEAP-1 antibodies are found to be safe upon human administration.
Example 41
Human Clinical Trial Adjunctive Therapy with Human Anti-STEAP-1 Antibody and Chemotherapeutic Agent
A phase I human clinical trial is initiated to assess the safety of six intravenous doses of a human anti-STEAP-1 antibody in connection with the treatment of a solid tumor, e.g., a cancer of a tissue listed in Table I. In the study, the safety of single doses of anti-STEAP-1 antibodies when utilized as an adjunctive therapy to an antineoplastic or chemotherapeutic agent as defined herein, such as, without limitation: cisplatin, topotecan, doxorubicin, adriamycin, taxol, or the like, is assessed. The trial design includes delivery of six single doses of an anti-STEAP-1 antibody with dosage of antibody escalating from approximately about 25 mg/m2 to about 275 mg/m2 over the course of the treatment in accordance with the following schedule:


















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






















mAb Dose
25
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 STEAP-1. Standard tests and follow-up are utilized to monitor each of these safety concerns. Patients are also assessed for clinical outcome, and particularly reduction in tumor mass as evidenced by MRI or other imaging.
The anti-STEAP-1 antibodies are demonstrated to be safe and efficacious, Phase II trials confirm the efficacy and refine optimum dosing.
Example 42
Human Clinical Trial: Monotherapy with Human Anti-STEAP-1 Antibody
Anti-STEAP-1 antibodies are safe in connection with the above-discussed adjunctive trial, a Phase II human clinical trial confirms the efficacy and optimum dosing for monotherapy. Such trial is accomplished, and entails the same safety and outcome analyses, to the above-described adjunctive trial with the exception being that patients do not receive chemotherapy concurrently with the receipt of doses of anti-STEAP-1 antibodies.
Example 43
Human Clinical Trial: Diagnostic Imaging with Anti-STEAP-1 Antibody
Once again, as the adjunctive therapy discussed above is safe within the safety criteria discussed above, a human clinical trial is conducted concerning the use of anti-STEAP-1 antibodies as a diagnostic imaging agent. The protocol is designed in a substantially similar manner to those described in the art, such as in Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991). The antibodies are found to be both safe and efficacious when used as a diagnostic modality.
Example 44
Homology Comparison of STEAP-1 to Known Sequences
The human STEAP protein does not exhibit a high degree of homology to any known human proteins. The prototype member of the STEAP family, STEAP-1v. 1, is a type IIIa membrane protein consisting of 339 amino acids. STEAP-1 is a six transmembrane domain protein, with intracellular N- and C-termini.
The STEAP-1 gene is somewhat homologous to a previously cloned gene, namely mouse TNFa-induced adipose-related protein (gi|16905133|). STEAP-1v.1 shares 40% identities and 63% homology with TIARP. In addition, STEAP-1 shows homology to rat pHyde protein, with 49% identity and 71% homology to that protein. The TIARP protein is a 6 transmembrane protein of unknown function which localizes to the cell surface (Moldes M et al, J. Biol. Chem. 2001, 276:33938). Rat pHyde is a protein expressed in Dunning rats prostate cancer lines. Overexpression of pHyde in DU145 cells reduces their tumor growth properties (Steiner M et al, Cancer Res 2000, 60:4419). In addition, STEAP1 shows significant homology to mouse six transmembrane epithelial antigen of the prostate (gi 20820492).
Motift analysis revealed the presence of several protein functional motifs (Table XXI). Prints prediction identified a Transforming protein P21 ras signature, and Fibronectin type III repeat signature, while Blocks prediction identified a Half-A-TPR repeat, and an arsenical pump membrane protein signature. The fibronectin type III repeat region is a 100 amino acid domain, which mediated binding to DNA, heparin, basement membrane and the cell surface. The predominant role of these repeats is binding to the cell surface, allowing cell adhesion and mediating signaling event. Half-A-TPR repeat is a motif found in several RNA processing proteins. The arsenical pump functions in the efflux of ion and small molecules (Walmsley A R et al. J Biol Chem 2001, 276:6378-91). The motifs can participate in tumor growth and progression by enhancing the initial stages of tumorigenesis, such as tumor take or establishment of a tumor, by allowing adhesion to basement membranes and surrounding cells, by mediating cell communication and transport of ions.
Accordingly, when STEAP-1 functions as a regulator of tumor establishment, tumor formation, tumor growth, cell signaling or as a modulator of transcription involved in activating genes associated with survival, invasion, tumorigenesis or proliferation, STEAP-1 is used for therapeutic, diagnostic, prognostic and/or preventative purposes. In addition, when a molecule, such as a variant or SNP of STEAP-1 is expressed in cancerous tissues, such as those listed in Table I, they are used for therapeutic, diagnostic, prognostic and/or preventative purposes.
Example 45
Regulation of Transcription
The localization of STEAP-1 coupled to the presence of RNA processing motifs within its sequence indicate that STEAP-1 modulates the transcriptional regulation of eukaryotic genes. Regulation of gene expression is confirmed, e.g., by studying gene expression in cells expressing or lacking STEAP-1. For this purpose, two types of experiments are performed.
In the first set of experiments, RNA from parental and STEAP-1-expressing cells are extracted and hybridized to commercially available gene arrays (Clontech) (Smid-Koopman E et al. Br J Cancer. 2000. 83:246). Resting cells as well as cells treated with FBS, androgen or growth factors are compared. Differentially expressed genes are identified in accordance with procedures known in the art. The differentially expressed genes are then mapped to biological pathways (Chen K et al. Thyroid. 2001. 11:41.).
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, STEAP-1 plays a role in gene regulation, and it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 46
Identification and Confirmation of Potential Signal Transduction Pathways
Many mammalian proteins have been reported to interact with signaling molecules and to participate in regulating signaling pathways. (J Neurochem. 2001; 76:217-223). Fibronectin in particular has been associated with the MAPK signaling cascade that control cell mitogenesis (Jiang F, Jia Y, Cohen I. Blood. 2002, 99:3579). In addition, the STEAP-1 protein contains several phosphorylation sites (see Table XXI) indicating an association with specific signaling cascades. Using immunoprecipitation and Western blotting techniques, proteins are identified that associate with STEAP-1 and mediate signaling events. Several pathways known to play a role in cancer biology can be regulated by STEAP-1, including phospholipid pathways such as PI3K, AKT, etc, adhesion and migration pathways, including FAK, Rho, Rac-1, β-catenin, etc, as well as mitogenic/survival cascades such as ERK, p38, etc (Cell Growth Differ. 2000, 11:279; J Biol Chem. 1999, 274:801; Oncogene. 2000, 19:3003, J. Cell Biol. 1997, 138:913.). In order to determine whether expression of STEAP-1 is sufficient to regulate specific signaling pathways not otherwise active in resting PC3 cells, the effect of these genes on the activation of the p38 MAPK cascade was investigated in the prostate cancer cell line PC3 (FIGS. 21A-B). Activation of the p38 kinase is dependent on its phosphorylation on tyrosine and serine residues. Phosphorylated p38 can be distinguished from the non-phosphorylated state by a Phospho-p38 mAb. This phospho-specific Ab was used to study the phosphorylation state of p38 in engineered PC3 cell lines.
PC3 cells stably expressing STEAP-1 neo were grown overnight in either 1% or 10% FBS. Whole cell lysates were analyzed by western blotting. PC3 cells treated with the known p38 activators, NaSaI or TNF, were used as a positive control. The results show that while expression of the control neo gene has no effect on p38 phosphorylation, expression of STEAP-1 in PC3 cells is sufficient to induce the activation of the p38 pathway (FIG. 21A). The results were verified using western blotting with an anti-p38 Ab, which shows equal protein loading on the gels (FIG. 21B).
In another set of experiments, the sufficiency of expression of STEAP-1 in the prostate cancer cell line PC3 to activate the mitogenic MAPK pathway, namely the ERK cascade, was examined (FIG. 22A-B). Activation of ERK is dependent on its phosphorylation on tyrosine and serine residues. Phosphorylated ERK can be distinguished from the non-phosphorylated state by a Phospho-ERK mAb. This phospho-specific Ab was used to study the phosphorylation state of ERK in engineered PC3 cell lines. PC3 cells, expressing an activated form of Ras, were used as a positive control.
The results show that while expression of the control neo gene has no effect on ERK phosphorylation, expression of STEAP-1 in PC3 cells is sufficient to induce an increase in ERK phosphorylation (FIG. 22A). These results were verified using anti-ERK western blotting (FIG. 22B) and confirm the activation of the ERK pathway by STEAP-1 and STEAP-2.
Since FBS contains several components that may contribute to receptor-mediated ERK activation, we examined the effect of STEAP-1 in low and optimal levels of FBS. PC3 cells expressing neo or STEAP-1 were grown in either 0.1% or 10% FBS overnight. The cells were analyzed by anti-Phospho-ERK western blotting. This experiment shows that STEAP-1 induces the phosphorylation of ERK in 0.1% FBS, and confirms that expression of STEAP-1 is sufficient to induce activation of the ERK signaling cascade in the absence of additional stimuli.
To confirm that STEAP-1 directly or indirectly activates known signal transduction pathways in cells, luciferase (luc) based transcriptional reporter assays are carried out in cells expressing individual genes. These transcriptional reporters contain consensus-binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways. The reporters and examples of these associated transcription factors, signal transduction pathways, and activation stimuli are listed below.

    
    
        1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK; growth/apoptosis/stress
        2. SRE-luc, SRF/TCF/ELK1; MAPK/SAPK; growth/differentiation
        3. AP-1-luc, FOS/JUN; MAPK/SAPK/PKC; growth/apoptosis/stress
        4. ARE-luc, androgen receptor; steroids/MAPK; growth/differentiation/apoptosis
        5. p53-luc, p53; SAPK; growth/differentiation/apoptosis
        6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress
        7. TCF-luc, TCF/Lef; β-catenin, Adhesion/invasion
    
    


Gene-mediated effects can be assayed in cells showing mRNA expression. Luciferase reporter plasmids can be introduced by lipid-mediated transfection (TFX-50, Promega). Luciferase activity, an indicator of relative transcriptional activity, is measured by incubation of cell extracts with luciferin substrate and luminescence of the reaction is monitored in a luminometer.
Signaling pathways activated by STEAP-1 are mapped and used for the identification and validation of therapeutic targets. When STEAP-1 is involved in cell signaling, it is used as target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 47
Involvement in Tumor Progression
Based on the documented role of transforming protein in transformation, and progression, the STEAP-1 gene can contribute to the growth and transformation of cancer cells. The role of STEAP-1 in tumor growth is confirmed in a variety of primary and transfected cell lines including prostate cell lines, as well as NIH 3T3 cells engineered to stably express STEAP-1. Parental cells lacking STEAP-1 and cells expressing STEAP-1 are evaluated for cell growth using a well-documented proliferation assay (Fraser S P, Grimes J A, Djamgoz M B. Prostate. 2000; 44:61, Johnson D E, Ochieng J, Evans S L. Anticancer Drugs. 1996, 7:288).
To confirm the role of STEAP-1 in the transformation process, its effect in colony forming assays is investigated. Parental NIH-3T3 cells lacking STEAP-1 are compared to NIH-3T3 cells expressing STEAP-1, using a soft agar assay under stringent and more permissive conditions (Song Z. et al. Cancer Res. 2000; 60:6730).
To confirm the role of STEAP-1 in invasion and metastasis of cancer cells, a well-established assay is used, e.g., a Transwell Insert System assay (Becton Dickinson) (Cancer Res. 1999; 59:6010). Control cells, including prostate, breast and kidney cell lines lacking STEAP-1 are compared to cells expressing STEAP-1. Cells are loaded with the fluorescent dye, calcein, and plated in the top well of the Transwell insert coated with a basement membrane analog. Invasion is determined by fluorescence of cells in the lower chamber relative to the fluorescence of the entire cell population.
STEAP-1 can also play a role in cell cycle and apoptosis. Parental cells and cells expressing STEAP-1 are compared for differences in cell cycle regulation using a well-established BrdU assay (Abdel-Malek Z A. J Cell Physiol. 1988, 136:247). In short, cells are grown under both optimal (full serum) and limiting (low serum) conditions are labeled with BrdU and stained with anti-BrdU Ab and propidium iodide. Cells are analyzed for entry into the G1, S, and G2M phases of the cell cycle. Alternatively, the effect of stress on apoptosis is evaluated in control parental cells and cells expressing STEAP-1, including normal and tumor prostate cells. Engineered and parental cells are treated with various chemotherapeutic agents, such as etoposide, taxol, etc, and protein synthesis inhibitors, such as cycloheximide. Cells are stained with annexin V-FITC and cell death is measured by FACS analysis. The modulation of cell death by STEAP-1 can play a critical role in regulating tumor progression and tumor load.
When STEAP-1 plays a role in cell growth, transformation, invasion or apoptosis, it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 48
Involvement in Angiogenesis
Angiogenesis or new capillary blood vessel formation is necessary for tumor growth (Hanahan D, Folkman J. Cell. 1996, 86:353; Folkman J. Endocrinology. 1998 139:441). Based on the effect of phsophodieseterase inhibitors on endothelial cells, STEAP-1 plays a role in angiogenesis (DeFouw L et al, Microvasc Res 2001, 62:263). Several assays have been developed to measure angiogenesis in vitro and in vivo, such as the tissue culture assays endothelial cell tube formation and endothelial cell proliferation. Using these assays as well as in vitro neo-vascularization, the role of STEAP-1 in angiogenesis, enhancement or inhibition, is confirmed.
For example, endothelial cells engineered to express STEAP-1 are evaluated using tube formation and proliferation assays. The effect of STEAP-1 is also confirmed in animal models in vivo. For example, cells either expressing or lacking STEAP-1 are implanted subcutaneously in immunocompromised mice. Endothelial cell migration and angiogenesis are evaluated 5-15 days later using immunohistochemistry techniques. STEAP-1 affects angiogenesis, and it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes
Example 49
Involvement in Protein-Protein Interactions
Fibronectin motifs have been shown to mediate interaction with other proteins, including cell surface protein. Using immunoprecipitation techniques as well as two yeast hybrid systems, proteins are identified that associate with STEAP-1. Immunoprecipitates from cells expressing STEAP-1 and cells lacking STEAP-1 are compared for specific protein-protein associations.
Studies are performed to confirm the extent of association of STEAP-1 with effector molecules, such as nuclear proteins, transcription factors, kinases, phosphates etc. Studies comparing STEAP-1 positive and STEAP-1 negative cells as well as studies comparing unstimulated/resting cells and cells treated with epithelial cell activators, such as cytokines, growth factors, androgen and anti-integrin Ab reveal unique interactions.
In addition, protein-protein interactions are confirmed using two yeast hybrid methodology (Curr Opin Chem. Biol. 1999, 3:64). A vector carrying a library of proteins fused to the activation domain of a transcription factor is introduced into yeast expressing a STEAP-1-DNA-binding domain fusion protein and a reporter construct. Protein-protein interaction is detected by colorimetric reporter activity. Specific association with effector molecules and transcription factors directs one of skill to the mode of action of STEAP-1, and thus identifies therapeutic, prognostic, preventative and/or diagnostic targets for cancer. This and similar assays are also used to identify and screen for small molecules that interact with STEAP-1.
Thus it is found that STEAP-1 associates with proteins and small molecules. Accordingly, STEAP-1 and these proteins and small molecules are used for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 50
Involvement of STEAP-1 in Small Molecule Transport and Cell-Cell Communication
Cell-cell communication is essential in maintaining organ integrity and homeostasis, both of which become deregulated during tumor formation and progression. Intercellular communications can be measured using two types of assays (J. Biol. Chem. 2000, 275:25207). In the first assay, cells loaded with a fluorescent dye are incubated in the presence of unlabeled recipient cells and the cell populations are examined under fluorescent microscopy. This qualitative assay measures the exchange of dye between adjacent cells. In the second assay system, donor and recipient cell populations are treated as above and quantitative measurements of the recipient cell population are performed by FACS analysis. Using these two assay systems, cells expressing STEAP-1 are compared to controls that do not express STEAP-1, and it is found that STEAP-1 enhances cell communications. FIG. 19 and FIG. 20 demonstrate that STEAP-1 mediates the transfer of the small molecule calcein between adjacent cells, and thereby regulates cell-cell communication in prostate cancer cells. In this experiment, recipient PC3 cells were labeled with dextran-Texas Red and donor PC3 cells were labeled with calcein AM (green). The donor (green) and recipient (red) cells were co-cultured at 37° C. and analyzed by microscopy for the co-localization of Texas red and calcein. The results demonstrated that while PC3 control cells (no detectable STEAP-1 protein expression) exhibit little calcein transfer, the expression of STEAP-1 allows the transfer of small molecules between cells (FIG. 19), whereby the initially red recipient cells take on a brownish color, and co-localize the red and green molecules. Small molecules and/or antibodies that modulate cell-cell communication mediated by STEAP-1 are used as therapeutics for cancers that express STEAP-1. FIG. 20 shows the time-dependent manner of cell-cell communication mediated by STEAP-1, where little transfer in PC3-STEAP-1 cells is seen at 6 hours, and much transfer is seen at 24 hours. FIG. 23 demonstrates that expression of STEAP-1 is necessary on both donor and recipient populations for the transfer of small molecules to take place. In this experiment, donor cells labeled with dextran red were incubated with recipient cells labeled with calcein (green). However, the donor and recipient populations were alternated, such that a PC3 donor population was incubated with PC3 recipient, or with PC3-STEAP-1 recipient populations. Similarly, a PC3-STEAP-1 donor population was co-cultured with a PC3 or a PC3-STEAP-1 recipient population. The results show that co-culturing of control PC3 and PC3 cells fail to mediate calcein transfer. Similarly, co-incubation of control PC3 and PC3-STEAP-1 does not allow the transfer of calcein. However, co-culturing PC3-STEAP-1 donor and PC3-STEAP-1 recipient cells mediates small molecule transfer as depicted by co-localization of green and red pigments in the same cells. Taken together, the data shown in FIGS. 19, 20 and 23 demonstrate that STEAP-1 mediates small molecule transfer and regulates cell-cell communication by forming inter-cellular communication channels that are similar in function to gap junctions.
Thus, because STEAP-1 functions in cell-cell communication and small molecule transport, it is used as a target or marker for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 51
Involvement of STEAP-1 in Ion Transport
The localization and topology of STEAP-1 supports its function as a transporter. To confirm that STEAP-1 functions as an ion channel, FACS analysis and fluorescent microscopy techniques were used (Gergely L, et al., Clin Diagn Lab Immunol. 1997; 4:70; Skryma R, et al., J. Physiol. 2000, 527: 71). Using FACS analysis and commercially available indicators (Molecular Probes), parental cells and cells expressing STEAP-1 were compared for their ability to transport calcium; prostate cancer PC3 cell lines were used in these studies. PC3 and PC3-STEAP-1 cells were loaded with calcium responsive indicators Fluo4 and Fura red, incubated in the presence or absence of calcium and lipophosphatidic acid (LPA), and analyzed by flow cytometry. FIG. 17 shows that STEAP-1 increased calcium flux in response to LPA. This data validates an important mechanism by which cancer cells are regulated. This is particularly true in the case of calcium, as calcium channel inhibitors have been reported to induce the death of certain cancer cells, including prostate cancer cell lines (Batra S, Popper L D, Hartley-Asp B. Prostate. 1991, 19: 299).
Furthermore, FIG. 18 shows that STEAP-1-mediated calcium transport regulates prostate cancer growth by regulating intracellular levels of calcium. In this study, control PC3 and PC3-STEAP-1 cells were grown in the presence or absence of ion channel inhibitors, namely amiloride, NDGA and TEA, which inhibit the transport of sodium, calcium and potassium, respectively. STEAP-1 expression rendered PC3 cells sensitive to treatment with the Ca++ channel inhibitor NDGA. While PC3 control cells demonstrated 100% growth in the presence of the inhibitor, NDGA, at 50 μM inhibited the proliferation of PC3-STEAP-1 cells by 42% and 10 example 51 μM NDGA inhibited proliferation 20%. These findings confirm the importance of STEAP-1 as regulator of calcium transport in the proliferation of cancer cells that express this protein. Thus, STEAP-1 is used in therapeutic modalities that control the proliferation of prostate cancer cells. Because STEAP-1 functions in ion transport, it is used as a target or marker for diagnostic, prognostic, preventative and therapeutic purposes.
Example 52
RNA Interference (RNAi)
RNA interference (RNAi) makes use of sequence specific double stranded RNA to prevent gene expression. Small interfering RNA (siRNA) are transfected into mammalian cells and thereby mediate sequence specific mRNA degradation. (Elbashir, et al, Nature, 2001; vol. 411: 494-498). The following siRNA oligonucleotide sequences were used:











STEAP-1 (1) target:
(SEQ ID NO: 96)




GACAUCACAAACCAAGAAG









(corresponding to nucleotides 16-34 of



STEAP-1 ORF)



 











STEAP-1 (2) target:
(SEQ ID NO: 97)




GUGGAUAGAUAUAAAACAA









(corresponding to nucleotides 893-911 of



STEAP-1 ORF)






The sense strand of STEAP-1 (1) is labeled at 3′ with fluorescein, 6-FAM (ABS 494 nm, EMM 525 nm, green). The siRNA is dissolved in RNA-free sterile buffer (100 mM KOAc, 30 mM HEPES KOH, 2mM MOAc, at pH 7.4) to make 20 μM stock (200×). The siRNa is transfected into LNCaP, 3T3-STEAP-1, and Rat-1-STEAP-1 cells seeded on 6-well plates with oligofectamine reagent (Invitrogen). The final concentration of siRNA was 100× nM. The following oligonucleotide was used as a control to rule out any non-specific effect of STEAP-1 siRNA oligonucleotides:











Control:
(SEQ ID NO: 98)




GCCUUCCCAUUGUGCUCCC









(corresponding to nucleotides 91-109 of ORF)






Protein expression was detected 24 hours after transfection by immunostaining followed by flow cytometry. In addition, confirmation of altered gene expression was performed by Western blotting. The results (FIG. 24) show that the introduction of STEAP-1 specific RNAi reduced the expression of STEAP-1 in recombinant 3T3 and Rat-1 cells. 3T3 and Rat-1 cells were permeabilized and stained with anti-STEAP-1 polyclonal antibodies. Whole cell immunostaining revealed that the STEAP-1 RNAi reduced STEAP-1 expression in Rat-1 and 3T3 cells. This reduction was confirmed by Western blot analysis where STEAP-1 protein was substantially reduced in STEAP-1 RNAi treated cells relative to control and untreated cells. Additionally, RNAi reduces the endogenous expression of STEAP-1 in the prostate cancer and LNCaP cell line as shown in FIG. 25.
Accordingly, the RNA oligonucleotide sequences are used in therapeutic and prophylactic applications. Moreover, the RNA oligonucleotide sequences are used to assess how modulating the expression of a STEAP-1 gene affects function of cancer cells and/or tissues.
Example 53
Modulation of STEAP-1 Function
Ion transport plays an important role regulating cell growth intracellular permeability, molecular trafficking and signal transduction (Minke B. Cell Mol. Neurobiol. 2001, 21:629; Golovina et al, Am J Physiol Heart Circ Physiol. 2001, 280:H746) these are functions that are especially relevant to the neoplastic condition. Cell-cell communication regulates homeostasis, cell proliferation and cell death (Evans W H, Martin P E. Mol Membr Biol. 2002 19:121; Carruba G, et al, Ann N Y Acad. Sci. 2002, 963:156) these functions too are especially relevant to the neoplastic condition.
Using control cell lines and cell lines expressing STEAP-1, inhibitors of STEAP-1 function are identified. For example, PC3 and PC3-STEAP-1 cells can be incubated in the presence and absence of mAb or small molecule inhibitors. The effect of these mAb or small molecule inhibitors are investigated using the ion flux, cell communication, proliferation and signaling assays described above.
Signal transduction and biological output mediated by transporters can be modulated through various mechanisms, including inhibition of receptor and ligand binding, ion antagonists, protein interactions, regulation of ion and small molecule transport, etc (Tang W et al, Front Biosci 2002, 7:1583). Using control cell lines and cell lines expressing STEAP-1, modulators (inhibitors or enhancers) of STEAP-1 function are identified. For example, PC3 and PC3-STEAP-1 cells are incubated in the presence and absence of mAb or small molecule modulators. In view of the functions of STEAP-1 disclosed herein, modulators that are ion channel blockers used in the context of the present invention include such compounds as amlodipine, azulene, dihydropyridines, thianines, nifedine, verapamil and their derivatives (Tanaka Y, Shigenobu K. Cardiovasc Drug Rev. 2001, 19:297; Djuric D, Mitrovic V, Jakovljevic V. Arzneimittelforschung. 2002, 52:365; Kourie J I, Wood H B. Prog Biophys Mol. Biol. 2000; 73:91); and, modulators that are inhibitors of cell communication used in the context of the present invention include such compounds as beta-glycyrrhetinic acid, retinoids, TPA (Krutovskikh V A et al, Oncogene. 2002, 21:1989; Rudkin et al, J Surg Res. 2002, 103:183; Ruch J et al, J Cell Biochem. 2001, 83:163). Accordingly, the effect(s) of mAb or small molecule inhibitors are investigated using the ion flux, cell communication, proliferation and signaling assays described Examples above.
When mAb and small molecules modulate, e.g., inhibit, the transport and tumorigenic function of STEAP-1, they are used for preventative, prognostic, diagnostic and/or therapeutic purposes.
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 STEAP-1 When Malignant

















Stomach



Rectum

















TABLE II







Amino Acid Abbreviations











SINGLE LETTER
THREE LETTER
FULL NAME







F
Phe
phenylalanine



L
Leu
leucine



S
Ser
serine



Y
Tyr
tyrosine



C
Cys
cysteine



W
Trp
tryptophan



P
Pro
proline



H
His
histidine



Q
Gln
glutamine



R
Arg
arginine



I
Ile
isoleucine



M
Met
methionine



T
Thr
threonine



N
Asn
asparagine



K
Lys
lysine



V
Val
valine



A
Ala
alanine



D
Asp
aspartic acid



E
Glu
glutamic acid



G
Gly
glycine

















TABLE III







Amino Acid Substitution Matrix



























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





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.






Table IV

HLA Class I/II Mostifs/Supermotifs








TABLE IV (A)







HLA Class I Supermotifs/Motifs













POSITION



POSITION
POSITION
C Terminus



2 (Primary
3 (Primary
(Primary



Anchor)
Anchor)
Anchor)














SUPERMOTIFS





A1
TILVMS

FWY


A2
LIVMATQ

IVMATL


A3
VSMATLI

RK


A24
YFWIVLMT

FIYWLM


B7
P

VILFMWYA


B27
RHK

FYLWMIVA


B44
ED

FWYLIMVA


B58
ATS

FWYLIVMA


B62
QLIVMP

FWYMIVLA


MOTIFS


A1
TSM

Y


A1

DEAS
Y


A2.1
LMVQIAT

VLIMAT


A3
LMVISATFCGD

KYRHFA


A11
VTMLISAGNCDF

KRYH


A24
YFWM

FLIW


A*3101
MVTALIS

RK


A*3301
MVALFIST

RK


A*6801
AVTMSLI

RK


B*0702
P

LMFWYAIV


B*3501
P

LMFWYIVA


B51
P

LIVFWYAM


B*5301
P

IMFWYALV


B*5401
P

ATIVLMFWY





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













TABLE IV (B)







HLA Class II Supermotif









1
6
9





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
















TABLE IV (C)





HLA Class II Motifs
























MOTIFS
1° anchor 1
2
3
4
5
1° anchor 6
7
8
9




















DR4
preferred
FMYLIVW
M
T

I
VSTCPALIM
MH

MH



deleterious



W


R

WDE


DR1
preferred
MFLIVWY


PAMQ

VMATSPLIC
M

AVM



deleterious

C
CH
FD
CWD

GDE
D



DR7
preferred
MFLIVWY
M
W
A

IVMSACTPL
M

IV



deleterious

C

G


GRD
N
G

















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





motif a preferred

LIVMFY







motif b preferred

LIVMFAY


DNQEST

KRH


DR Supermotif

MFLIVWY




VMSTACPLI





Italicized residues indicate less preferred or “tolerated” residues













TABLE IV (D)







HLA Class I Supermotifs

















SUPER-












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





A1


1° Anchor






1° Anchor





TILVMS






FWY


A2


1° Anchor






1° Anchor





LIVMATQ






LIVMAT


A3
preferred

1° Anchor
YFW


YFW
YFW
P
1° Anchor





VSMATLI
(4/5)


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



deleterious
DE (3/5);

DE










P (5/5)

(4/5)








A24


1° Anchor






1° Anchor





YFWIVLMT






FIYWLM


B7
preferred
FWY (5/5)
1° Anchor
FWY




FWY
1° Anchor




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




(3/5)
VILFMWYA



deleterious
DE (3/5);



DE
G
QN
DE





P(5/5);



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





G(4/5);












A(3/5);












QN(3/5)










B27


1° Anchor






1° Anchor





RHK






FYLWMIVA


B44


1° Anchor






1° Anchor





ED






FWYLIMVA


B58


1° Anchor






1° Anchor





ATS






FWYLIVMA


B62


1° Anchor






1° Anchor





QLIVMP






FWYMIVLA





Italicized residues indicate less preferred or “tolerated” residues













TABLE IV (E)





HLA Class I Motifs






















POSITION:
1
2
3
4
5





A1
preferred
GFYW
1° Anchor
DEA
YFW



9-mer


STM






deleterious
DE

RHKLIVMP
A
G


A1
preferred
GRHK
ASTCLIVM
1° Anchor
GSTC



9-mer



DEAS





deleterious
A
RHKDEPYFW

DE
PQN


A1
preferred
YFW
1° Anchor
DEAQN
A
YFWQN


10-mer


STM






deleterious
GP

RHKGLIVM
DE
RHK


A1
preferred
YFW
STCLIVM
1° Anchor
A
YFW


10-mer



DEAS





deleterious
RHK
RHKDEPYFW


P


A2.1
preferred
YFW
1° Anchor
YFW
STC
YFW


9-mer


LMIVQAT






deleterious
DEP

DERKH





















9




POSITION:
6
7
8
or C-terminus
C-terminus





A1
preferred
P
DEQN
YFW
1° Anchor



9-mer

A


Y




deleterious
ASTC






A1
preferred

LIVM
DE
1° Anchor



9-mer

RHK


Y




deleterious

PG
GP




A1
preferred

PASTC
GDE
P
1° Anchor


10-mer

QNA



Y



deleterious

RHKYFW
RHK
A



A1
preferred

PG
G
YFW
1° Anchor


10-mer

G



Y



deleterious


PRHK
QN



A2.1
preferred

A
P
1° Anchor



9-mer

RKH


VLIMAT




deleterious

DERKH

















POSITION:
1
2
3
4
5





A2.1
preferred
AYFW
1° Anchor
LVIM
G



10-mer


LMIVQAT






deleterious
DEP

DE
RKHA
P


A3
preferred
RHK
1° Anchor
YFW
PRHKYFW
A





LMVISATFCGD






deleterious
DEP

DE




A11
preferred
A
1° Anchor
YFW
YFW
A





VTLMISAGNCDF






deleterious
DEP






A24
preferred
YFWRHK
1° Anchor

STC



9-mer


YFWM






deleterious
DEG

DE
G
QNP


A24
preferred

1° Anchor

P
YFWP


10-mer


YFWM






deleterious


GDE
QN
RHK


A3101
preferred
RHK
1° Anchor
YFW
P






MVTALIS






deleterious
DEP

DE

ADE


A3301
preferred

1° Anchor
YFW







MVALFIST






deleterious
GP

DE

















POSITION:
6
7
8
9
C-terminus





A2.1
preferred
G

FYWLVIM

1° Anchor


10-mer





VLIMAT



deleterious

RKH
DERKH
RKH



A3
preferred
YFW

P
1° Anchor








KYRHFA




deleterious







A11
preferred
YFW
YFW
P
1° Anchor








KRYH




deleterious

A
G




A24
preferred

YFW
YFW
1° Anchor



9-mer




FLIW




deleterious
DERHK
G
AQN




A24
preferred

P


1° Anchor


10-mer





FLIW



deleterious
DE
A
QN
DEA



A3101
preferred
YFW
YFW
AP
1° Anchor








RK




deleterious
DE
DE
DE




A3301
preferred

AYFW

1° Anchor








RK




deleterious

















POSITION:
1
2
3
4
5





A6801
preferred
YFWSTC
1° Anchor


YFWLIVM





AVTMSLI






deleterious
GP

DEG

RHK


B0702
preferred
RHKFWY
1° Anchor
RHK

RHK





P






deleterious
DEQNP

DEP
DE
DE


B3501
preferred
FWYLIVM
1° Anchor
FWY







P






deleterious
AGP



G


B51
preferred
LIVMFWY
1° Anchor
FWY
STC
FWY





P






deleterious
AGPDERHKSTC



DE


B5301
preferred
LIVMFWY
1° Anchor
FWY
STC
FWY





P






deleterious
AGPQN






B5401
preferred
FWY
1° Anchor
FWYLIVM

LIVM





P






deleterious
GPQNDE

GDESTC

RHKDE

















POSITION:
6
7
8
9
C-terminus





A6801
preferred

YFW
P
1° Anchor








RK




deleterious


A




B0702
preferred
RHK
RHK
PA
1° Anchor








LMFWYAIV




deleterious
GDE
QN
DE




B3501
preferred

FWY

1° Anchor








LMFWYIVA




deleterious
G






B51
preferred

G
FWY
1° Anchor








LIVFWYAM




deleterious
G
DEQN
GDE




B5301
preferred

LIVMFWY
FWY
1° Anchor








IMFWYALV




deleterious
G
RHKQN
DE




B5401
preferred

ALIVM
FWYAP
1° Anchor








ATIVLMFWY




deleterious
DE
QNDGE
DE





Italicized residues indicate less preferred or “tolerated” residues. The information in this Table is specific for 9-mers unless otherwise specified.














TABLE V-V1-HLA-A1







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 3; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Start
Subsequence
Score













32
TGETSMLKR
11.250



 


180
LSYPMRRSY
7.500


 


22
LEEDDYLHK
4.500


 


9
NQEELWKMK
2.700


 


80
IIASLTFLY
2.500


 


122
SITLLALVY
2.500


 


7
ITNQEELWK
2.500


 


31
DTGETSMLK
2.500


 


277
WIDIKQFVW
2.500


 


324
WEDVTKINK
2.500


 


99
ATSHQQYFY
2.500


 


244
SLTWREFHY
2.500


 


153
WLDKWMLTR
2.500


 


21
NLEEDDYLH
1.800


 


100
TSHQQYFYK
1.500


 


286
YTPPTFMIA
1.250


 


84
LTFLYTLLR
1.250


 


108
KIPILVINK
1.000


 


307
ILFLPCLRK
1.000


 


208
WIEHDVWRM
0.900


 


178
YSLSYPMRR
0.750


 


78
AAIIASLTF
0.500


 


51
HADEFDCPS
0.500


 


301
VLIFKSILF
0.500


 


148
KKFPHWLDK
0.500


 


296
FLPIVVLIF
0.500


 


215
RMEIYVSLG
0.450


 


297
LPIVVLIFK
0.250


 


247
WREFHYIQS
0.225


 


203
NKEDAWIEH
0.225


 


138
IYQLHNGTK
0.200


 


140
QLHNGTKYK
0.200


 


268
HALIFAWNK
0.200


 


236
TSIPSVSDS
0.150


 


64
TQELFPQWH
0.135


 


265
GTIHALIFA
0.125


 


174
LHAIYSLSY
0.125


 


264
LGTIHALIF
0.125


 


210
EHDVWRMEI
0.125


 


94
VIHPLATSH
0.100


 


60
ELQHTQELF
0.100


 


306
SILFLPCLR
0.100


 


240
SVSDSLTWR
0.100


 


68
FPQWHLPIK
0.100


 


116
KVLPMVSIT
0.100


 


98
LATSHQQYF
0.100


 


308
LFLPGLRKK
0.100


 


91
LREVIHPLA
0.090


 


139
VQLHNGTKY
0.075


 


241
VSDSLTWRE
0.075


 


256
KLGIVSLLL
0.050


 


125
LLALVYLPG
0.050


 


227
LAILALLAV
0.050


 


88
YTLLREVIH
0.050


 


293
IAVFLPIVV
0.050


 


223
GIVGLAILA
0.050


 


123
ITLLALVYL
0.050


 


224
IVGLAILAL
0.050


 


97
PLATSHQQY
0.050


 


170
FFAVLHAIY
0.050


 


261
SLLLGTIHA
0.050


 


63
HTQELFPQW
0.050


 


112
LVINKVLPM
0.050


 


258
GIVSLLLGT
0.050


 


171
FAVLHAIYS
0.050


 


81
IASLTFLYT
0.050


 


49
TAHADEFDC
0.050


 


135
IAAIVQLHN
0.050


 


266
TIHALIFAW
0.050


 


232
LLAVTSIPS
0.050


 


221
SLGIVGLAI
0.050


 


165
GLLSFFFAV
0.050


 


117
VLPMVSITL
0.050


 


120
MVSITLLAL
0.050


 


226
GLAILALLA
0.050


 


323
GWEDVTKIN
0.045


 


35
TSMLKRPVL
0.030


 


121
VSITLLALV
0.030


 


167
LSFFFAVLH
0.030


 


82
ASLTFLYTL
0.030


 


305
KSILFLPCL
0.030


 


34
ETSMLKRPV
0.025


 


19
RRNLEEDDY
0.025


 


129
VYLPGVIAA
0.025


 


24
EDDYLHKDT
0.025


 


182
YPMRRSYRY
0.025


 


71
WHLPIKIAA
0.025


 


285
WYTPPTFMI
0.025


 


291
FMIAVFLPI
0.025


 


163
QFGLLSFFF
0.025


 


29
HKDTGETSM
0.025


 


54
EFDCPSELQ
0.025


 


55
FDCPSELQH
0.025


 


211
HDVWRMEIY
0.025


 


161
RKQFGLLSF
0.025


 


142
HNGTKYKKF
0.025


 


10
QEELWKMKP
0.022


 


172
AVLHAIYSL
0.020


 


321
RHGWEDVTK
0.020


 


131
GVIAAIVQL
0.020

















TABLE V-V2-HLA-A1







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 5; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Start
Subsequence
Score













1
WREFHYIQV
0.225



 


3
EFHYIQVNN
0.001


 


2
REFHYIQVN
0.001


 


4
FHYIQVNNI
0.001

















TABLE V-V3-HLA-A1







9mers: STEAP-1








Each peptide is a portion of SEQ


ID NO: 7; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Start
Subsequence
Score













20
LWDPCLTRF
2.500



 


13
KSDVPESLW
0.750


 


16
VPESLWDPC
0.225


 


1
WREFHYIQI
0.225


 


19
SLWDPCLTR
0.050


 


4
FHYIQIIHK
0.050


 


18
ESLWDPCLT
0.015


 


22
DPCLTRFKG
0.013


 


25
LTRFKGLNL
0.013


 


8
QIIHKKSDV
0.010


 


6
YIQIIHKKS
0.010


 


5
HYIQIIHKK
0.010


 


21
WDPCLTRFK
0.010


 


14
SDVPESLWD
0.003


 


3
EFHYIQIIH
0.003


 


28
FKGLNLIQS
0.003


 


15
DVPESLWDP
0.001


 


24
CLTRFKGLN
0.001


 


23
PCLTRFKGL
0.001


 


2
REFHYIQII
0.001


 


12
KKSDVPESL
0.001


 


27
RFKGLNLIQ
0.000


 


7
IQIIHKKSD
0.000


 


17
PESLWDPCL
0.000


 


9
IIHKKSDVP
0.000


 


11
HKKSDVPES
0.000


 


26
TRFKGLNLI
0.000


 


10
IHKKSDVPE
0.000

















TABLE V-V4-HLA-A1







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 9; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Start
Subsequence
Score













5
GLLSLFFAV
0.050



 


8
SLFFAVLHA
0.050


 


7
LSLFFAVLH
0.030


 


3
QFGLLSLFF
0.025


 


6
LLSLFFAVL
0.020


 


2
KQFGLLSLF
0.015


 


4
FGLLSLFFA
0.013


 


1
RKQFGLLSL
0.003


 


9
LFFAVLHAI
0.001

















TABLE VI-V1-HLA-A1







10mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 3; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Start
Subsequence
Score













21
NLEEDDYLHK
90.000



 


277
WIDIKQFVWY
50.000


 


241
VSDSLTWREF
7.500


 


323
GWEDVTKINK
4.500


 


243
DSLTWREFHY
3.750


 


121
VSITLLALVY
3.750


 


99
ATSHQQYFYK
2.500


 


173
VLHAIYSLSY
2.500


 


79
AIIASLTFLY
2.500


 


215
RMEIYVSLGI
2.250


 


180
LSYPMRRSYR
1.500


 


210
EHDVWRMEIY
1.250


 


286
YTPPTFMIAV
1.250


 


31
DTGETSMLKR
1.250


 


296
FLPIVVLIFK
1.000


 


153
WLDKWMLTRK
1.000


 


98
LATSHQQYFY
1.000


 


306
SILFLPCLRK
1.000


 


6
DITNQEELWK
1.000


 


247
WREFHYIQSK
0.900


 


77
IAAIIASLTF
0.500


 


138
IVQLHNGTKY
0.500


 


300
VVLIFKSILF
0.500


 


263
LLGTIHALIF
0.500


 


107
YKIPILVINK
0.300


 


116
KVLPMVSITL
0.500


 


83
SLTFLYTLLR
0.500


 


179
SLSYPMRRSY
0.500


 


66
ELFPQWHLPI
0.500


 


10
QEELWKMKPR
0.450


 


140
QLHNGTKYKK
0.400


 


310
LPCLRKKILK
0.250


 


295
VFLPIVVLIF
0.250


 


91
LREVIHPLAT
0.225


 


195
WAYQQVQQNK
0.200


 


307
ILFLPCLRKK
0.200


 


176
AIYSLSYPMR
0.200


 


137
AIVQLHNGTK
0.200


 


305
KSILFLPCLR
0.150


 


236
TSIPSVSDSL
0.150


 


64
TQELFPQWHL
0.135


 


58
PSELQHTQEL
0.135


 


48
QTAHADEFDC
0.125


 


96
HPLATSHQQY
0.125


 


54
EFDCPSELQH
0.125


 


265
GTIHALIFAW
0.125


 


94
VIHPLATSHQ
0.100


 


51
HADEFDCPSE
0.100


 


208
WIEHDVWRME
0.090


 


167
LSFFFAVLHA
0.075


 


162
KQFGLLSFFF
0.075


 


260
VSLLLGTIHA
0.075


 


220
VSLGIVGLAI
0.075


 


9
NQEELWKMKP
0.068


 


80
IIASLTFLYT
0.050


 


226
GLAILALLAV
0.050


 


292
MIAVFLPIVV
0.050


 


223
GIVGLAILAL
0.050


 


169
FFFAVLHAIY
0.050


 


34
ETSMLKRPVL
0.050


 


30
KDTGETSMLK
0.050


 


217
EIYVSLGIVG
0.050


 


124
TLLALVYLPG
0.050


 


8
TNQEELWKMK
0.050


 


252
YIQSKLGIVS
0.050


 


111
ILVINKVLPM
0.050


 


256
KLGIVSLLLG
0.050


 


301
VLIFKSILFL
0.050


 


231
ALLAVTSIPS
0.050


 


134
VIAAIVQLHN
0.050


 


128
LVYLPGVIAA
0.050


 


287
TPPTFMIAVF
0.050


 


181
SYPMRRSYRY
0.050


 


52
ADEFDCPSEL
0.045


 


139
VQLHNGTKYK
0.030


 


100
TSHQQYFYKI
0.030


 


35
TSMLKRPVLL
0.030


 


7
ITNQEELWKM
0.025


 


4
RKDITNQEEL
0.025


 


29
HKDTGETSML
0.025


 


204
KEDAWIEHDV
0.025


 


235
VTSIPSVSDS
0.025


 


23
EEDDYLHKDT
0.025


 


285
WYTPPTFMIA
0.025


 


152
HWLDKWMLTR
0.025


 


157
WMLTRKQFGL
0.025


 


24
EDDYLHKDTG
0.025


 


324
WEDVTKINKT
0.025


 


177
IYSLSYPMRR
0.025


 


63
HTQELFPQWH
0.025


 


32
TGETSMLKRP
0.022


 


232
LLAVTSIPSV
0.020


 


43
LLHLHQTAHA
0.020


 


227
LAILALLAVT
0.020


 


130
YLPGVIAAIV
0.020


 


262
LLLGTIHALI
0.020


 


294
AVFLPIVVLI
0.020


 


224
IVGLAILALL
0.020


 


166
LLSFFFAVLH
0.020


 


127
ALVYLPGVIA
0.020

















TABLE VI-V2-HLA-A1







10mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 5; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Start
Subsequence
Score













2
WREFHYIQVN
0.045



 


3
REFHYIQVNN
0.001


 


4
EFHYIQVNNI
0.001


 


1
TWREFHYIQV
0.000

















TABLE VI-V3-HLA-A1







10mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 7; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Start
Subsequence
Score













21
LWDPCLTRFK
5.000



 


19
ESLWDPCLTR
0.750


 


17
VPESLWDPCL
0.450


 


14
KSDVPESLWD
0.375


 


4
EFHYIQIIHK
0.050


 


2
WREFHYIQII
0.045


 


16
DVPESLWDPC
0.010


 


5
FHYIQIIHKK
0.010


 


20
SLWDPCLTRF
0.010


 


25
CLTRFKGLNL
0.005


 


3
REFHYIQIIH
0.003


 


22
WDPCLTRFKG
0.003


 


26
LTRFKGLNLI
0.003


 


23
DPCLTRFKGL
0.003


 


8
IQIIHKKSDV
0.002


 


24
PCLTRFKGLN
0.001


 


9
QIIHKKSDVP
0.001


 


7
YIQIIHKKSD
0.001


 


15
SDVPESLWDP
0.001


 


6
HYIQIIHKKS
0.001


 


13
KKSDVPESLW
0.001


 


1
TWREFHYIQI
0.000


 


28
RFKGLNLIQS
0.000


 


27
TRFKGLNLIQ
0.000


 


10
IIHKKSDVPE
0.000


 


12
HKKSDVPESL
0.000


 


18
PESLWDPCLT
0.000


 


11
IHKKSDVPES
0.000

















TABLE VI-V4-HLA-A1







10mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 9; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Start
Subsequence
Score













8
LSLFFAVLHA
0.075



 


3
KQFGLLSLFF
0.075


 


10
LFFAVLHAIY
0.050


 


6
GLLSLFFAVL
0.020


 


7
LLSLFFAVLH
0.020


 


5
FGLLSLFFAV
0.013


 


9
SLFFAVLHAI
0.010


 


2
RKQFGLLSLF
0.005


 


4
QFGLLSLFFA
0.003


 


1
TRKQFGLLSL
0.000

















TABLE VII-V1-HLA-A2







9mers: STEAP-1



Each peptide is a portion of SEQ ID


NO: 3; each start position is


specified, the length of peptide is 9


amino acids, and the end position


for each peptide is the start position


plus eight.









Start
Subsequence
Score













165
GLLSFFFAV
10776.470



 


86
FLYTLLREV
470.951


 


262
LLLGTIHAL
309.050


 


302
LIFKSILFL
233.719


 


158
MLTRKQFGL
210.633


 


192
LLNWAYQQV
177.358


 


130
YLPGVIAAI
110.379


 


166
LLSFFFAVL
107.503


 


256
KLGIVSLLL
74.768


 


291
FMIAVFLPI
65.854


 


292
MIAVFLPIV
58.258


 


36
SMLKRPVLL
57.085


 


270
LIFAWNKWI
49.627


 


309
FLPCLRKKI
47.991


 


113
VINKVLPMV
37.393


 


79
AIIASLTFL
37.157


 


117
VLPMVSITL
36.316


 


164
FGLLSFFFA
33.454


 


83
SLTFLYTLL
32.044


 


42
VLLHLHQTA
31.249


 


90
LLREVIHPL
26.027


 


76
KIAAIIASL
23.646


 


123
ITLLALVYL
23.608


 


245
LTWREFHYI
21.365


 


212
DVWRMEIYV
20.836


 


219
YVSLGIVGL
19.776


 


261
SLLLGTIHA
18.382


 


300
VVLIFKSIL
18.318


 


263
LLGTIHALI
17.736


 


172
AVLHAIYSL
15.907


 


126
LALVYLPGV
13.975


 


65
QELFPQWHL
11.527


 


221
SLGIVGLAI
10.433


 


272
FAWNKWIDI
9.405


 


116
KVLPMVSIT
7.963


 


120
MVSITLLAL
7.309


 


224
IVGLAILAL
7.309


 


294
AVFLPTVVL
6.522


 


201
QQNKEDAWI
5.818


 


20
RNLEEDDYL
5.701


 


252
YIQSKLGIV
5.393


 


157
WMLTRKQFG
5.038


 


226
GLAILALLA
4.968


 


82
ASLTFLYTL
4.510


 


27
YLHKDTGET
4.456


 


133
GVIAAIVQL
4.299


 


305
KSILFLPCL
4.266


 


287
TPPTFMIAV
3.968


 


233
LAVTSIPSV
3.777


 


227
LAILALLAV
3.777


 


228
AILALLAVT
3.575


 


208
WIEHDVWRM
3.469


 


103
QQYFYKIPI
3.385


 


299
IVVLIFKSI
2.949


 


258
GIVSLLLGT
2.357


 


8
TNQEELWKM
2.294


 


176
AIYSLSYPM
2.186


 


319
KIRHGWEDV
2.119


 


230
LALLAVTSI
2.096


 


118
LPMVSITLL
2.017


 


225
VGLAILALL
2.017


 


81
IASLTFLYT
1.839


 


312
CLRKKILKI
1.824


 


109
IPILVINKV
1.775


 


121
VSITLLALV
1.775


 


112
LVINKVLPM
1.700


 


59
SELQHTQEL
1.537


 


237
SIPSVSDSL
1.439


 


162
KQFGLLSFF
1.238


 


137
AIVQLHNGT
1.025


 


279
DIKQFVWYT
0.970


 


128
LVYLPGVIA
0.970


 


265
GTIHALIFA
0.873


 


150
FPHWLDKWM
0.820


 


72
HLPIKIAAI
0.816


 


276
KWIDIKQFV
0.762


 


105
YFYKIPILV
0.711


 


223
GIVGLAILA
0.683


 


293
IAVFLPIVV
0.673


 


127
ALVYLPGVI
0.556


 


53
DEFDCPSEL
0.473


 


259
IVSLLLGTI
0.464


 


244
SLTWREFHY
0.432


 


49
TAHADEFDC
0.400


 


216
MEIYVSLGI
0.345


 


296
FLPTVVLIF
0.323


 


131
LPGVIAAIV
0.316


 


214
WRMEIYVSL
0.311


 


169
FFFAVLHAI
0.284


 


217
EIYVSLGIV
0.281


 


255
SKLGIVSLL
0.252


 


283
FVWYTPPTF
0.249


 


286
YTPPTFMIA
0.247


 


222
LGIVGLAIL
0.224


 


199
QVQQNKEDA
0.213


 


35
TSMLKRPVL
0.212


 


69
PQWHLPIKI
0.193


 


191
KLLNWAYQQ
0.193


 


173
VLHAIYSLS
0.190


 


125
LLALVYLPG
0.190

















TABLE VII-V2-HLA-A2







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 5; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Start
Subsequence
Score













4
FHYIQVNNI
0.245



 


2
REFHYIQVN
0.005


 


1
WREFHYIQV
0.004


 


3
EFHYIQVNN
0.000

















TABLE VII-V3-HLA-A2







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 7; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Start
Subsequence
Score













8
QIIRKKSDV
9.563



 


2
REFHYIQII
0.708


 


19
SLWDPCLTR
0.507


 


12
KKSDVPESL
0.209


 


6
YIQIIHKKS
0.047


 


25
LTRFKGLNL
0.038


 


24
CLTRFKGLN
0.037


 


23
PCLTRFKGL
0.022


 


18
ESLWDPCLT
0.013


 


26
TRFKGLNLI
0.012


 


17
PESLWDPCL
0.009


 


15
DVPESLWDP
0.007


 


16
VPESLWDPC
0.003


 


7
IQIIHKKSD
0.003


 


28
FKGLNLIQS
0.001


 


9
IIHKKSDVP
0.001


 


13
KSDVPESLW
0.001


 


1
WREFHYIQI
0.001


 


4
FHYIQIIHK
0.000


 


22
DPCLTRFKG
0.000


 


14
SDVPESLWD
0.000


 


21
WDPCLTRFK
0.000


 


20
LWDPCLTRF
0.000


 


3
EFHYIQIIH
0.000


 


10
IHKKSDVPE
0.000


 


11
HKKSDVPES
0.000


 


5
HYIQIIHKK
0.000


 


27
RFKGLNLIQ
0.000

















TABLE VII-V4-HLA-A2







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 9; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Start
Subsequence
Score













5
GLLSLFFAV
2835.913



 


6
LLSLFFAVL
107.503


 


8
SLFFAVLHA
42.278


 


4
FGLLSLFFA
40.497


 


2
KQFGLLSLF
0.225


 


9
LFFAVLHAI
0.105


 


1
RKQFGLLSL
0.068


 


7
LSLFFAVLH
0.012


 


3
QFGLLSLFF
0.000

















TABLE VIII-V1-HLA-A2







10mers: STEAP-1



Each peptide is a portion of SEQ ID


NO: 3; each start position is


specified, the length of peptide is 10


amino acids, and the end position for


each peptide is the start position plus


nine.









Start
Subsequence
Score













157
WMLTRKQFGL
1523.030



 


191
KLLNWAYQQV
1351.048


 


291
FMIAVFLPIV
828.006


 


301
VLIFKSILFL
459.398


 


164
FGLLSFFFAV
323.593


 


125
LLALVYLPGV
271.948


 


232
LLAVTSIPSV
271.948


 


165
GLLSFFFAVL
233.977


 


261
SLLLGTIHAL
181.794


 


89
TLLREVIHPL
181.794


 


226
GLAILALLAV
159.970


 


130
YLPGVLAAIV
131.175


 


244
SLTWREFHYI
109.023


 


283
FVWYTPPTFM
91.283


 


117
VLPMVSITLL
83.527


 


108
KIPILVINKV
76.985


 


262
LLLGTIHALI
65.622


 


269
ALIFAWNKWI
57.380


 


229
ILALLAVTSI
40.792


 


309
FLPCLRKKIL
40.289


 


286
YTPPTFMIAV
38.837


 


122
SITLLALVYL
37.157


 


86
FLYTLLREVI
30.852


 


116
KVLPMVSITL
24.206


 


120
MVSITLLALV
23.795


 


112
LVINKVLPMV
23.795


 


221
SLGIVGLAIL
20.145


 


111
ILVINKVLPM
19.425


 


80
IIASLTFLYT
18.203


 


200
VQQNKEDAWI
9.890


 


281
KQFVWYTPPT
9.784


 


158
MLTRKQFGLL
8.970


 


253
IQSKLGIVSL
8.469


 


43
LLHLHQTAHA
8.446


 


294
AVFLPIVVLI
7.769


 


224
IVGLAILALL
7.309


 


223
GIVGLAILAL
6.756


 


292
MIAVFLPIVV
6.666


 


103
QQYFYKIPIL
6.536


 


278
IDIKQFVWYT
6.343


 


128
LVYLPGVIAA
5.439


 


299
IVVLIFKSIL
4.951


 


150
FPHWLDKWML
4.811


 


275
NKWIDIKQFV
4.410


 


81
IASLTFLYTL
4.408


 


78
AAIIASLTFL
3.753


 


76
KIAAIIASLT
3.586


 


171
FAVLHAIYSL
3.139


 


162
KQFGLLSFFF
3.121


 


37
MLKRPVLLHL
2.923


 


66
ELFPQWHLPI
2.895


 


90
LLREVIHPLA
2.632


 


127
ALVYLPGVIA
2.037


 


7
ITNQEELWKM
2.023


 


145
TKYKKFPHWL
1.984


 


204
KEDAWIEHDV
1.893


 


248
REFHYIQSKL
1.537


 


209
IEHDVWRMEI
1.414


 


304
FKSILFLPCL
1.193


 


64
TQELFPQWHL
1.040


 


237
SIPSVSDSLT
1.025


 


296
FLPIVVLIFK
0.867


 


119
PMVSITLLAL
0.781


 


82
ASLTFLYTLL
0.774


 


264
LGTIHALIFA
0.697


 


324
WEDVTKINKT
0.613


 


256
KLGIVSLLLG
0.600


 


219
YVSLGIVGLA
0.598


 


35
TSMLKRPVLL
0.516


 


215
RMEIYVSLGI
0.482


 


166
LLSFFFAVLH
0.481


 


293
IAVFLPIVVL
0.476


 


167
LSFFFAVLHA
0.469


 


42
VLLHLHQTAH
0.469


 


36
SMLKRPVLLH
0.458


 


258
GIVSLLLGTI
0.429


 


220
VSLGIVGLAI
0.428


 


124
TLLALVYLPG
0.414


 


48
QTAHADEFDC
0.400


 


68
FPQWHLPIKI
0.313


 


71
WHLPIKIAAI
0.284


 


216
MEIYVSLGIV
0.282


 


231
ALLAVTSIPS
0.276


 


33
GETSMLKRPV
0.270


 


198
QQVQQNKEDA
0.247


 


163
QFGLLSFFFA
0.209


 


260
VSLLLGTIHA
0.204


 


225
VGLAILALLA
0.204


 


118
LPMVSITLLA
0.204


 


277
WIDIKQFVWY
0.197


 


257
LGIVSLLLGT
0.190


 


302
LIFKSILFLP
0.172


 


27
YLHKDTGETS
0.168


 


227
LAILALLAVT
0.166


 


72
HLPIKIAAII
0.145


 


318
LKIRHGWEDV
0.137


 


100
TSHQQYFYKI
0.129


 


173
VLHAIYSLSY
0.127


 


75
IKIAAIIASL
0.116


 


40
RPVLLHLHQT
0.112

















TABLE VIII-V2-HLA-A2







10mers: STEAP-1



Each peptide is a portion of SEQ ID


NO: 5; each start position is


specified, the length of peptide is 10


amino acids, and the end position


for each peptide is the start position


plus nine.









Start
Subsequence
Score













3
REFHYIQVNN
0.005



 


1
TWREFHYIQV
0.005


 


4
EFHYIQVNNI
0.001


 


2
WREFHYIQVN
0.000

















TABLE VIII-V3-HLA-A2







10mers: STEAP-1



Each peptide is a portion of SEQ ID


NO: 7; each start position is


specified, the length of peptide is 10


amino acids, and the end position


for each peptide is the start position


plus nine.









Start
Subsequence
Score













25
CLTRFKGLNL
21.362



 


8
IQIIHKKSDV
11.988


 


20
SLWDPCLTRF
5.833


 


16
DVPESLWDPC
0.111


 


26
LTRFKGLNLI
0.043


 


23
DPCLTRFKGL
0.020


 


17
VPESLWDPCL
0.015


 


7
YIQIIHKKSD
0.009


 


10
IIHKKSDVPE
0.003


 


18
PESLWDPCLT
0.003


 


22
WDPCLTREKG
0.003


 


3
REFHYIQIIH
0.002


 


13
KKSDVPESLW
0.001


 


14
KSDVPESLWD
0.001


 


1
TWREFHYIQI
0.001


 


9
QIIHKKSDVP
0.000


 


2
WREFHYIQII
0.000


 


15
SDVPESLWDP
0.000


 


5
FHYIQIIHKK
0.000


 


19
ESLWDPCLTR
0.000


 


24
PCLTRFKGLN
0.000


 


12
HKKSDVPESL
0.000


 


11
IHKKSDVPES
0.000


 


28
RFKGLNLIQS
0.000


 


27
TRFKGLNLIQ
0.000


 


4
EFHYIQIIHK
0.000


 


21
LWDPCLTRFK
0.000


 


6
HYIQIIHKKS
0.000

















TABLE VIII-V4-HLA-A2







10mers: STEAP-1



Each peptide is a portion of SEQ ID


NO: 9; each start position is


specified, the length of peptide is 10


amino acids, and the end position


for each peptide is the start position


plus nine.









Start
Subsequence
Score












6
GLLSLFFAVL
233.977


 


9
SLFFAVLHAI
88.783


 


5
FGLLSLFFAV
85.156


 


3
KQFGLLSLFF
0.538


 


7
LLSLFFAVLH
0.481


 


8
LSLFFAVLHA
0.469


 


4
QFGLLSLFFA
0.253


 


1
TRKQFGLLSL
0.000


 


2
RKQFGLLSLF
0.000


 


10
LFFAVLHAIY
0.000

















TABLE IX-VI-A3







9mers: STEAP-1



Each peptide is a portion of SEQ ID


NO: 3; each start position is


specified, the length of peptide is 9


amino acids, and the end position


for each peptide is the start position


plus eight.









Start
Subsequence
Score













307
ILFLPCLRK
300.000



 


153
WLDKWMLTR
36.000


 


108
KIPILVINK
36.000


 


165
GLLSFFFAV
24.300


 


296
FLPIVVLIF
18.000


 


244
SLTWREFHY
12.000


 


140
QLHNGTKYK
10.000


 


291
FMIAVFLPI
8.100


 


301
VLIFKSILF
6.000


 


84
LTFLYTLLR
6.000


 


12
ELWKMKPRR
6.000


 


256
KLGIVSLLL
5.400


 


166
LLSFFFAVL
5.400


 


268
HALIFAWNK
2.700


 


90
LLREVIHPL
2.025


 


162
KQFGLLSFF
2.025


 


262
LLLGTIHAL
2.025


 


130
YLPGVIAAI
2.025


 


7
ITNQEELWK
2.000


 


37
MLKRPVLLH
1.800


 


221
SLGIVGLAI
1.800


 


158
MLTRKQFGL
1.800


 


117
VLPMVSITL
1.800


 


312
CLRKKILKI
1.800


 


248
REFHYIQSK
1.350


 


297
LPIVVLIFK
1.350


 


226
GLAILALLA
1.200


 


80
IIASLTFLY
1.200


 


283
FVWYTPPTF
1.000


 


148
KKFPHWLDK
0.900


 


31
DTGETSMLK
0.900


 


72
HLPIKIAAI
0.900


 


21
NLEEDDYLH
0.900


 


263
LLGTIHALI
0.900


 


302
LIFKSILFL
0.900


 


36
SMLKRPVLL
0.900


 


191
KLLNWAYQQ
0.810


 


122
SITLLALVY
0.800


 


294
AVFLPIVVL
0.675


 


60
ELQHTQELF
0.600


 


306
SILFLPCLR
0.600


 


261
SLLLGTIHA
0.600


 


138
IVQLHNGTK
0.600


 


99
ATSHQQYFY
0.600


 


240
SVSDSLTWR
0.600


 


83
SLTFLYTLL
0.600


 


103
QYYFYKIPI
0.540


 


9
NQEELWKMK
0.450


 


270
LIFAWNKWI
0.450


 


245
LTWREFHYI
0.450


 


76
KIAAIIASL
0.405


 


172
AVLHAIYSL
0.405


 


133
GVIAAIVQL
0.405


 


97
PLATSHQQY
0.400


 


176
AIYSLSYPM
0.300


 


68
FPQWHLPIK
0.300


 


100
TSHQQYFYK
0.300


 


309
FLPCLRKKI
0.300


 


269
ALIFAWNKW
0.300


 


42
VLLHLHQTA
0.300


 


127
ALVYLPGVI
0.270


 


300
VVLIFKSIL
0.270


 


124
TLLALVYLP
0.270


 


223
GIVGLAILA
0.270


 


299
IVVLIFKSI
0.203


 


116
KVLPMVSIT
0.203


 


43
LLHLHQTAH
0.200


 


183
PMRRSYRYK
0.200


 


192
LLNWAYQQV
0.200


 


178
YSLSYPMRR
0.180


 


139
VQLHNGTKY
0.180


 


224
IVGLAILAL
0.180


 


120
MVSITLLAL
0.180


 


182
YPMRRSYRY
0.180


 


208
WIEHDVWRM
0.180


 


112
LVINKVLPM
0.180


 


89
TLLREVIHP
0.180


 


215
RMEIYVSLG
0.180


 


128
LVYLPGVIA
0.150


 


86
FKYTLLREV
0.150


 


258
GIVSLLLGT
0.135


 


265
GTIHALIFA
0.135


 


316
KILKIRHGW
0.135


 


79
AILASLTFL
0.135


 


324
WEDVTKINK
0.120


 


22
LEEDDYLHK
0.120


 


273
AWNKWIDIK
0.090


 


141
LHNGTKYKK
0.090


 


237
SIPSVSDSL
0.090


 


292
MIAVFLPTV
0.090


 


286
YTPPTFMIA
0.090


 


119
PMVSITLLA
0.090


 


219
YVSLGIVGL
0.090


 


266
TIHALIFAW
0.090


 


134
VIAAIVQLH
0.090


 


15
KMKPRRNLE
0.090


 


144
GTKYKKFPH
0.090


 


279
DIKQFVWYT
0.081


 


232
LLAVTSIPS
0.080


 


123
ITLLALVYL
0.068

















TABLE IX-V2-HLA-A3







9mers: STEAP-1



Each peptide is a portion of SEQ ID


NO: 5; each start position is


specified, the length of peptide is 9


amino acids, and the end position


for each peptide is the start position


plus eight.









Start
Subsequence
Score












4
FHYIQVNNI
0.009


 


2
REFHYIQVN
0.001


 


1
WREFHYIQV
0.000


 


3
EFHYIQVNN
0.000

















TABLE IX-V3-HLA-A3







9mers: STEAP-1



Each peptide is a portion of SEQ ID


NO: 7; each start position is


specified, the length of peptide is 9


amino acids, and the end position


for each peptide is the start position


plus eight.









Start
Subsequence
Score













19
SLWDPCLTR
60.000



 


4
FHYIQIIHK
0.300


 


5
HYIQIIHKK
0.068


 


25
LTRFKGLNL
0.060


 


2
REFHYIQII
0.041


 


8
QIIHKKSDV
0.030


 


24
CLTRFKGLN
0.018


 


20
LWDPCLTRF
0.005


 


13
KSDVPESLW
0.005


 


26
TRFKGLNLI
0.005


 


15
DVPESLWDP
0.004


 


6
YIQIIHKKS
0.003


 


12
KKSDVPESL
0.003


 


21
WDPCLTRFK
0.002


 


16
VPESLWDPC
0.002


 


9
IIHKKSDVP
0.002


 


23
PCLTRFKGL
0.001


 


1
WREFHYIQI
0.001


 


17
PESLWDPCL
0.001


 


7
IQIIHKKSD
0.000


 


18
ESLWDPCLT
0.000


 


22
DPCLTRFKG
0.000


 


3
EFHYIQIIH
0.000


 


28
FKGLNLIQS
0.000


 


11
HKKSDVPES
0.000


 


14
SDVPESLWD
0.000


 


27
RFKGLNLIQ
0.000


 


10
IHKKSDVPE
0.000

















TABLE IX-V4-HLA-A3







9mers: STEAP-1



Each peptide is a portion of SEQ ID


NO: 9; each start position is


specified, the length of peptide is 9


amino acids, and the end position


for each peptide is the start position


plus eight.









Start
Subsequence
Score












5
GLLSLFFAV
8.100


 


8
SLFFAVLHA
6.000


 


6
LLSLFFAVL
5.400


 


2
KQFGLLSLF
2.025


 


9
LFFAVLHAI
0.007


 


7
LSLFFAVLH
0.005


 


3
QFGLLSLFF
0.004


 


1
RKQFGLLSL
0.002


 


4
FGLLSLFFA
0.001

















TABLE X-V1-HLA-A3







10mers: STEAP-1



Each peptide is a portion of SEQ ID


NO: 3; each start position is


specified, the length of peptide is 10


amino acids, and the end position


for each peptide is the start position


plus nine.









Start
Subsequence
Score













296
FLPIVVLIFK
90.000



 


140
QLHNGTKYKK
60.000


 


21
NLEEDDYLHK
60.000


 


153
WLDKWMLTRK
30.000


 


165
GLLSFFFAVL
24.300


 


83
SLTFLYTLLR
24.000


 


173
VLHAIYSLSY
24.000


 


307
ILFLPCLRKK
15.000


 


306
SILFLPCLRK
9.000


 


66
ELFPQWHLPI
8.100


 


312
CLRKKILKIR
4.500


 


272
FAWNKWIDIK
4.500


 


162
KQFGLLSFFF
4.050


 


263
LLGTIHALIF
4.000


 


99
ATSHQQYFYK
3.000


 


86
FLYTLLREVI
3.000


 


157
WMLTRKQFGL
2.700


 


37
MLKRPVLLHL
2.700


 


36
SMLKRPVLLH
2.700


 


301
VLIFKSILFL
2.700


 


262
LLLGTIHALI
2.025


 


89
TLLREVIHPL
2.025


 


176
AIYSLSYPMR
2.000


 


229
ILALLAVTSI
1.800


 


79
AIIASLTFLY
1.800


 


226
GLAILALLAV
1.800


 


244
SLTWREFHYI
1.800


 


111
ILVINKVLPM
1.800


 


215
RMEIYVSLGI
1.800


 


195
WAYQQVQQNK
1.500


 


261
SLLLGTIHAL
1.350


 


294
AVFLPIVVLI
1.350


 


269
ALIFAWNKWI
1.350


 


291
FMIAVFLPIV
1.350


 


116
KVLPMVSITL
1.215


 


223
GIVGLAILAL
1.215


 


6
DITNQEELWK
1.200


 


277
WIDIKQFVWY
1.200


 


117
VLPMVSITLL
0.900


 


137
AIVQLHNGTK
0.900


 


72
HLPIKIAAII
0.900


 


221
SLGIVGLAIL
0.900


 


125
LLALVYLPGV
0.900


 


191
KLLNWAYQQV
0.900


 


128
LVYLPGVIAA
0.600


 


300
VVLIFKSILF
0.600


 


166
LLSFFFAVLH
0.600


 


183
PMRRSYRYKL
0.540


 


158
MLTRKQFGLL
0.540


 


130
YLPGVIAAIV
0.450


 


139
VQLHNGTKYK
0.450


 


90
LLREVIHPLA
0.450


 


127
ALVYLPGVIA
0.450


 


133
GVIAAIVQLH
0.405


 


138
IVQLHNGTKY
0.400


 


179
SLSYPMRRSY
0.400


 


310
LPCLRKKILK
0.400


 


42
VLLHLHQTAH
0.300


 


309
FLPCLRKKIL
0.300


 


232
LLAVTSIPSV
0.300


 


31
DTGETSMLKR
0.270


 


107
YKIPILVINK
0.270


 


119
PMVSITLLAL
0.270


 


299
IVVLIFKSIL
0.270


 


265
GTIHALIFAW
0.203


 


97
PLATSHQQYF
0.200


 


43
LLHLHQTAHA
0.200


 


267
IHALIFAWNK
0.180


 


30
KDTGETSMLK
0.180


 


256
KLGIVSLLLG
0.180


 


15
KMKPRRNLEE
0.180


 


283
FVWYTPPTFM
0.150


 


180
LSYPMRRSYR
0.150


 


224
IVGLAILALL
0.135


 


103
QQYFYKIPIL
0.135


 


281
KQFVWYTPPT
0.135


 


286
YTPPTFMIAV
0.135


 


108
KIPILVINKV
0.135


 


258
GIVSLLLGTI
0.121


 


323
GWEDVTKINK
0.120


 


231
ALLAVTSIPS
0.120


 


98
LATSHQQYFY
0.120


 


102
HQQYFYKIPI
0.108


 


247
WREFHYIQSK
0.090


 


319
KIRHGWEDVT
0.090


 


305
KSILFLPCLR
0.090


 


124
TLLALVYLPG
0.090


 


122
SITLLALVYL
0.090


 


144
GTKYLLFPHW
0.090


 


64
TQELFPQWHL
0.081


 


81
IASLTFLYTL
0.081


 


100
TSHQQYFYKI
0.081


 


112
LVINKVLPMV
0.068


 


302
LIFKSILFLP
0.068


 


96
HPLATSHQQY
0.060


 


192
LLNWAYQQVQ
0.060


 


206
DAWIEHHDVR
0.060


 


80
IIASLTFLYT
0.060


 


317
ILKIRHGWED
0.060


 


188
YRYKLLNWAY
0.060

















TABLE X-V2-HLA-A3







10mers: STEAP-1



Each peptide is a portion of SEQ ID


NO: 5; each start position is


specified, the length of peptide is 10


amino acids, and the end position


for each peptide is the start position


plus nine.









Start
Subsequence
Score













1
TWREFHYIQV
0.001



 


4
EFHYIQVNNI
0.001


 


3
REFHYIQVNN
0.000


 


2
WREFHYIQVN
0.000

















TABLE X-V3-HLA-A3







10mers: STEAP-1



Each peptide is a portion of SEQ ID


NO: 7; each start position is


specified, the length of peptide is 10


amino acids, and the end position


for each peptide is the start position


plus nine.









Start
Subsequence
Score













20
SLWDPCLTRF
15.000



 


25
CLTRFKGLNL
1.200


 


5
FHYIQIIHKK
0.225


 


26
LTRFKGLNLI
0.045


 


19
ESLWDPCLTR
0.027


 


17
VPESLWDPCL
0.018


 


4
EFHYIQIIHX
0.018


 


8
IQIIHKKSDV
0.009


 


3
REFHYIQIIH
0.009


 


16
DVPESLWDPC
0.009


 


21
LWDPCLTRFK
0.003


 


9
QIIHKKSDVP
0.003


 


2
WREFHYIQII
0.003


 


23
DPCLTRFKGL
0.003


 


10
IIHKKSDVPE
0.002


 


1
TWREFHYIQI
0.002


 


7
YIQIIHKKSD
0.001


 


13
KKSDVPESLW
0.001


 


12
HKKSDVPESL
0.001


 


14
KSDVPESLWD
0.001


 


15
SDVPESLWDP
0.000


 


27
TRFKGLNLIQ
0.000


 


28
RFKGLNLIQS
0.000


 


11
IHKKSDVPES
0.000


 


22
WDPCLTRFKG
0.000


 


6
HYIQIIHKKS
0.000


 


18
PESLWDPCLT
0.000


 


24
PCLTRFKGLN
0.000

















TABLE X-V4-HLA-A3







10mers: STEAP-1



Each peptide is a portion of SEQ ID


NO: 9; each start position is


specified, the length of peptide is 10


amino acids, and the end position


for each peptide is the start position


plus nine.









Start
Subsequence
Score













6
GLLSLFEAVL
24.300



 


9
SLFFAVLHAI
6.750


 


3
KQFGLLSLFF
2.700


 


7
LLSLFFAVLH
0.600


 


10
LFFAVLHAIY
0.020


 


8
LSLFFAVLHA
0.009


 


2
RKQFGLLSLF
0.003


 


5
FGLLSLFFAV
0.003


 


1
TRKQFGLLSL
0.002


 


4
QFGLLSLFFA
0.001

















TABLE XI-V1-HLA-A11







9mers: STEAP-1



Each peptide is a portion of SEQ ID


NO: 3; each start position is


specified, the length of peptide is 9


amino acids, and the end position


for each peptide is the start position


plus eight.









Start
Subsequence
Score













108
KIPILVINK
2.400



 


138
IVQLHNGTK
2.000


 


7
ITNQEELWK
2.000


 


307
LLFLPCLRK
1.600


 


84
LTFLYTLLR
0.800


 


268
HALIFAWNK
0.600


 


297
LPIVVLLFK
0.600


 


31
DTGETSMLK
0.600


 


196
AYQQVQQNK
0.400


 


240
SVSDSLTWR
0.400


 


248
REFHYIQSK
0.360


 


9
NQEELWKMK
0.300


 


148
KKFPHWLDK
0.240


 


140
QLHNGTKYK
0.200


 


68
FPQWHLPIK
0.200


 


153
WLDKWMLTR
0.160


 


22
LEEDDYLHK
0.120


 


306
SILFLPCLR
0.120


 


324
WEDVTKINK
0.120


 


144
GTKYKKFPH
0.090


 


133
GVIAAIVQL
0.090


 


265
GTIHALIFA
0.090


 


177
IYSLSYPMR
0.080


 


181
SYPMRRSYR
0.080


 


321
RHGWEDVTK
0.060


 


311
PCLRKKILK
0.060


 


100
TSHQQYFYK
0.060


 


172
AVLHAIYSL
0.060


 


112
LV1NKVLPM
0.060


 


165
GLLSFFFAV
0.054


 


12
ELWKMKPRR
0.048


 


294
AVFLP1VVL
0.040


 


141
LHNGTKYKK
0.040


 


128
LVYLPGVIA
0.040


 


283
FVWYTPPTF
0.040


 


120
MVSITLLAL
0.040


 


224
LYGLAILAL
0.040


 


162
KQFGLLSFF
0.036


 


223
GIVGLAILA
0.036


 


308
LFLPCLRKK
0.030


 


300
VVLIFKSIL
0.030


 


189
RYKLLNWAY
0.024


 


212
DVWRMEIYV
0.024


 


103
QQYFYKIPI
0.024


 


256
KLGIVSLLL
0.024


 


226
GLAILALLA
0.024


 


219
YVSLGIVGL
0.020


 


154
LDKWMLTRK
0.020


 


273
AWNKWIDIK
0.020


 


259
IVSLLLGTI
0.020


 


99
ATSHQQYFY
0.020


 


286
YTPPTFMIA
0.020


 


199
QVQQNKEDA
0.020


 


245
LTWREFHYI
0.020


 


316
KILKIRHGW
0.018


 


302
LIFKSILFL
0.016


 


176
AIYSLSYPM
0.016


 


299
IVVLIFKSI
0.015


 


123
ITLLALVYL
0.015


 


88
YTLLREVIH
0.015


 


178
YSLSYPMRR
0.012


 


244
SLTWREFHY
0.012


 


291
FMIAVFLPI
0.012


 


80
IIASLTFLY
0.012


 


277
WIDIKQFVW
0.012


 


319
KIRHGWEDV
0.012


 


146
KYKKFPHWL
0.012


 


285
WYTPPTFMI
0.012


 


266
TIHALIFAW
0.012


 


261
SLLLGTIHA
0.012


 


301
VLIFKSILF
0.012


 


76
KIAAIIASL
0.012


 


129
VYLPGVIAA
0.012


 


158
MLTRKQFGL
0.012


 


251
HYIQSKLGI
0.012


 


327
VTKINKTEI
0.010


 


63
HTQELFPQW
0.010


 


159
LTRKQFGLL
0.010


 


116
KVLPMVSIT
0.009


 


139
VQLHNGTKY
0.009


 


187
SYRYKLLNW
0.008


 


122
SITLLALVY
0.008


 


106
FYKIPILVI
0.008


 


32
TGETSMLKR
0.008


 


312
CLRKKILKI
0.008


 


105
YFYKIPILV
0.008


 


208
WIEHDVWRM
0.008


 


37
MLKRPVLLH
0.008


 


182
YPMRRSYRY
0.008


 


117
VLPMVSITL
0.008


 


168
SFFFAVLHA
0.008


 


104
QYFYKIPIL
0.008


 


296
FLPIVVLIF
0.008


 


21
NLEEDDYLH
0.008


 


272
FAWNKWIDI
0.008


 


221
SLGIVGLAI
0.008


 


207
AWIEHDVWR
0.006


 


262
LLLGTIHAL
0.006


 


36
SMLKRPVLL
0.006


 


163
QFGLLSFFF
0.006

















TABLE XI-V2-HLA-A11







9mers: STEAP-1



Each peptide is a portion of SEQ ID


NO: 5; each start position is


specified, the length of peptide is 9


amino acids, and the end position


for each peptide is the start position


plus eight.









Start
Subsequence
Score













4
FHYIQVNNI
0.000



 


1
WREFHYIQV
0.000


 


2
REFHYIQVN
0.000


 


3
EFHYIQVNN
0.000

















TABLE XI-V3-HLA-A11







9mers: STEAP-1



Each peptide is a portion of SEQ ID


NO: 7; each start position is


specified, the length of peptide is 9


amino acids, and the end position


for each peptide is the start position


plus eight.









Start
Subsequence
Score













5
HYIQIIHKK
0.600



 


19
SLWDPCLTR
0.320


 


4
FHYIQIIIK
0.080


 


25
LTRFKGLNL
0.020


 


8
QIIHKKSDV
0.006


 


2
REFHYIQII
0.004


 


21
WDPCLTRFK
0.002


 


15
DVPESLWDP
0.001


 


27
RFKGLNLTQ
0.001


 


3
EFHYIQIIH
0.001


 


12
KKSDVPESL
0.001


 


13
KSDVPESLW
0.001


 


7
IQIIHKXSD
0.000


 


26
TRFKGLNLI
0.000


 


24
CLTRFKGLN
0.000


 


9
IIHKKSDVP
0.000


 


1
WREEHYIQI
0.000


 


6
YIQIIHKKS
0.000


 


20
LWDPCLTRF
0.000


 


16
VPESLWDPC
0.000


 


22
DPCLTRFKG
0.000


 


23
PCLTRFKGL
0.000


 


14
SDVPESLWD
0.000


 


17
PESLWDPGL
0.000


 


28
FKGLNLIQS
0.000


 


11
HKKSDVPES
0.000


 


10
IHKKSDVPE
0.000


 


18
ESLWDPCLT
0.000
















TABLE XI-V4-HLA-A11







9mers: STEAP-1


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


each start position is


specified, the length of peptide is 9


amino acids, and the end position


for each peptide is the start position


plus eight.









Start
Subsequence
Score












5
GLLSLFFAV
0.054


 


2
KQFGLLSLF
0.036


 


8
SLFFAVLHA
0.016


 


6
LLSLFFAVL
0.004


 


9
LFFAVLHAI
0.004


 


3
QFGLLSLFF
0.004


 


1
RKQFGLLSL
0.001


 


4
FGLLSLFFA
0.001


 


7
LSLFFAVLH
0.000
















TABLE XII-V1-HLA-A11







10mers: STEAP-1


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


each start position is


specified, the length of peptide is 10


amino acids, and the end position


for each peptide is the start position


plus nine.









Start
Subsequence
Score












99
ATSHQQYFYK
3.000


 


306
SILFLPCLRK
1.200


 


296
FLPIVVLIFK
0.800


 


21
NLEEDDYLHK
0.800


 


140
QLHNGTKYKK
0.800


 


137
AIVQLHNGTK
0.600


 


139
VQLHNGTKYK
0.450


 


195
WAYQQVQQNK
0.400


 


153
WLDKWMLTRK
0.400


 


272
FAWNXWIDIX
0.400


 


310
LPGLRXKILK
0.400


 


6
DITNQEELWK
0.240


 


67
LFPQWHLPIK
0.200


 


116
KVLPMVSITL
0.180


 


177
IYSLSYPMRR
0.160


 


83
SLTFLYTLLR
0.160


 


176
AIYSLSYPMR
0.160


 


265
GTLHALIFAW
0.135


 


30
KDTGETSMLK
0.120


 


323
GWEDVTKINK
0.120


 


31
DTGETSMLKR
0.120


 


162
KQFGLLSFFF
0.108


 


133
GVIAAIVQLH
0.090


 


307
ILFLPCLRKK
0.080


 


128
LVYLPGVIAA
0.080


 


107
YKIPILVINK
0.060


 


300
VVLIFKSILF
0.060


 


147
YKKFPHWLDK
0.040


 


294
AVFLPIVVLI
0.040


 


182
YPMRRSYRYK
0.040


 


267
IHALIFAWNK
0.040


 


312
CLRXKILKIR
0.040


 


283
FVWYTPPTFM
0.040


 


223
GIVGLAILAL
0.036


 


144
GTKYKKFPHW
0.030


 


299
IVVLIFKSIL
0.030


 


112
LVINKVLPMV
0.030


 


206
DAWIEHDVWR
0.024


 


226
GLAILALLAV
0.024


 


215
RMEIYVSLGI
0.024


 


120
MVSITLLALV
0.020


 


8
TNQEELWKMK
0.020


 


320
IRHGWEDVTK
0.020


 


7
TNQEELWKMK
0.020


 


286
YTPPTFMIAV
0.020


 


138
IVQLHNGTKY
0.020


 


199
QVQQNKEDAW
0.020


 


219
YVSLGIVGLA
0.020


 


247
WREFHYIQSK
0.020


 


259
IVSLLLGTTH
0.020


 


224
IVGLAILALL
0.020


 


305
KSILFLPCLR
0.020


 


157
WMLTRKQFGL
0.018


 


79
AIIASLTFLY
0.018


 


191
KLLNWAYQQV
0.018


 


258
GIVSLLLGTI
0.018


 


165
GLLSFFFAVL
0.018


 


104
QYFYKIPILV
0.016


 


152
HWLDKWMLTR
0.012


 


102
HQQYFYKIPI
0.012


 


36
SMLKRPVLLH
0.012


 


108
KIPILVINKV
0.012


 


64
TQELFPQWHL
0.012


 


103
QQYFYKIPIL
0.012


 


301
FLIFKSILFL
0.012


 


111
ILVINKVLPM
0.012


 


63
HTQELFPQWH
0.010


 


198
QQVQQNKEDA
0.009


 


93
EVIHPLATSH
0.009


 


285
WYTPPTFMIA
0.008


 


181
SYPMRRSYRY
0.008


 


86
FLYTLLREVI
0.008


 


37
MLKRPVLLHL
0.008


 


292
MIAVFLPIVV
0.008


 


180
LSYPMRRSYR
0.008


 


173
VLHAIYSLSY
0.008


 


118
LPMVSITLLA
0.008


 


105
YFYKIPILVI
0.008


 


263
LLGTIHALIF
0.008


 


290
TFMIAVFLPI
0.008


 


200
VQQNKEDAWI
0.006


 


171
FAVLHAIYSL
0.006


 


73
LPIKIAAIIA
0.006


 


262
LLLGTIHALI
0.006


 


129
VYLPGVIAAI
0.006


 


291
FMIAVFLPIV
0.006


 


253
IQSKLGIVSL
0.006


 


42
VLLHLHQTAH
0.006


 


163
QFGLLSFFFA
0.006


 


326
DVTKINKTEI
0.006


 


89
TLLREVIHPL
0.006


 


251
HYIQSKLGIV
0.006


 


295
VFLPIVVLIF
0.006


 


10
QEELWKMKPR
0.006


 


127
ALVYLPGVIA
0.006


 


261
SLLLGTIHAL
0.006


 


218
IYVSLGIVGL
0.006


 


175
HAIYSLSYPM
0.006


 


11
EELWKMKPRR
0.005


 


66
ELFPQWHLPI
0.005

















TABLE XII-V2-HLA-A11







10mers: STEAP-1



Each peptide is a portion of SEQ ID


NO: 5; each start position is


specified, the length of peptide is 10


amino acids, and the end position


for each peptide is the start position


plus nine.









Start
Subsequence
Score













4
EFHYIQVNNI
0.001



 


1
TWREFHYIQV
0.000


 


3
REFHYIQVNN
0.000


 


2
WREFHYIQVN
0.000

















TABLE XII-V3-HLA-A3







10mers: STEAP- 1



Each peptide is a portion of SEQ ID


NO: 7; each start position is


specified, the length of peptide is 10


amino acids, and the end position


for each peptide is the start position


plus nine.









Start
Subsequence
Score













4
EFHYIQIIHK
0.120



 


5
FHYIQIIHKK
0.040


 


26
LTRFKGLNLI
0.010


 


8
IQIIHKKSDV
0.009


 


25
CLTRFKGLNL
0.008


 


20
SLWDPCLTRF
0.008


 


3
REFHYIQIIH
0.007


 


19
ESLWDPCLTR
0.004


 


21
LWDPCLTRFK
0.002


 


17
VPESLWDPCL
0.002


 


28
RFKGLNLIQS
0.001


 


9
QIIHKKSDVP
0.001


 


13
KKSDVPESLW
0.001


 


16
DVPESLWDPC
0.001


 


10
IIHKKSDVPE
0.000


 


1
TWREFHYIQI
0.000


 


6
HYIQIIHKKS
0.000


 


23
DPCLTRFKGL
0.000


 


2
WREFHYIQII
0.000


 


12
HKKSDVPESL
0.000


 


7
YIQIIHKKSD
0.000


 


14
KSDVPESLWD
0.000


 


27
TRFKGLNLIQ
0.000


 


22
WDPCLTRFKG
0.000


 


15
SDVPESLWDP
0.000


 


24
PCLTRFKGLN
0.000


 


11
IHKKSDVPES
0.000


 


18
PESLWDPCLT
0.000

















TABLE XII-V4-HLA-A3







10mers: STEAP-1



Each peptide is a portion of SEQ ID


NO: 7; each start position is


specified, the length of peptide is 10


amino acids, and the end position


for each peptide is the start position


plus nine.









Start
Subsequence
Score













6
GLLSLFFAVL
24.300



 


9
SLFFAVLHAI
6.750


 


3
KQFGLLSLFF
2.700


 


7
LLSLFFAVLH
0.600


 


10
LFFAVLHAIY
0.020


 


8
LSLFFAVLHA
0.009


 


2
RKQFGLLSLF
0.003


 


5
FGLLSLFFAV
0.003


 


1
TRKQFGLLSL
0.002


 


4
QFGLLSLFFA
0.001
















TABLE XIII-V1-HLA-A24







9mers: STEAP-1


Each peptide is a portion of SEQ ID


NO: 3; each start position is


specified, the length of peptide is 9


amino acids, and the end position


for each peptide is the start position


plus eight.









Start
Subsequence
Score












146
KYKKFPHWL
400.000


 


104
QYFYKIPIL
200.000


 


251
HYIQSKLGI
75.000


 


87
LYTLLREVI
60.000


 


285
WYTPPTFMI
60.000


 


106
FYKIPILVI
50.000


 


249
EFHYIQSKL
30.800


 


20
RNLEEDDYL
14.400


 


295
VFLPIVVLI
12.600


 


189
RYKLLNWAY
12.000


 


163
QFGLLSFFF
12.000


 


305
KSILFLPCL
12.000


 


256
KLGIVSLLL
11.200


 


76
KIAAIIASL
11.200


 


129
VYLPGVIAA
9.000


 


225
VGLAILALL
8.400


 


237
SIPSVSDSL
8.400


 


117
VLPMVSITL
8.400


 


67
LFPQWHLPI
7.500


 


169
FFFAVLHAI
7.000


 


90
LLREVIHPL
6.720


 


82
ASLTFLYTL
6.000


 


222
LGIVGLAIL
6.000


 


118
LPMVSITLL
6.000


 


35
TSMLKRPVL
6.000


 


262
LLLGTIHAL
6.000


 


79
AIIASLTFL
6.000


 


156
KWMLTRKQF
6.000


 


36
SMLKRPVLL
6.000


 


133
GVIAAIVQL
6.000


 


300
VVLIFKSIL
6.000


 


172
AVLHAIYSL
6.000


 


123
ITLLALVYL
6.000


 


187
SYRYKLLNW
5.000


 


159
LTRKQFGLL
4.800


 


166
LLSFFFAVL
4.800


 


162
KQFGLLSFF
4.800


 


83
SLTFLYTLL
4.800


 


296
FLPIVVLIF
4.200


 


302
LIFKSILFL
4.000


 


310
LPCLRKKIL
4.000


 


158
MLTRKQFGL
4.000


 


224
IVGLAILAL
4.000


 


294
AVFLPIVVL
4.000


 


120
MVSITLLAL
4.000


 


219
YVSLGIVGL
4.000


 


254
QSKLGIVSL
4.000


 


60
ELQHTQELF
3.000


 


301
VLIFKSILF
3.000


 


78
AAIIASLTF
3.000


 


299
IVVLIFKSI
2.520


 


291
FMIAVFLPI
2.520


 


98
LATSHQQYF
2.400


 


47
HQTAHADEF
2.200


 


142
HNGTKYKKF
2.200


 


73
LPIKIAAII
2.100


 


130
YLPGVIAAI
2.100


 


283
FVWYTPPTF
2.000


 


264
LGTIHALIF
2.000


 


309
FLPCLRKKI
1.980


 


127
ALVYLPGVI
1.800


 


149
KFPHWLDKW
1.650


 


230
LALLAVTSI
1.500


 


201
QQNKEDAWI
1.500


 


72
HLPIKIAAI
1.500


 


263
LLGTIHALI
1.400


 


221
SLGIVGLAI
1.400


 


322
HGWEDVTKI
1.320


 


5
KDITNQEEL
1.320


 


196
AYQQVQQNK
1.260


 


245
LTWRLEFHYI
1.200


 


259
IVSLLLGTI
1.200


 


327
VTKINKTEI
1.100


 


312
CLRKKILKI
1.100


 


214
WRMEIYVSL
1.008


 


270
LIFAWNKWI
1.000


 


272
FAWNKWIDI
1.000


 


103
QQYFYKIPI
1.000


 


8
TNQEELWKM
0.990


 


218
IYVSLGIVG
0.900


 


255
SKLGIVSLL
0.840


 


110
PILVINKVL
0.840


 


185
RRSYRYKLL
0.800


 


30
KDTGETSML
0.800


 


59
SELQHTQEL
0.792


 


208
WIEHDVWRM
0.750


 


282
QFVWYTPPT
0.750


 


181
SYPMRRSYR
0.750


 


112
LVINKVLPM
0.750


 


26
DYLHKDTGE
0.750


 


105
YFYKIPILV
0.700


 


38
LKRPVLLHL
0.672


 


331
NKTEICSQL
0.672


 


276
KWIDIKQFV
0.605


 


170
FFAVLHAIY
0.600


 


176
AIYSLSYPM
0.600


 


150
FPHWLDKWM
0.600


 


161
RKQFGLLSF
0.600


 


14
WKMKPRRNL
0.600


 


65
QELFPQWHL
0.600
















TABLE XIII-V2-HLA-A24







9mers: STEAP-1


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


each start position is specified, the length


of peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.









Start
Subsequence
Score





3
EFHYIQVNN
0.700


 


4
FHYIQVNNI
0.120


 


2
REFHYIQVN
0.024


 


1
WREFHYIQV
0.015
















TABLE XIII-V3-HLA-A24







9mers: STEAP-1


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


each start position is specified, the length


of peptide is 9 amino acids, and the end


position for each peptide is the start


position plus nine.











Start
Subsequence
Score






25
LTRFKGLNL
4.000


 



10
LWDPCLTRF
2.400


 



12
KKSDVPESL
1.344


 



 5
HYIQIIHKK
1.155


 



23
PCLTRFKGL
0.720


 



 2
REFHYIQII
0.240


 



 6
YIQIIHKKS
0.231


 



13
KSDVPESLW
0.200


 



16
VPESLWDPC
0.180


 



 8
QIIHKKSDV
0.150


 



 1
WREFHYIQI
0.150


 



18
ESLWDPCLT
0.150


 



27
RFKGLNLIQ
0.120


 



26
TRFKGLNLI
0.120


 



24
CLTRFKGLN
0.200


 



 3
EFHYIQIIH
0.070


 



17
PESLWDPCL
0.040


 



15
DVPESLWDP
0.018


 



 7
IQIIHKKSD
0.015


 



19
SLWDPCLTR
0.012


 



11
HDDSDVPES
0.011


 



22
DPCLTRFKG
0.011


 



 9
IIHKKSDVP
0.010


 



28
FKGLNLIQS
0.010


 



21
WDPCLTRFK
0.002


 



14
SDVPESLWD
0.002


 



10
IHKKSDVPE
0.001


 



 4
FHYIQIIHK
0.001
















TABLE XIII-V4-HLA-A24







9mers: STEAP-1


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


each start position is specified, the length


of peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.









Start
Subsequence
Score












3
QFGLLSLFF
12.000


 


9
LFFAVLHAI
7.000


 


2
KQFGLLSLF
4.800


 


6
LLSLFFAVL
4.800


 


1
RKQFGLLSL
1.200


 


5
GLLSLFFAV
0.216


 


4
FGLLSLFFA
0.150


 


8
SLFFAVLHA
0.100


 


7
LSLFFAVLH
0.015
















TABLE XIV-V1-HLA-A24







10mers: STEAP-1


Each peptide is a portion of SEQ ID


NO: 3; each start position is


specified, the length of peptide is 10


amino acids, and the end position


for each peptide is the start position


plus nine.









Start
Subsequence
Score












218
IYVSLGIVGL
300.000


 


129
VYLLPGVIAAI
126.000


 


295
VFLPIVVLIF
25.200


 


116
KVLPMVSITL
20.160


 


282
QFVWYTPPTF
15.000


 


290
TFMIAVFLPI
12.600


 


308
LFLPCLRKKI
11.880


 


236
TSIPSVSDSL
10.080


 


149
KFPHWLDKWM
9.000


 


89
TLLREVIHPL
8.400


 


109
IPILVINKVL
8.400


 


26
DYLHKDTGET
8.250


 


181
SYPMRRSYRY
7.500


 


251
HYIQSKLGIV
7.500


 


187
SYRYKLLNWA
7.200


 


285
WYTPPTRMIA
7.200


 


82
ASLTFLYTLL
7.200


 


165
GLLSFFFAVL
7.200


 


104
QYFYKIPILV
7.000


 


106
FYKIPILVIN
7.000


 


168
SFFFAVLHAI
7.000


 


261
SLLLGTIHAL
6.000


 


293
IAVFLPIVVL
6.000


 


299
IVVLIFKSIL
6.000


 


157
WMLTRKQFG
6.000


 


223
GIVGLAILAL
6.000


 


171
FAVLHAIYSL
6.000


 


309
FLPCLRKKIL
6.000


 


35
TSMLKRPVLL
6.000


 


117
VLPMVSITLL
6.000


 


78
AAIIASLTFL
6.000


 


301
VLIFKSILFL
6.000


 


64
TQELFPQWHL
6.000


 


213
VWRMEIYVSL
5.600


 


254
QSKLGIVSLL
5.600


 


37
MLKRPVLLHL
5.600


 


330
INKTEICSQL
5.600


 


224
IVGLAILALL
5.600


 


271
IFAWNKWIDI
5.000


 


105
YFYKIPILVI
5.000


 


158
MLTRKQFGLL
4.800


 


162
KQFGLLSFFF
4.800


 


215
RMEIYVSLGI
4.200


 


103
QQYFYKIPIL
4.000


 


253
IQSKLGIVSL
4.000


 


122
SITLLALVYL
4.000


 


81
IASLTFLYTL
4.000


 


34
ETSMLKRPVL
4.000


 


13
LWKMKPRRNL
4.000


 


221
SLGIVGLAIL
4.000


 


150
FPHWLDKWML
4.000


 


287
TPPTFMIAVF
3.000


 


300
VVLIFKSILF
3.000


 


241
VSDSLTWREF
2.200


 


262
LLLGTIHALI
2.100


 


72
HLPLKIAAII
2.100


 


220
VSLGIVGLAI
2.100


 


274
WNKWIDIKQF
2.000


 


263
LLGTIHALIF
2.000


 


77
IAAIIASLTF
2.000


 


68
FPQWHLPIKI
1.980


 


258
GIVSLLLGTI
1.800


 


126
LALVYLPGVI
1.800


 


102
HQQYFYKIPI
1.500


 


200
VQQNKEDAWI
1.500


 


269
ALIFAWNKWI
1.500


 


294
AVFLPIVVLI
1.400


 


100
TSHQQYFYKI
1.320


 


248
REFHYIQSKL
1.232


 


86
FLYTLLREVI
1.200


 


244
SLTWREFHYI
1.200


 


66
ELFPQWHLPI
1.200


 


19
RENLEEDDYL
1.200


 


326
DVTKINKTEI
1.100


 


146
KYKKFPHWLD
1.000


 


189
RYKLLNWAYQ
1.000


 


284
VWYTPPTFMI
1.000


 


114
INKVLPMVSI
1.000


 


229
ILALLAVTSI
1.000


 


196
AYQQVQQNKE
0.990


 


85
TFLYTLLREV
0.990


 


207
AWIEHDVWRM
0.900


 


175
HAIYSLSYPM
0.900


 


4
RKDITNQEEL
0.880


 


255
SKLGIVSLLL
0.840


 


75
IKIAAIIASL
0.840


 


7
ITNQEELWKM
0.825


 


58
PSELQHTQEL
0.792


 


111
ILVINKVLPM
0.750


 


161
RKQFGLLSFF
0.720


 


303
IFKSILFLPC
0.720


 


52
ADEFDCPSEL
0.660


 


119
PMVSITLLAL
0.600


 


163
QFGLLSFFFA
0.600


 


169
FFFAVLHAIY
0.600


 


132
PGVIAAIVQL
0.600


 


288
PPTFMIAVFL
0.560


 


108
KIPILVINKV
0.554


 


170
FFAVLHAIYS
0.500


 


87
LYTLLREVIH
0.500
















TABLE XIV-V2-HLA-A24







10mers: STEAP-1


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


each start position is specified, the length


of peptide is 10 amino acids, and the end


position for each peptide is the start


position plus nine.









Start
Subsequence
Score





4
EFHYIQVNNI
6.000


 


1
TWREFHYIQV
0.120


 


3
REFHYIQVNN
0.028


 


2
WREFHYIQVN
0.018
















TABLE XIV-V3-HLA-A24







10mers: STEAP-1


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


each start position is specified, the length


of peptide is 10 amino acids, and the end


position for each peptide is the start


position plus nine.









Start
Subsequence
Score












 6
HYIQIIHKKS
11.550


 


17
VPESLWDPCL
6.000


 


23
DPCLTRFKGL
4.800


 


25
CLTRFKGLNL
4.000


 


20
SLWDPCLTRF
2.400


 


 1
TWREFHYIQI
1.200


 


26
LTRFKGLNLI
1.200


 


28
RFKGLNLIQS
1.000


 


12
HKKSDVPESL
0.560


 


16
DVPESLWDPC
0.216


 


 2
WREFHYIQII
0.180


 


 8
IQIIHKKSDV
0.150


 


 4
EFHYIQIIHK
0.050


 


13
KKSDVPESLW
0.024


 


14
KSDVPESLWD
0.020


 


19
ESLWDPCLTR
0.015


 


24
PCLTRFKGLN
0.015


 


 9
QIIHKKSDVP
0.015


 


 7
YIQIIHKKSD
0.015


 


21
LWDPCLTRFK
0.014


 


11
IHKKSDVPES
0.011


 


10
IIHIKKSDVPE
0.010


 


 3
REFHYIQIIH
0.003


 


15
SDVPESLWDP
0.002


 


22
WDPCLTRFKG
0.002


 


 5
FHYIQIIHKK
0.002


 


27
TRFKGLNLIQ
0.001


 


18
PESLWDPCLT
0.001
















TABLE XIV-V4-HLA-A24







10mers: STEAP-1


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


each start position is specified, the length


of peptide is 10 amino acids, and the end


position for each peptide is the start


position plus nine.











Start
Subsequence
Score






 6
GLLSLFFAVL
7.200


 



 3
KQFGLLSLFF
4.800


 



 9
SLFFAVLHAI
1.400


 



 2
RKQFGLLSLF
0.720


 



10
LFFAVLHAIY
0.600


 



 4
QFGLLSLFFA
0.500


 



 1
TRKQFGLLSL
0.400


 



 5
FGLLSLFFAV
0.216


 



 8
LSLFFAVLHA
0.150


 



 7
LLSLFFAVLH
0.010
















TABLE XV-V1-HLA-B7







9mers: STEAP-1


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


each start position is specified, the length


of peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.











Start
Subsequence
Score














118
LPMVSITLL
240.000


 



294
AVFLPIVVL
90.000


 



310
LPCLRKKIL
80.000


 



172
AVLHAIYSL
60.000


 



159
LTRKQFGLL
40.000


 



 90
LLREVIHPL
40.000


 



120
MVSITLLAL
20.000


 



219
YVSLGIVGL
20.000


 



224
IVGLAILAL
20.000


 



150
FPHWLDKWM
20.000


 



133
GVIAAIVQL
20.000


 



300
VVLIFKSIL
20.000


 



 35
TSMLKRPVL
18.000


 



 79
AIIASLTFL
12.000


 



 82
ASLTFLYTL
12.000


 



 73
LPIKIAAII
8.000


 



184
MRRSYRYKL
6.000


 



112
LVINKVLPM
5.000


 



 38
LKRPVLLHL
4.000


 



312
CLRKKILKI
4.000


 



305
KSILFLPCL
4.000


 



225
VGLAILALL
4.000


 



158
MLTRKQFGL
4.000


 



254
QSKLGIVSL
4.000


 



262
LLLGTIHAL
4.000


 



123
ITLLALVYL
4.000


 



287
TPPTFMIAV
4.000


 



131
LPGVIAAIV
4.000


 



166
LLSFFFAVL
4.000


 



302
LIFKSILFL
4.000


 



222
LGIVGLAIL
4.000


 



117
VLPMVSITL
4.000


 



256
KLGIVSLLL
4.000


 



237
SIPSVSDSL
4.000


 



 76
KIAAIIASL
4.000


 



 83
SLTFLYTLL
4.000


 



 36
SMLKRPVLL
4.000


 



 20
RNLEEDDYL
4.000


 



109
IPILVINKV
4.000


 



176
AIYSLSYPM
3.000


 



 14
WKMKPRRNL
2.700


 



319
KIRHGWEDV
2.000


 



259
IVSLLLGTI
2.000


 



 17
KPRRNLEED
2.000


 



299
IVVLIFKSI
2.000


 



238
IPSVSDSLT
2.000


 



182
YPMRRSYRY
1.200


 



272
FAWNKWIDI
1.200


 



214
WRMEIYVSL
1.200


 



230
LALLAVTSI
1.200


 



127
ALVYLPGVI
1.200


 



  8
TNQEELWKM
1.000


 



212
DVWRMEIYV
1.000


 



128
LVYLPGVIA
0.750


 



227
LAILALLAV
0.600


 



293
IAVFLPIVV
0.600


 



126
LALVYLPGV
0.600


 



309
FLPCLRKKI
0.600


 



 65
QELFPQWHL
0.600


 



233
LAVTSIPSV
0.600


 



199
QVQQNKEDA
0.500


 



116
KVLPMVSIT
0.500


 



331
NKTEICSQL
0.400


 



291
FMIAVFLPI
0.400


 



146
KYKKFPHWL
0.400


 



322
HGWEDVTKI
0.400


 



263
LLGTIHALI
0.400


 



245
LTWREFHYI
0.400


 



221
SLGIVGLAI
0.400


 



 59
SELQHTQEL
3.400


 



289
PTFMIAVFL
0.400


 



 30
KDTGETSML
0.400


 



 53
DEFDCPSEL
0.400


 



201
QQNKEDAWI
0.400


 



270
LIFAWNKWI
0.400


 



249
EFHYIQSKL
0.400


 



110
PILVINKVL
0.400


 



104
QYFYKIPIL
0.400


 



103
QQYFYKIPI
0.400


 



  5
KDITNQEEL
0.400


 



185
RRSYRYKLL
0.400


 



 72
HLPIKIAAI
0.400


 



130
YLPGVIAAI
0.400


 



327
VTKINKTEI
0.400


 



255
SKLGIVSLL
0.400


 



234
AVTSIPSVS
0.300


 



 49
TAHADEFDC
0.300


 



 77
IAAIIASLT
0.300


 



228
AILALLAVT
0.300


 



 81
IASLTFLYT
0.300


 



137
AIVQLHNGT
0.300


 



208
WIEHDVWRM
0.300


 



252
YIQSKLGIV
0.200


 



217
EIYVSLGIV
0.200


 



 68
FPQWHLPIK
0.200


 



 40
RPVLLHLHQ
0.200


 



121
VSITLLALV
0.200


 



292
MIAVFLPIV
0.200


 



 57
CPSELQHTQ
0.200


 



192
LLNWAYQQV
0.200
















TABLE XV-V2-HLA-B7







9mers: STEAP-1


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


each start position is specified, the length


of peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.









Start
Subsequence
Score





4
FHYIQVNNI
0.040


 


1
WREFHYIQV
0.006


 


2
REFHYIQVN
0.002


 


3
EFHYIQVNN
0.002
















TABLE XV-V3-HLA-B7







9mers: STEAP-1


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


each start position is specified, the length


of peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.











Start
Subsequence
Score














25
LTRFKGLNL
40.000


 



16
VPESLWDPC
0.600


 



12
KKSDVPESL
0.400


 



23
PCLTRFKGL
0.400


 



22
DPCLTRFKG
0.300


 



 8
QIIHKKSDV
0.200


 



18
ESLWDPGLT
0.100


 



15
DVPESLWDP
0.050


 



17
PESLWDPCL
0.040


 



26
TRFKGLNLI
0.040


 



 2
REFHYIQII
0.040


 



 6
YIQIIHKKS
0.020


 



24
CLTRFKGLN
0.020


 



19
SLWDPGLTR
0.015


 



 1
WREFHYIQI
0.012


 



 7
IQIIHKKSD
0.010


 



 9
IIHKKSDVP
0.010


 



13
KSDVPESLW
0.009


 



11
HKKSDVPES
0.002


 



28
FKGLNLIQS
0.002


 



 4
FHYIQIIHK
0.001


 



21
WDPCLTRFK
0.001


 



27
RFKGLNLIQ
0.001


 



 5
HYIQILHKK
0.001


 



 3
EFHYIQIIH
0.001


 



10
IHKKSDVPE
0.001


 



14
SDVPESLWD
0.001


 



20
LWDPCLTRF
0.001
















TABLE XV-V4-HLA-B7







9mers: STEAP-1


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


each start position is specified, the length


of peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.









Start
Subsequence
Score





6
LLSLFFAVL
4.000


 


1
RKQFGLLSL
0.400


 


5
GLLSLFFAV
0.200


 


4
FGLLSLFFA
0.100


 


8
SLFFAVLHA
0.100


 


9
LFFAVLHAI
0.040


 


2
KQFGLLSLF
0.020


 


7
LSLFFAVLH
0.010


 


3
QFGLLSLFF
0.002
















TABLE XVI-V1-HLA-B7







10mers: STEAP-1


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


each start position is specified, the length


of peptide is 10 amino acids, and the end


position for each peptide is the start


position plus nine.











Start
Subsequence
Score














109
IPILVLNKVL
80.000


 



150
FPHWLDKWML
80.000


 



 78
AAIIASLTFL
36.000


 



116
KVLPMVSITL
20.000


 



299
IVVLIFKSIL
20.000


 



224
IYGLAILALL
20.000


 



293
IAVFLPIVVL
18.000


 



 82
ASLTFLYTLL
12.000


 



 81
IASLTFLYTL
12.000


 



171
FAVLHAIYSL
12.000


 



 35
TSMLKRPVLL
12.000


 



 68
FPQWHLPIKI
8.000


 



288
PPTFMIAVFL
8.000


 



283
FVWYTPPTFM
7.500


 



183
PMRRSYRYKL
6.000


 



294
AVFLPIVVLI
6.000


 



118
LPMVSITLLA
6.000


 



 34
ETSMLKRPVL
6.000


 



261
SLLLGTIHAL
4.000


 



165
GLLSFFFAVL
4.000


 



117
VLPMVSITLL
4.000


 



213
VWRMEIYVSL
4.000


 



184
MRRSYRYKLL
4.000


 



254
QSKLGIVSLL
4.000


 



330
INKTEICSQL
4.000


 



221
SLGIVGLAIL
4.000


 



309
FLPCLRKKIL
4.000


 



236
TSIPSVSDSL
4.000


 



 37
MLKRPVLLHL
4.000


 



301
VLIFKSILFL
4.000


 



122
SITLLALVYL
4.000


 



253
IQSKLGIVSL
4.000


 



103
QQYFYKIPIL
4.000


 



223
GIVGLAILAL
4.000


 



157
WMLTRKQFGL
4.000


 



 89
TLLREVIHPL
4.000


 



158
MLTRKQFGLL
4.000


 



175
HAIYSLSYPM
3.000


 



326
DVTKINKTEI
2.000


 



 17
KPRRNLEEDD
2.000


 



 73
LPIKIAAIIA
2.000


 



 40
RPVLLHLHQT
2.000


 



 64
TQELFPQWHL
1.800


 



269
ALIFAWNKWI
1.200


 



126
LALVYLPGVI
1.200


 



 90
LLREVIHPLA
1.000


 



111
ILVINKVLPM
1.000


 



  7
ITNQEELWKM
1.000


 



319
KIRHGWEDVT
1.000


 



112
LVTNKVLPMV
1.000


 



120
MVSITLLALV
1.000


 



 13
LWKMKPRRNL
0.900


 



136
AAIVQLHNGT
0.900


 



114
INKVLPMVSI
0.600


 



182
YPMRRSYRYK
0.600


 



 86
FLYTLLREVI
0.600


 



219
YVSLGIVGLA
0.500


 



128
LVYLPGVIAA
0.500


 



127
ALVYLPGVIA
0.450


 



220
VSLGIVGLAI
0.400


 



 66
ELFPQWHLPI
0.400


 



145
TKYKKFPHWL
0.400


 



255
SKLGIVSLLL
0.400


 



304
FKSILFLPCL
0.400


 



200
VQQNKEDAWI
0.400


 



218
IYVSLGIVGL
0.400


 



287
TPPTFMIAVF
0.400


 



297
LPIVVLIFKS
0.400


 



258
GIVSLLLGTI
0.400


 



262
LLLGTIHALI
0.400


 



244
SLTWREFHYI
0.400


 



238
IPSVSDSLTW
0.400


 



229
ILALLAVTSI
0.400


 



 75
IKIAAIIASL
0.400


 



100
TSHQQYFYKI
0.400


 



 96
HPLATSHQQY
0.400


 



119
PMVSITLLAL
0.400


 



102
HQQYFYKIPI
0.400


 



248
REFHYIQSKL
0.400


 



 19
RRNLEEDDYL
0.400


 



132
PGVIAAIVQL
0.400


 



 72
HLPIKIAAII
0.400


 



 52
ADEFDCPSEL
0.360


 



207
AWIEHDVWRM
0.300


 



172
AVLHAIYSLS
0.300


 



227
LAILALLAVT
0.300


 



234
AVTSIPSVSD
0.225


 



232
LLAVTSIPSV
0.200


 



226
GLAILALLAV
0.200


 



130
YLPGVIAAIV
0.200


 



108
KIPILVINKV
0.200


 



291
FMIAVFLPIV
0.200


 



164
FGLLSFFFAV
0.200


 



159
LTRKQFGLLS
0.200


 



 57
CPSELQHTQE
0.200


 



125
LLALVYLPGV
0.200


 



292
MIAVFLPIVV
0.200


 



286
YTPPTFMIAV
0.200


 



310
LPCLRKKILK
0.200


 



191
KLLNWAYQQV
0.200
















TABLE XVI-V2-HLA-B7







10mers: STEAP-1


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


each start position is specified, the length


of peptide is 10 amino acids, and the end


position for each peptide is the start


position plus nine.









Start
Subsequence
Score





1
TWREFHYIQV
0.200


 


4
EFHYIQVNNI
0.040


 


3
REFHYIQVNN
0.002


 


2
WREFHYIQVN
0.001
















TABLE XVI-V3-HLA-B7







10mers: STEAP-1


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


each start position is specified, the length


of peptide is 10 amino acids, and the end


position for each peptide is the start


position plus nine.











Start
Subsequence
Score














23
DPCLTRFKGL
80.000


 



17
VPESLWDPCL
24.000


 



25
CLTRFKGLNL
4.000


 



26
LTRFKGLNLI
4.000


 



16
DVPESLWDPC
0.500


 



 1
TWREFHYIQI
0.400


 



12
HKKSDVPESL
0.400


 



 8
IQIIHKKSDV
0.200


 



20
SLWDPCLTRF
0.020


 



19
ESLWDPCLTR
0.015


 



 2
WREFHYIQII
0.012


 



10
IIHKKSDVPE
0.010


 



 9
QIIHKKSDVP
0.010


 



 7
YIQIIHKKSD
0.010


 



13
KKSDVPESLW
0.003


 



14
KSDVPESLWD
0.003


 



11
IHKKSDVPES
0.002


 



24
PCLTRFKGLN
0.002


 



 6
HYIQIIHKKS
0.002


 



28
RFKGLNLIQS
0.002


 



22
WDPCLTRFKG
0.002


 



18
PESLWDPCLT
0.001


 



 3
REFHYIQIIH
0.001


 



 5
FHYIQIIHKK
0.001


 



15
SDVPESLWDP
0.001


 



27
TRFKGLNLIQ
0.001


 



 4
EFHYIQIIHK
0.001


 



21
LWDPCLTRFK
0.000
















TABLE XVI V4-HLA-B7







10mers: STEAP-1


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


each start position is specified, the length


of peptide is 10 amino acids, and the end


position for each peptide is the start


position plus nine.











Start
Subsequence
Score






 6
GLLSLFFAVL
4.000


 



 9
SLFFAVLHAI
0.400


 



 1
TRKQFGLLSL
0.400


 



 5
FGLLSLFFAV
0.200


 



 8
LSLFFAVLHA
0.100


 



 3
KQFGLLSLFF
0.020


 



 7
LLSLFFAVLH
0.010


 



 4
QFGLLSLFFA
0.010


 



 2
RKQFGLLSLF
0.002


 



10
LFFAVLHAIY
0.002
















TABLE XVII-V1-HLA-B35







9mers: STEAP-1


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


each start position is specified, the length


of peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.











Start
Subsequence
Score














150
FPHWLDKWM
40.000


 



182
YPMRRSYRY
40.000


 



310
LPGLRKKIL
20.000


 



118
LPMVSITLL
20.000


 



254
QSKLGIVSL
15.000


 



305
KSILFLPCL
10.000


 



180
LSYPMRRSY
10.000


 



 73
LPIKIAAII
8.000


 



 90
LLREVIHPL
6.000


 



 20
RNLEEDDYL
6.000


 



  8
TNQEELWKM
6.000


 



 82
ASLTFLYTL
5.000


 



 35
TSMLKRPVL
5.000


 



131
LPGVIAAIV
4.000


 



109
IPILVINKV
4.000


 



287
TPPTFMIAV
4.000


 



 78
AAIIASLTF
3.000


 



159
LTRKQFGLL
3.000


 



 98
LATSHQQYF
3.000


 



206
DAWIEHDVW
2.250


 



122
SITLLALVY
2.000


 



176
AIYSLSYPM
2.000


 



162
KQFGLLSFF
2.000


 



112
LVINKVLPM
2.000


 



 76
KIAAIIASL
2.000


 



139
VQLHNGTKY
2.000


 



 80
ILASLTFLY
2.000


 



244
SLTWREFHY
2.000


 



238
IPSVSDSLT
2.000


 



256
KLGIVSLLL
2.000


 



 99
ATSHQQYFY
2.000


 



288
PPTFMIAVF
2.000


 



319
KIRHGWEDV
1.200


 



189
RYKLLNWAY
1.200


 



 17
KPRRNLEED
1.200


 



272
FAWNKWIDI
1.200


 



327
VTKINKTEI
1.200


 



312
CLRKKILKI
1.200


 



230
LALLAVTSI
1.200


 



322
HGWEDVTKI
1.200


 



262
LLLGTIHAL
1.000


 



172
AVLHAIYSL
1.000


 



166
LLSFFFAVL
1.000


 



219
YVSLGIVGL
1.000


 



224
IVGLAILAL
1.000


 



133
GVIAAIVQL
1.000


 



 47
HQTAHADEF
1.000


 



222
LGIVGLAIL
1.000


 



 63
HTQELFPQW
1.000


 



294
AVFLPIVVL
1.000


 



186
RSYRYKLLN
1.000


 



 60
ELQHTQELF
1.000


 



264
LGTIHALIF
1.000


 



316
KILKIRHGW
1.000


 



283
FVWYTPPTF
1.000


 



225
VGLAILALL
1.000


 



158
MLTRKQFGL
1.000


 



121
VSITLLALV
1.000


 



 79
AIIASLTFL
1.000


 



 36
SMLKRPVLL
1.000


 



300
VVLIFKSIL
1.000


 



296
FLPIVVLIF
1.000


 



117
VLPMVSITL
1.000


 



301
VLIFKSILF
1.000


 



237
SIPSVSDSL
1.000


 



123
ITLLALVYL
1.000


 



142
HNGTKYKKF
1.000


 



120
MVSITLLAL
1.000


 



302
LIFKSILFL
1.000


 



 83
SLTFLYTLL
1.000


 



208
WIEHDVWRM
0.900


 



201
QQNKEDAWI
0.600


 



146
KYKKFPHWL
0.600


 



233
LAVTSIPSV
0.600


 



293
IAVFLPIVV
0.600


 



 19
RRNLEEDDY
0.600


 



245
LTWREFHYI
0.600


 



126
LALVYLPGV
0.600


 



227
LAILALLAV
0.600


 



200
VQQNKEDAW
0.500


 



220
VLSGIVGLA
0.500


 



269
ALIFAWNKW
0.500


 



236
TSIPSVSDS
0.500


 



  6
DITNQEELW
0.500


 



266
TIHALIFAW
0.500


 



 49
TAHADEFDC
0.450


 



221
SLGIVGLAI
0.400


 



259
IVSLLLGTI
0.400


 



 72
HLPIKIAAI
0.400


 



263
LLGTIHALI
0.400


 



130
YLPGVIAAI
0.400


 



299
IVVLIFKSI
0.400


 



103
QQYFYKIPI
0.400


 



309
FLPCLRKKI
0.400


 



 40
RPVLLHLHQ
0.400


 



270
LIFAWNKWI
0.400


 



127
ALVYLPGVI
0.400


 



291
FLIAVFLPI
0.400


 



 57
CPSELQHTQ
0.400


 



239
PSVSDSLTW
0.375
















TABLE XVII-V2-HLA-B35







9mers: STEAP-1


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


each start position is specified, the length


of peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.









Start
Subsequence
Score





4
FHYIQVNNI
0.040


 


2
REFHYIQVN
0.020


 


3
EFHYIQVNN
0.010


 


1
WREFHYIQV
0.006
















TABLE XVII-V3-HLA-B35







9mers: STEAP-1


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


each start position is specified, the length


of peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.











Start
Subsequence
Score






25
LTRFKGLNL
3.000


 



13
KSDVPESLW
1.500


 



18
ESLWDPCLT
0.750


 



16
VPESLWDPC
0.600


 



12
KKSDVPESL
0.400


 



 8
QIIHKKSDV
0.200


 



22
DPCLTRFKG
0.200


 



 6
YIQIIHKKS
0.100


 



24
CLTRFKGLN
0.100


 



23
PCLTRFKGL
0.100


 



 2
REFHYIQII
0.080


 



11
HKKSDVPES
0.045


 



26
TRFKGLNLI
0.040


 



20
LWDPCLTRF
0.030


 



19
SLWDPCLTR
0.020


 



15
DVPESLWDP
0.020


 



 1
WREFHYIQI
0.012


 



 9
IIHKKSDVP
0.010


 



28
FKGLNLIQS
0.010


 



17
PESLWDPCL
0.010


 



 7
IQIIHKKSD
0.010


 



27
RFKGLNLIQ
0.006


 



10
IHKKSDVPE
0.003


 



14
SDVPESLWD
0.002


 



 3
EFHYIQIIH
0.001


 



 5
HYIQIIHKK
0.001


 



 4
FHYIQIIHK
0.001


 



21
WDPCLTRFK
0.001
















TABLE XVII-V4-HLA-B35







9mers: STEAP-1


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


each start position is specified, the length


of peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.









Start
Subsequence
Score





2
KQFGLLSLF
2.000


 


6
LLSLFFAVL
1.000


 


1
RKQFGLLSL
0.200


 


5
GLLSLFFAV
0.200


 


8
SLFFAVLHA
0.100


 


4
FGLLSLFFA
0.100


 


3
QFGLLSLFF
0.100


 


7
LSLFFAVLH
0.050


 


9
LFFAVLHAI
0.040
















TABLE XVIII-V1-HLA-B35







10mers: STEAP-1


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


each start position is specified, the length


of peptide is 10 amino acids, and the end


position for each peptide is the start


position plus nine.











Start
Subsequence
Score














 96
HPLATSHQQY
40.000


 



150
FPHWLDKWML
30.000


 



109
IPILVINKVL
20.000


 



287
TPPTFMIAVF
20.000


 



238
IPSVSDSLTW
15.000


 



254
QSKLGIVSLL
15.000


 



243
DSLTWREFHY
10.000


 



121
VSITLLALVY
10.000


 



 68
FPQWHLPIKI
8.000


 



175
HAIYSLSYPM
6.000


 



 98
LATSHQQYFY
6.000


 



236
TSIPSVSDSL
5.000


 



 35
TSMLKRPVLL
5.000


 



186
RSYRYKLLNW
5.000


 



 82
ASLTFLYTLL
5.000


 



274
WNKWIDIKQF
4.500


 



 40
RPVLLHLHQT
4.000


 



171
FAVLHAIYSL
3.000


 



330
INKTEICSQL
3.000


 



 81
IASLTFLYTL
3.000


 



 78
AAIIASLTFL
3.000


 



 37
MLKRPVLLHL
3.000


 



 77
IAAIIASLTF
3.000


 



  7
ITNQEELWKM
3.000


 



293
IAVFLPIVVL
3.000


 



179
SLSYPMRRSY
2.000


 



297
LPIVVLIFKS
2.000


 



 73
LPIKIAAIIA
2.000


 



283
FVWYTPPTFM
2.000


 



162
KQFGLLSFFF
2.000


 



116
KVLPMVSITL
2.000


 



220
VSLGIVGLAI
2.000


 



138
IVQLHNGTKY
2.000


 



118
LPMVSITLLA
2.000


 



288
PPTFMIAVFL
2.000


 



100
TSHQQYFYKI
2.000


 



111
ILVINKVLPM
2.000


 



 79
AIIASLTFLY
2.000


 



173
VLHAIYSLSY
2.000


 



241
VSDSLTWREF
1.500


 



268
HALIFAWNKW
1.500


 



144
GTKYKKFPHW
1.500


 



114
INKVLPMVSI
1.200


 



 17
KPRRNLEEDD
1.200


 



 28
LHKDTGETSM
1.200


 



126
LALVYLPGVI
1.200


 



301
VLIFKSILFL
1.000


 



253
IQSKLGIVSL
1.000


 



103
QQYFYKIPIL
1.000


 



224
IVGLAILALL
1.000


 



122
SITLLALVYL
1.000


 



263
LLGTIHALIF
1.000


 



221
SLGIVGLAIL
1.000


 



223
GIVGLAILAL
1.000


 



158
MLTRKQFGLL
1.000


 



 89
TLLREVIHPL
1.000


 



157
WMLTRKQFGL
1.000


 



299
IVVLIFKSIL
1.000


 



165
GLLSFFFAVL
1.000


 



261
SLLLGTIHAL
1.000


 



309
FLPCLRKKIL
1.000


 



300
VVLIFKSILF
1.000


 



 34
ETSMLKRPVL
1.000


 



117
VLPMVSITLL
1.000


 



200
VQQNKEDAWI
0.600


 



277
WIDIKQFVWY
0.600


 



 90
LLREVIHPLA
0.600


 



207
AWIEHDVWRM
0.600


 



244
SLTWREFHYI
0.600


 



319
KIRHGWEDVT
0.600


 



178
YSLSYPMRRS
0.500


 



167
LSFFFAVLHA
0.500


 



265
GTIHALIFAW
0.500


 



260
VSLLLGTIHA
0.500


 



199
QVQQNKEDAW
0.500


 



269
ALIFAWNKWI
0.400


 



191
KLLNWAYQQV
0.400


 



149
KFPHWLDKWM
0.400


 



294
AVFLPIVVLI
0.400


 



 86
FLYTLLREVI
0.400


 



 66
ELFPQWHLPI
0.400


 



258
GIVSLLLGIT
0.400


 



262
LLLGTIHALI
0.400


 



326
DVTKINKTEI
0.400


 



 57
CPSELQHTQE
0.400


 



102
HQQYFYKIPI
0.400


 



 72
HLPIKIAAII
0.400


 



108
KIPILVINKV
0.400


 



229
ILALLAVTSI
0.400


 



327
VTKINKTEIC
0.300


 



213
VWRMEIYVSL
0.300


 



227
LAILALLAVT
0.300


 



184
MRRSYRYKLL
0.300


 



159
LTRKQFGLLS
0.300


 



233
LAVTSIPSVS
0.300


 



136
AAIVQLHNGT
0.300


 



 64
TQELFPQWHL
0.300


 



160
TRKQFGLLSF
0.300


 



 13
LWKMKPRRNL
0.300


 



183
PMRRSYRYKL
0.300
















TABLE XVIII-V2-HLA-B35







10mers: STEAP-1


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


each start position is specified, the length


of peptide is 10 amino acids, and the end


position for each peptide is the start


position plus nine.


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


each start position is specified, the length


of peptide is 10 amino acids, and the end


position for each peptide is the start


position plus nine.









Start
Subsequence
Score





1
TWREFHYIQV
0.120


 


4
EFHYIQVNNI
0.040


 


3
REFHYIQVNN
0.020


 


2
WREFHYIQVN
0.003
















TABLE XVIII-V3-HLA-B35







10mers: STEAP-1


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


each start position is specified, the length


of peptide is 10 amino acids, and the end


position for each peptide is the start


position plus nine.











Start
Subsequence
Score














23
DPCLTRFKGL
20.000


 



17
VPESLWDPCL
6.000


 



20
SLWDPCLTRF
2.000


 



26
LTRFKGLNLI
1.200


 



25
CLTRFKGLNL
1.000


 



12
HKKSDVPESL
0.300


 



 1
TWREFHYIQI
0.240


 



 8
IQIIHKKSDV
0.200


 



16
DVPESLWDPC
0.200


 



13
KKSDVPESLW
0.200


 



28
RFKGLNLIQS
0.060


 



19
ESLWDPCLTR
0.050


 



11
IHKKSDVPES
0.045


 



14
KSDVPESLWD
0.045


 



 2
WREFHYIQII
0.012


 



 6
HYIQIIHKKS
0.010


 



 9
QIIHKKSDVP
0.010


 



24
PGLTRFKGLN
0.010


 



10
IIHKKSDVPE
0.010


 



 7
YIQIIHKKSD
0.010


 



 3
REFHYIQIIH
0.002


 



18
PESLWDPCLT
0.002


 



22
WDPCLTRFKG
0.001


 



 5
FHYIQIIHKK
0.001


 



27
TRFKGLNLIQ
0.001


 



 4
EFHYIQIIHK
0.001


 



15
SDVPESLWDP
0.001


 



21
LWDPCLTRFK
0.000
















TABLE XVIII-V4-HLA-B35







10mers: STEAP-1


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


each start position is specified, the length


of peptide is 10 amino acids, and the end


position for each peptide is the start


position plus nine.











Start
Subsequence
Score






 3
KQFGLLSLFF
2.000


 



 6
GLLSLFFAVL
1.000


 



 8
LSLFFAVLHA
0.500


 



 9
SLFFAVLHAI
0.400


 



 1
TRKQFGLLSL
0.300


 



10
LFFAVLHAIY
0.200


 



 2
RKQFGLLSLF
0.200


 



 5
FGLLSLFFAV
0.200


 



 4
QFGLLSLFFA
0.010


 



 7
LLSLFFAVLH
0.010
















TABLE XIX







Frequently Occurring Motifs











avrg. %




Name
identity
Description
Potential Function





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





transcription factor, nuclear location





probable


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




terminal)/b6/petB
superoxide


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





and include a conserved intradomain





disulfide bond.


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





containing a Trp-Asp motif. Function in





signal transduction and protein interaction


PDZ
23%
PDZ domain
may function in targeting signaling





molecules to sub-membranous sites


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





protein interactions


Pkinase
23%
Protein kinase domain
conserved catalytic core common to both





serine/threonine and tyrosine protein





kinases containing an ATP binding site and





a catalytic site


PH
16%
PH domain
pleckstrin homology involved in





intracellular signaling or as constituents of





the cytoskeleton


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





extracellular domain of membrane-bound





proteins or in secreted proteins


Rvt
49%
Reverse transcriptase




(RNA-dependent DNA




polymerase)


Ank
25%
Ank repeat
Cytoplasmic protein, associates integral





membrane proteins to the cytoskeleton


Oxidored_q1
32%
NADH-
membrane associated. Involved in proton




Ubiquinone/plastoquinone
translocation across the membrane




(complex I), various chains


Efhand
24%
Ef hand
calcium-binding domain, consists of a 12





residue loop flanked on both sides by a 12





residue alpha-helical domain


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





catalytic aspartyl residue


Collagen
42%
Collagen triple helix repeat
extracellular structural proteins involved in




(20 copies)
formation of connective tissue. The





sequence consists of the G-X-Y and the





polypeptide chains forms a triple helix.


fn3
20%
Fibronectin type III domain
Located in the extracellular ligand-binding





region of receptors and is about 200 amino





acid residues long with two pairs of





cysteines involved in disulfide bonds


7tm_1
19%
7 transmembrane receptor
seven hydrophobic transmembrane regions,




(rhodopsin family)
with the N-terminus located extracellularly





while the C-terminus is cytoplasmic.





Signal through G proteins
















TABLE XX





Motifs and Post-translational Modifications


of STEAP-1















N-glycosylation site









143-146
NGTK
(SEQ ID NO: 61)


331-334
NKTE
(SEQ ID NO: 62)


 







Protein kinase C phosphorylation site








  3-5
SrK


160-162
TrK


187-189
SyR


246-248
TwR


 







Casem kinase II phosphorylation site









  3-6
SrkD
(SEQ ID NO: 63)


  8-11
TnqE
(SEQ ID NO: 64)


240-243
SvsD
(SEQ ID NO: 65)


246-249
TwrE
(SEQ ID NO: 66)


 







Tyrosine kinase phosphorylation site









 19-27
RRNLEEDDY
(SEQ ID NO: 67)


 







N-myristoylation site









133-138
GVIAAI
(SEQ ID NO: 68)


265-270
GTIHAL
(SEQ ID NO: 69)


 







Bipartite nuclear targeting sequence









  4-20
RKDITNQEELWKMKLPRR
(SEQ ID NO: 70)
















TABLE XXI







Properties of STEAP-1










Bioinformatic




Program
Outcome













ORF
ORF finder
1193 bp


Protein length

339 aa


Transmembrane
TM Pred
6 TM at aa 73-91, 120-141, 163-181,


region

218-236, 253-274, 286-304



HMMTop
6 TM at aa 73-90, 117-139, 164-182,




220-238, 257-274, 291-309



Sosui
6 TM at aa 70-92, 114-136, 163-184,




219-241, 255-273, 292-313



TMHMM
6 TM at aa 73-95, 117-139, 164-182,




218-240, 252-274, 289-311


Signal Peptide
Signal P
potential cleavage between aa 136




and 137


pI
pI/MW tool
9.2 pI


Molecular weight
pI/MW tool
39.8 kD


Localization
PSORT
60% plama membrane, 40% golgi,




30% endoplasmic reticulum



PSORT II
66% endoplasmic reticulum, 11%




mitochondria, 11% plasma membrane


Motifs
Pfam
none



Prints
Transforming protein P21 ras




signature, Fibronectin type III




repeat signature



Blocks
Half-A-TPR repeat, Arsenical pump




membrane protein signature, M




protein repeat
















TABLE XXII-V1-HLA-A1







9mers: STEAP-1


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


each start position is specified, the length


of peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.











Pos
123456789
score






 99
ATSHQQYFY
24


 



122
SITLLALVY
24


 



180
LSYPMRRSY
24


 



174
LHAIYSLSY
22


 



278
IDIKQFVWY
20


 



139
VQLHNGTKY
19


 



 32
TGETSMLKR
18


 



244
SLTWREFHY
18


 



 80
IIASLTFLY
17


 



153
WLDKWMLTR
17


 



182
YPMRRSYRY
17


 



 22
LEEDDYLHK
16


 



 58
PSELQHTQE
16


 



 97
PLATSHQQY
16


 



170
FFAVLHAIY
16


 



189
RYKLLNWAY
16


 



 19
RRNLEEDDY
15


 



 84
LTFLYTLLR
15


 



211
HDVWRMEIY
15


 



241
VSDSLTWRE
15


 



286
YTPPTFMIA
15


 



203
NKEDAWIEH
14


 



215
RMEIYVSLG
14


 



247
WREFHYIQS
14


 



324
WEDVTKINK
14


 



 21
NLEEDDYLH
13


 



 23
EEDDYLHKD
13


 



 54
EFDCPSELQ
13


 



159
LTRKQFGLL
13


 



186
RSYRYKLLN
13


 



  7
ITNQEELWK
12


 



 10
QEELWKMKP
12


 



 29
HKDTGETSM
12


 



 38
LKRPVLLHL
12


 



 91
LREVIHPLA
12


 



129
VYLPGVIAA
12


 



221
SLGIVGLAI
12


 



291
FMIAVFLPI
12


 



323
GWEDVTKIN
12


 



  4
RKDITNQEE
11


 



  9
NQEELWKMK
11


 



 37
MLKRPVLLH
11


 



 52
ADEFDCPSE
11


 



 55
FDCPSELQH
11


 



208
WIEHDVWRM
11


 



210
EHDVWRMEI
11


 



256
KLGIVSLLL
11


 



277
WIDIKQFVW
11


 



296
FLPIVVLIF
11
















TABLE XXII-V2-HLA-A1







9mers: STEAP-1


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


each start position is specified, the length


of peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.









Pos
123456789
score





2
WREFHYIQV
14
















TABLE XXII-V3-HLA-A1







9mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 7; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score












13
KSDVPESLW
17


 


1
WREFHYIQI
14


 


20
LWDPCLTRF
14


 


25
LTRFKGLNL
13


 


14
SDVPESLWD
12


 


16
VPESLWDPC
11


 


19
SLWDPCLTR
10


 


27
RFKGLNLIQ
8
















TABLE XXII-V4-HLA-A1







9mers: STEAP-1


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


each start position is specified, the length


of peptide is 9 amino acids, and the end


position for each peptide is the start


position plus eight.









Pos
123456789
score












8
SLFFAVLHA
10


 


1
RKQFGLLSL
8


 


3
QFGLLSLFF
6


 


7
LSLFFAVLH
6


 


6
LLSLFFAVL
5


 


5
GLLSLFFAV
4
















TABLE XXIII-V1-HLA-A0201







9mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 3; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight









Pos
123456789
score












262
LLLGTIHAL
32


 


76
KIAAIIASL
29


 


86
FLYTLLREV
29


 


90
LLREVIHPL
29


 


130
YLPGVIAAI
29


 


72
HLPIKIAAI
26


 


79
AIIASLTFL
26


 


292
MIAVFLPIV
26


 


302
LIFKSILFL
26


 


312
CLRKKILKI
26


 


113
VINKVLPMV
25


 


126
LALVYLPGV
25


 


165
GLLSFFFAV
25


 


172
AVLHAIYSL
25


 


219
YVSLGIVGL
25


 


252
YIQSKLGIV
25


 


36
SMLKRPVLL
24


 


123
ITLLALVYL
24


 


166
LLSFFFAVL
24


 


227
LAILALLAV
24


 


294
AVGLPIVVL
24


 


117
VLPMVSITL
23


 


133
GVIAAIVQL
23


 


192
LLNWAYQQV
23


 


228
AILALLAVT
23


 


233
LAVTSIPSV
23


 


82
ASLTFLYTL
22


 


221
SLGIVGLAI
22


 


222
LGIVGLAIL
22


 


224
IVGLAILAL
22


 


230
LALLAVTSI
22


 


237
SIPSVSDSL
22


 


256
KLGIVSLLL
22


 


261
SLLLGTIHA
22


 


263
LLGTIHALI
22


 


83
SLTFLYTLL
21


 


109
IPILVINKV
21


 


158
MLTRKQFGL
21


 


259
IVSLLLGTI
21


 


309
FLPCLRKKI
21


 


319
KIRHGWEDV
21


 


27
YLHKDTGET
20


 


42
VLLHLHQTA
20


 


120
MVSITLLAL
20


 


121
VSITLLALV
20


 


127
ALVYLPGVI
20


 


217
EIYVSLGIV
20


 


255
SKLGIVSLL
20


 


258
GIVSLLLGT
20


 


291
FMIAVFLPI
20


 


38
LKRPVLLHL
19


 


225
VGLAILALL
19


 


226
GLAILALLA
19


 


229
ILALLAVTS
19


 


254
QSKLGIVSL
19


 


295
VFLPIVVLI
19


 


305
KSILFLPCL
19


 


59
SELQHTQEL
18


 


112
LVINKVLPM
18


 


124
TLLALVYLP
18


 


270
LIFAWNKWI
18


 


300
VVLIFKSIL
18


 


116
KVLPMVSIT
17


 


118
LPMVSITLL
17


 


137
AIVQLHNGT
17


 


176
AIYSLSYPM
17


 


214
WRMEIYVSL
17


 


322
HGWEDVTKI
17


 


89
TLLREVIHP
16


 


104
QYFYKIPIL
16


 


110
PILVINKVL
16


 


129
VYLPGVIAA
16


 


159
LTRKQFGLL
16


 


169
FFFAVLHAI
16


 


223
GIVGLAILA
16


 


231
ALLAVTSIP
16


 


245
LTWREFHYI
16


 


269
ALIFAWNKW
16


 


293
IAVFLPIVV
16


 


296
FLPIVVLIF
16


 


5
KDITNQEEL
15


 


37
MLKRPVLLH
15


 


80
IIASLTFLY
15


 


125
LLALVYLPG
15


 


134
VIAAIVQLH
15


 


208
WIEHDVWRM
15


 


265
GTIHALIFA
15


 


272
FAWNKWIDI
15


 


279
DIKQFVWYT
15


 


299
IVVLIFKSI
15


 


307
ILFLPCLRK
15

















TABLE XXIII-V2-HLA-A0201







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 5; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













5
FHYIQVNNI
15



 


2
WREFHYIQV
8

















TABLE XXIII-V3-HLA-A0201







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 7; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













8
QIIHKKSDV
21



 


19
SLWDPCLTR
18


 


25
LTRFKGLNL
17


 


26
TRFKGLNLI
16


 


2
REFHYIQII
14


 


12
KKSDVPESL
14


 


6
YIQIIHKKS
11


 


9
IIHKKSDVP
11


 


24
CLTRFKGLN
10
















TABLE XXIII-V4-HLA-0201







9mers: STEAP-1


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


each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score












5
GLLSLFFAV
25


 


6
LLSLFFAVL
25


 


8
SLFFAVLHA
21


 


1
RKQFGLLSL
18


 


9
LFFAVLHAI
17
















TABLE XXIV-V1-HLA-A202







9mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 3; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight









Pos
123456789
score






NoResultsfound.

















TABLE XXIV-V2-HLA-A0202







9mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 5; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight









Pos
123456789
score






NoResultsfound.

















TABLE XXIV-V3-HLA-A0202







9mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 7; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight


Each peptide is a portion of SEQ


ID NO: 7; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight









Pos
123456789
score






NoResultsfound.

















TABLE XXIV-V4-HLA-A0202







9mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 9; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight









Pos
123456789
score






NoResultsfound.

















TABLE XXV-V1-A0203







9mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 3; each start position is


specified, the length of peptide is


9 amino adds, and the end


position for each peptide is the


start position plus eight









Pos
123456789
score






NoResultsfound.

















TABLE XXV-V2-HLA-A0203







9mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 5; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight









Pos
123456789
score










No Results found.
















TABLE XXV-V3-HLA-A0203







9mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 7; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight









Pos
123456789
score










No Results found.
















TABLE XXV-V4-HLA-A0203







9mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 9; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight









Pos
123456789
score










No Results found.

















TABLE XXVI-V1-HLA-A3







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 3; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













138
IVQLHNGTK
28



 


122
SITLLALVY
26


 


307
ILFLPCLRK
26


 


140
QLHNGTKYK
25


 


229
ILALLAVTS
25


 


108
KIPILVINK
22


 


128
LVYLPGVIA
22


 


133
GVIAAIVQL
22


 


191
KLLNWAYQQ
22


 


228
AILALLAVT
22


 


294
AVFLPIVVL
22


 


321
RHGWEDVTK
22


 


37
MLKRPVLLH
21


 


94
VIHPLATSH
21


 


116
KVLPMVSIT
21


 


153
WLDKWMLTR
21


 


231
ALLAVTSIP
21


 


234
AVTSIPSVS
21


 


93
EVIHPLATS
20


 


97
PLATSHQQY
20


 


221
SLGIVGLAI
20


 


76
KIAAIIASL
49


 


127
ALVYLPGVI
19


 


148
KKFPHWLDK
19


 


244
SLTWREFHY
19


 


283
FVWYTPPTF
19


 


296
FLPIVVLIF
19


 


78
AAIIASLTF
18


 


112
LVINKVLPM
18


 


226
GLAILALLA
18


 


240
SVSDSLTWR
18


 


256
KLGIVSLLL
18


 


259
IVSLLLGTI
18


 


301
VLIFKSILF
18


 


317
ILKIRHGWE
18


 


43
LLHLHQTAH
17


 


79
AIIASLTFL
17


 


80
IIASLTFLY
17


 


111
ILVINKVLP
17


 


130
YLPGVIAAI
17


 


166
LLSFFFAVL
17


 


172
AVLHAIYSL
17


 


219
YVSLGIVGL
17


 


224
IVGLAILAL
17


 


312
CLRKKILKI
17


 


7
ITNQEELWK
16


 


41
PVLLHLHQT
16


 


42
VLLHLHQTA
16


 


72
HLPIKIAAI
16


 


125
LLALVYLPG
16


 


180
LSYPMRRSY
16


 


183
PMRRSYRYK
16


 


207
AWIEHDVWR
16


 


248
REFHYIQSK
16


 


261
SLLLGTIHA
16


 


268
HALIFAWNK
16


 


269
ALIFAWNKW
16


 


278
IDIKQFVWY
16


 


300
VVLIFKSIL
16


 


306
SILFLPCLR
16


 


311
PCLRKKILK
16


 


319
KIRHGWEDV
16


 


21
NLEEDDYLH
15


 


89
TLLREVIHP
15


 


90
LLREVIHPL
15


 


117
VLPMVSITL
15


 


120
MVSITLLAL
15


 


134
VIAAIVQLH
15


 


161
RKQFGLLSF
15


 


173
VLHAIYSLS
15


 


176
AIYSLSYPM
15


 


262
LLLGTIHAL
15


 


299
IVVLIFKSI
15


 


308
LFLPCLRKK
15


 


12
ELWKMKPRR
14


 


22
LEEDDYLHK
14


 


45
HLHQTAHAD
14


 


60
ELQHTQELF
14


 


86
FLYTLLREV
14


 


110
PILVINKVL
14


 


124
TLLALVYLP
14


 


158
MLTRKQFGL
14


 


186
RSYRYKLLN
14


 


196
AYQQVQQNK
14


 


277
WIDIKQFVW
14


 


297
LPIVVLIFK
14


 


316
KILKIRHGW
14


 


326
DVTKINKTE
14


 


55
FDCPSELQH
13


 


66
ELFPQWHLP
13


 


74
PIKIAAIIA
13


 


88
YTLLREVIH
13


 


165
GLLSFFFAV
13


 


174
LHAIYSLSY
13


 


189
RYKLLNWAY
13


 


192
LLNWAYQQV
13


 


212
DVWRMEIYV
13


 


217
EIYVSLGIV
13


 


223
GIVGLAILA
13


 


263
LLGTIHALI
13


 


273
AWNKWIDIK
13
















TABLE XXVI-V2-HLA-A3







9mers: STEAP-1


Each pepfide is a portion of SEQ


ID NO: 5; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













3
REFHYIQVN
8



 


5
FHYIQVNNI
5


 


1
TWREFHYIQ
4


 


4
EFHYIQVNN
4


 


2
WREFHYIQV
3

















TABLE XXVI-V3-HLA-A3







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 7; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













19
SLWDPCLTR
23



 


8
QIIHKKSDV
17


 


4
FHYIQIIHK
15


 


9
IIHKKSDVP
15


 


24
CLTRFKGLN
14


 


5
HYIQIIHKK
13


 


15
DVPESLWDP
11
















TABLE XXVI-V4-HLA-A3







9mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 9; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













6
LLSLFFAVL
18



 


8
SLFFAVLHA
14


 


5
GLLSLFFAV
13


 


7
LSLFFAVLH
13


 


2
KQFGLLSLF
12


 


1
RKQFGLLSL
11


 


3
QFGLLSLFF
10

















TABLE XXVII-V1-HLA-A26







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 3; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight









Pos
123456789
score













133
GVIAAIVQL
26



 


302
LIFKSILFL
26


 


76
KIAAIIASL
25


 


79
AIIASLTFL
25


 


112
LVINKVLPM
25


 


294
AVFLPIVVL
25


 


60
ELQHTQELF
24


 


159
LTRKQFGLL
24


 


219
YVSLGIVGL
24


 


80
IIASLTFLY
23


 


93
EVIHPLATS
23


 


120
MVSITLLAL
23


 


123
ITLLALVYL
23


 


172
AVLHAIYSL
23


 


208
WIEHDVWRM
23


 


224
IVGLAILAL
23


 


262
LLLGTIHAL
23


 


296
FLPIVVLIF
23


 


90
LLREVIHPL
22


 


97
PLATSHQQY
22


 


249
EFHYIQSKL
22


 


279
DIKQFVWYT
22


 


83
SLTFLYTLL
21


 


99
ATSHQQYFY
21


 


122
SITLLALVY
21


 


166
LLSFFFAVL
21


 


217
EIYVSLGIV
21


 


237
SIPSVSDSL
21


 


283
FVWYTPPTF
21


 


289
PTFMIAVFL
21


 


301
VLIFKSILF
21


 


162
KQFGLLSFF
20


 


176
AIYSLSYPM
20


 


31
DTGETSMLK
19


 


53
DEFDCPSEL
19


 


63
HTQELFPQW
19


 


66
ELFPQWHLP
19


 


170
FFAVLHAIY
19


 


244
SLTWREFHY
19


 


278
IDIKQFVWY
19


 


300
VVLIFKSIL
19


 


110
PILVINKVL
18


 


158
MLTRKQFGL
18


 


286
YTPPTFMIA
18


 


6
DITNQEELW
17


 


12
ELWKMKPRR
17


 


34
ETSMLKRPV
17


 


108
KIPILVINK
17


 


113
VINKVLPMV
17


 


116
KVLPMVSIT
17


 


117
VLPMVSITL
17


 


130
YLPGVIAAI
17


 


163
QFGLLSFFF
17


 


258
GIVSLLLGT
17


 


326
DVTKINKTE
17


 


72
HLPIKIAAI
16


 


104
QYFYKIPIL
16


 


134
VIAAIVQLH
16


 


142
HNGTKYKKF
16


 


212
DVWRMEIYV
16


 


214
WRMEIYVSL
16


 


228
AILALLAVT
16


 


240
SVSDSLTWR
16


 


252
YIQSKLGIV
16


 


256
KLGIVSLLL
16


 


265
GTIHALIFA
16


 


266
TIHALIFAW
16


 


292
MIAVFLPIV
16


 


298
PIVVLIFKS
16


 


8
TNQEELWKM
15


 


38
LKRPVLLHL
15


 


41
PVLLHLHQT
15


 


161
RKQFGLLSF
15


 


222
LGIVGLAIL
15


 


254
QSKLGIVSL
15


 


255
SKLGIVSLL
15


 


259
IVSLLLGTI
15


 


275
NKWIDIKQF
15


 


288
PPTFMIAVF
15


 


299
IVVLIFKSI
15


 


305
KSILFLPCL
15


 


312
CLRKKILKI
15


 


56
DCPSELQHT
14


 


86
FLYTLLREV
14


 


94
VIHPLATSH
14


 


153
WLDKWMLTR
14


 


165
GLLSFFFAV
14


 


192
LLNWAYQQV
14


 


211
HDVWRMEIY
14


 


245
LTWREFHYI
14


 


23
EEDDYLHKD
13


 


37
MLKRPVLLH
13


 


54
EFDCPSELQ
13


 


89
TLLREVIHP
13


 


118
LPMVSITLL
13


 


124
TLLALVYLP
13


 


125
LLALVYLPG
13


 


137
AIVQLHNGT
13


 


168
SFFFAVLHA
13


 


169
FFFAVLHAI
13


 


173
VLHAIYSLS
13


 


179
SLSYPMRRS
13


 


180
LSYPMRRSY
13


 


189
RYKLLNWAY
13


 


223
GIVGLAILA
13


 


225
VGLAILALL
13


 


295
VFLPIVVLI
13


 


308
LFLPCLRKK
13


 


2
ESRKDITNQ
12


 


30
KDTGETSML
12


 


78
AAIIASLTF
12


 


82
ASLTFLYTL
12


 


84
LTFLYTLLR
12


 


98
LATSHQQYF
12


 


144
GTKYKKFPH
12


 


149
KFPHWLDKW
12


 


174
LHAIYSLSY
12


 


185
RRSYRYKLL
12


 


234
AVTSIPSVS
12


 


235
VTSIPSVSD
12


 


270
LIFAWNKWI
12


 


277
WIDIKQFVW
12


 


303
IFKSILFLP
12


 


316
KILKIRHGW
12


 


325
EDVTKINKT
12


 


327
VTKINKTEI
12


 


331
NTKEICSQL
12

















TABLE XXVII-V2-HLA-A26







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 5; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score












4
EFHYIQVNN
14


 


3
REFHYIQVN
7


 


5
FHYIQVNNI
6

















TABLE XXVII-V3-HLA-A26







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 7; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













15
DVPESLWDP
24



 


25
LTRFKGLNL
20


 


20
LWDPCLTRF
18


 


8
QIIHKKSDV
14


 


23
PCLTRFKGL
13


 


3
EFHYIQIIH
12


 


9
IIHKKSDVP
12


 


6
YIQIIHKKS
11

















TABLE XXVII-V4-HLA-26







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 9; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













6
LLSLFFAVL
21



 


2
KQFGLLSLF
20


 


3
QFGLLSLFF
17


 


5
GLLSLFFAV
15


 


8
SLFFAVLHA
15


 


1
RKQFGLLSL
13


 


9
LFFAVLHAI
13

















TABLE XXVIII-V1-HLA-B0702







mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 3; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













118
LPMVSITLL
22



 


310
LPCLRKKIL
20


 


238
IPSVSDSLT
19


 


73
LPIKIAAII
18


 


131
LPGVIAAIV
18


 


288
PPTFMIAVF
18


 


294
AVFLPIVVL
18


 


109
IPILVINKV
17


 


287
TPPTFMIAV
17


 


14
WKMKPRRNL
16


 


150
FPHWLDKWM
16


 


38
LKRPVLLHL
15


 


79
AIIASLTFL
15


 


120
MVSITLLAL
15


 


166
LLSFFFAVL
15


 


224
IVGLAILAL
15


 


30
KDTGETSML
14


 


35
TSMLKRPVL
14


 


76
KIAAIIASL
14


 


159
LTRKQFGLL
14


 


219
YVSLGIVGL
14


 


256
KLGIVSLLL
14


 


302
LIFKSILFL
14


 


17
KPRRNLEED
13


 


36
SMLKRPVLL
13


 


40
RPVLLHLHQ
13


 


65
QELFPQWHL
13


 


90
LLREVIHPL
13


 


123
ITLLALVYL
13


 


133
GVIAAIVQL
13


 


184
MRRSYRYKL
13


 


185
RRSYRYKLL
13


 


214
WRMEIYVSL
13


 


255
SKLGIVSLL
13


 


262
LLLGTIHAL
13


 


289
PTFMIAVFL
13


 


57
CPSELQHTQ
12


 


81
IASLTFLYT
12


 


82
ASLTFLYTL
12


 


106
FYKIPILVI
12


 


128
LVYLPGVIA
12


 


146
KYKKFPHWL
12


 


172
AVLHAIYSL
12


 


225
VGLAILALL
12


 


254
QSKLGIVSL
12


 


305
KSILFLPCL
12


 


5
KDITNQEEL
11


 


20
RNLEEDDYL
11


 


59
SELQHTQEL
11


 


68
FPQWHLPIK
11


 


83
SLTFLYTLL
11


 


96
HPLATSHQQ
11


 


104
QYFYKIPIL
11


 


110
PILVINKVL
11


 


115
NKVLPMVSI
11


 


182
YPMTTSYRY
11


 


221
SLGIVGLAI
11


 


222
LGIVGLAIL
11


 


228
AILALLAVT
11


 


249
EFHYIQSKL
11


 


284
VWYTPPTFM
11


 


285
WYTPPTFMI
11


 


291
FMIAVFLPI
11


 


297
LPIVVLIFK
11


 


312
CLRKKILKI
11


 


34
ETSMLKRPV
10


 


53
DEFDCPSEL
10


 


78
AAIIASLTF
10


 


92
REVIHPLAT
10


 


112
LVINKVLPM
10


 


117
VLPMVSITL
10


 


130
YLPGVIAAI
10


 


151
PHWLDKWML
10


 


158
MLTRKQFGL
10


 


162
KQFGLLSFF
10


 


237
SIPSVSDSL
10


 


295
VFLPIVVLI
10


 


300
VVLIFKSIL
10


 


320
IRHGWEDVT
10


 


331
NKTEICSQL
10

















TABLE XXVIII-V2-HLA-B0702







9mers:STEAP-1



Each peptide is a portion of SEQ


ID NO: 5; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













5
FHYIQVNNI
7



 


2
WREFHYIQV
6


 


4
EFHYIQVNN
3

















TABLE XXVIII-V3-HLA-B0702







9mers: STEAP-1



Each pepUde is a portion of SEQ


ID NO: 7; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













6
LLSLFFAVL
15



 


1
RKQFGLLSL
13


 


3
QFGLLSLFF
10


 


2
KQFGLLSLF
9


 


8
SLFFAVLHA
9


 


5
GLLSLFFAV
8


 


9
LFFAVLHAI
8


 


4
FGLLSLFFA
7

















TABLE XXVIII-V4-HLA-B0702







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 9; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score












6
LLSLFFAVL
15


 


1
RKQFGLLSL
13


 


3
QFGLLSLFF
10


 


2
KQFGLLSLF
9


 


8
SLFFAVLHA
9


 


5
GLLSLFFAV
8


 


9
LFFAVLHAI
8


 


4
FGLLSLFFA
7

















TABLE XXIX-V1-HLA-B08







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 3; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













312
CLRKKILKI
29



 


158
MLTRKQFGL
26


 


310
LPCLRKKIL
26


 


72
HLPIKIAAI
25


 


90
LLREVIHPL
25


 


317
ILKIRHGWE
25


 


254
QSKLGIVSL
23


 


301
VLIFKSILF
23


 


104
QYFYKIPIL
22


 


144
GTKYKKFPH
22


 


35
TSMLKRPVL
21


 


36
SMLKRPVLL
21


 


272
FAWNKWIDI
21


 


13
LWKMKPRRN
20


 


146
KYKKFPHWL
20


 


185
RRSYRYKLL
20


 


187
SYRYKLLNW
19


 


15
KMKPRRNLE
18


 


83
SLTFLYTLL
18


 


106
FYKIPILVI
18


 


118
LPMVSITLL
18


 


142
HNGTKYKKF
18


 


262
LLLGTIHAL
18


 


327
VTKINKTEI
18


 


37
MLKRPVLLH
17


 


110
PILVINKVL
17


 


117
VLPMVSITL
17


 


38
LKRPVLLHL
16


 


159
LTRKQFGLL
16


 


166
LLSFFFAVL
16


 


184
MRRSYRYKL
16


 


237
SIPSVSDSL
16


 


244
SLTWREFHY
16


 


256
KLGIVSLLL
16


 


76
KIAAIIASL
15


 


221
SLGIVGLAI
15


 


279
DIKQFVWYT
15


 


302
LIFKSILFL
15


 


60
ELQHTQELF
14


 


74
PIKIAAIIA
14


 


79
AIIASLTFL
14


 


130
YLPGVIAAI
14


 


182
YPMRRSYRY
14


 


224
IVGLAILAL
14


 


252
YIQSKLGIV
14


 


277
WIDIKQFVW
14


 


296
FLPIVVLIF
14


 


309
FLPCLRKKI
14


 


11
EELWKMKPR
13


 


17
KPRRNLEED
13


 


59
SELQHTQEL
13


 


123
ITLLALVYL
13


 


127
ALVYLPGVI
13


 


133
GVIAAIVQL
13


 


189
RYKLLNWAY
13


 


214
WRMEIYVSL
13


 


219
YVSLGIVGL
13


 


249
EFHYIQSKL
13


 


255
SKLGIVSLL
13


 


288
PPTFMIAVF
13


 


294
AVFLPIVVL
13


 


325
EDVTKINKT
13

















TABLE XXIX-V2-HLA-B08







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 5; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













1
TWREFHYIQ
8



 


5
FHYIQVNNI
8


 


4
EFHYIQVNN
4

















TABLE XXIX-V3-HLA-B08







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 7; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













25
LTRFKGLNL
26



 


23
PCLTRFKGL
19


 


9
IIHKKSDVP
15


 


8
QIIHKKSDV
14


 


11
HKKSDVPES
13


 


10
IHKKSDVPE
12

















TABLE XXIX-V4-HLA-B08







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 9; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













6
LLSLFFAVL
17



 


1
RKQFGLLSL
10


 


8
SLFFAVLHA
9


 


5
GLLSLFFAV
8

















TABLE XXX-V1-HLA-B1510







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 3; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













151
PHWLDKWML
21



 


219
YVSLGIVGL
16


 


294
AVFLPIVVL
16


 


14
WKMKPRRNL
15


 


35
TSMLKRPVL
15


 


36
SMLKRPVLL
14


 


71
WHLPIKIAA
14


 


123
ITLLALVYL
14


 


133
GVIAAIVQL
14


 


210
EHDVWRMEI
14


 


224
IVGLAILAL
14


 


254
QSKLGIVSL
14


 


53
DEFDCPSEL
13


 


110
PILVINKVL
13


 


166
LLSFFFAVL
13


 


214
WRMEIYVSL
13


 


262
LLLGTIHAL
13


 


267
IHALIFAWN
13


 


321
RHGWEDVTK
13


 


5
KDITNQEEL
12


 


28
LHKDTGETS
12


 


38
LKRPVLLHL
12


 


50
AHADEFDCP
12


 


59
SELQHTQEL
12


 


76
KIAAIIASL
12


 


90
LLREVIHPL
12


 


95
IHPLATSHQ
12


 


104
QYFYKIPIL
12


 


118
LPMVSITLL
12


 


120
MVSITLLAL
12


 


141
LHNGTKYKK
12


 


146
KYKKFPHWL
12


 


222
LGIVGLAIL
12


 


255
SKLGIVSLL
12


 


289
PTFMIAVFL
12


 


302
LIFKSILFL
12


 


20
RNLEEDDYL
11


 


30
KDTGETSML
11


 


62
QHTQELFPQ
11


 


65
QELFPQWHL
11


 


82
ASLTFLYTL
11


 


101
SHQQYFYKI
11


 


117
VLPMVSITL
11


 


159
LTRKQFGLL
11


 


172
AVLHAIYSL
11


 


174
LHAIYSLSY
11


 


184
MRRSYRYKL
11


 


185
RRSYRYKLL
11


 


249
EFHYIQSKL
11


 


300
VVLIFKSIL
11


 


305
KSILFLPCL
11


 


310
LPCLRKKIL
11


 


331
NKTEICSQL
11


 


44
LHLHQTAHA
10


 


46
LHQTAHADE
10


 


79
AIIASLTFL
10


 


83
SLTFLYTLL
10


 


158
MLTRKQFGL
10


 


208
WIEHDVWRM
10


 


225
VGLAILALL
10


 


237
SIPSVSDSL
10


 


242
SDSLTWREF
10


 


250
FHYIQSKLG
10


 


256
KLGIVSLLL
10


 


283
FYWYTPPTF
9


 


288
PPTFMIAVF
9

















TABLE XXX-V2-HLA-B1510







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 5; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score












5
FHYIQVNNI
11

















TABLE XXX-V3-B1510







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 7; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













10
IHKKSDVPE
15



 


12
KKSDVPESL
13


 


17
PESLWDPCL
12


 


4
FHYIQIIHK
11


 


20
LWDPCLTRF
10


 


23
PCLTRFKGL
10


 


25
LTRFKGLNL
10
















TABLE XXX-V4-HLA-B1510







9mers: STEAP-1


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


each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight









Pos
123456789
score












6
LLSLFFAVL
13


 


1
RKQFGLLSL
11


 


2
KQFGLLSLF
1


 


3
QFGLLSLFF
6
















TABLE XXXI-V1-HLA-B2705







9mers: STEAP-1


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


each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide isthe


start position plus eight.









Pos
123456789
score












19
RRNLEEDDY
24


 


185
RRSYRYKLL
24


 


214
WRMEIYVSL
24


 


313
LRKKILKIR
24


 


184
MRRSYRYKL
23


 


39
KRPVLLHLH
22


 


248
REFHYIQSK
21


 


294
AVFLPIVVL
21


 


133
GVIAAIVQL
20


 


162
KQFGLLSFF
20


 


302
LIFKSILFL
20


 


20
RNLEEDDYL
19


 


161
RKQFGLLSF
19


 


307
ILFLPCLRK
19


 


76
KIAAIIASL
18


 


104
QYFYKIPIL
18


 


172
AVLHAIYSL
18


 


196
AYQQVQQNK
18


 


254
QSKLGIVSL
18


 


321
RHGWEDVTK
18


 


5
KOITNQEEL
17


 


79
AIIASLTFL
17


 


108
KIPILVINK
17


 


148
KKFPHWLDK
17


 


188
YRYKLLNWA
17


 


219
YVSLGIVGL
17


 


255
SKLGIVSLL
17


 


262
LLLGTIHAL
17


 


305
KSILFLPCL
17


 


314
RKKILKIRH
17


 


12
ELWKMKPRR
16


 


30
KDTGETSML
16


 


65
GELFPQWHL
16


 


78
AAIIASLTF
16


 


82
ASLTFLYTL
16


 


118
LPMVSITLL
16


 


123
ITLLALVYL
16


 


141
LHNGTKYKK
16


 


189
RYKLLNWAY
16


 


207
AWIEHDVWR
16


 


222
LGIVGLAIL
16


 


224
IVGLAILAL
16


 


249
EFHYIQSKL
16


 


268
HALIFAWNK
16


 


273
AWNKWIDIK
16


 


289
PTFMIAVFL
16


 


308
LFLPCLRKK
16


 


7
ITNQEELWK
15


 


14
WKMKPRRNL
15


 


36
SMLKRPVLL
15


 


53
DEFDCPSEL
15


 


59
SELQHTQEL
15


 


84
LTFLYTLLR
15


 


90
LLREVIHPL
15


 


110
PILVINKVL
15


 


154
LDKWMLTRK
15


 


178
YSLSYPMRR
15


 


225
VGLAILALL
15


 


256
KLGIVSLLL
15


 


288
PPTFMIAVF
15


 


297
LPIVVLIFK
15


 


301
VLIFKSILF
15


 


311
PCLRKKILK
15


 


312
CLRKKILKI
15


 


8
TNQEELWKM
14


 


32
TGETSMLKR
14


 


35
TSMLKRPVL
14


 


38
LKRPVLLHL
14


 


94
VIHPLATSH
14


 


117
VLPMVSITL
14


 


138
IVQLHNGTK
14


 


139
VQLHNGTKY
14


 


146
KYKKFPHWL
14


 


153
WLDKWMLTR
14


 


156
KWMLTRKQF
14


 


176
AIYSLSYPM
14


 


177
IYSLSYPMR
14


 


240
SVSDSLTWR
14


 


260
VSLLLGTIH
14


 


275
NKWIDIKQF
14


 


278
IDIKQFVWY
14


 


300
VVLIFKSIL
14


 


320
IRHGWEDVT
14


 


324
WEDVTKINK
14


 


331
NKTEICSQL
14


 


3
SRKDITNQE
13


 


11
EELWKMKPR
13


 


29
HKDTGETSM
13


 


31
DTGETSMLK
13


 


43
LLHLHQTAH
13


 


73
LPIKIAAII
13


 


83
SLTFLYTLL
13


 


112
LVINKVLPM
13


 


122
SITLLALVY
13


 


130
YLPGVIAAI
13


 


142
HNGTKYKKF
13


 


144
GTKYKKFPH
13


 


151
PHWLDKWML
13


 


158
MLTRKQFGL
13


 


160
TRKQFGLLS
13


 


163
QFGLLSFFF
13


 


167
LSFFFAVLH
13


 


230
LALLAVTSI
13


 


237
SIPSVSDSL
13


 


251
HYIQSKLGI
13


 


284
VWYTPPTFM
13


 


296
FLPIVVLIF
13


 


306
SILFLPCLR
13


 


9
NQEELWKMK
12


 


47
HQTAHADEF
12


 


55
FDCPSELQH
12


 


60
ELQHTQELF
12


 


68
FPQWHLPIK
12


 


88
YTLLREVIH
12


 


91
LREVIHPLA
12


 


98
LATSHQQYF
12


 


99
ATSHQQYFY
12


 


100
TSHQQYFYK
12


 


106
FYKIPILVI
12


 


120
MVSITLLAL
12


 


140
QLHNGTKYK
12


 


159
LTRKQFGLL
12


 


166
LLSFFFAVL
12


 


180
LSYPMRRSY
12


 


182
YPMRRSYRY
12


 


208
WIEHDVWRM
12


 


242
SDSLTWREF
12


 


247
WREFHYIQS
12


 


264
LGTIHALIF
12


 


283
FVWYTPPTF
12


 


295
VFLPIVVLI
12


 


310
LPCLRKKIL
12


 


322
HGWEDVTKI
12


 


18
PRRNLEEDD
11


 


21
NLEEDDYLH
11


 


22
LEEDDYLHK
11


 


37
MLKRPVLLH
11


 


72
HLPIKIAAI
11


 


101
SHQQYFYKI
11


 


109
IPILVINKV
11


 


115
NKVLPMVSI
11


 


169
FFFAVLHAI
11


 


174
LHAIYSLSY
11


 


181
SYPMRRSYR
11


 


183
PMRRSYRYK
11


 


201
QQNKEDAWI
11


 


216
MEIYVSLGI
11


 


259
IVSLLLGTI
11


 


299
IVVLIFKSI
11

















TABLE XXXI-V2-HLA-B2705







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 5; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













2
WREFHYIQV
12



 


5
FHYIQVNNI
12


 


3
REFHYIQVN
11


 


4
EFHYIQVNN
6

















TABLE XXXI-V3-HLA-B2705







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 7; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













26
TRFKGLNLI
25



 


5
HYIQIIHKK
20


 


1
WREFHYIQI
18


 


2
REFHYIQII
16


 


4
FHYIQIIHK
16


 


20
LWDPCLTRF
16


 


12
KKSDVPESL
14


 


25
LTRFKGLNL
14


 


17
PESLWDPCL
13


 


19
SLWDPCLTR
13


 


3
EFHYIQIIH
12


 


23
PCLTRFKGL
12

















TABLE XXXI-V4-HLA-B2705







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 9; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













2
KQFGLLSLF
20



 


1
RKQFGLLSL
19


 


3
QFGLLSLFF
14


 


6
LLSLFFAVL
13


 


7
LSLFFAVLH
12


 


9
LFFAVLHAI
11

















TABLE XXXII-V1-HLA-B2709







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 3; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













185
RRSYRYKLL
24



 


184
MRRSYRYKL
22


 


214
WRMEIYVSL
22


 


20
RNLEEDDYL
16


 


133
GVIAAIVQL
16


 


289
PTFMIAVFL
15


 


19
RRNLEEDDY
14


 


82
ASLTFLYTL
14


 


123
ITLLALVYL
14


 


165
GLLSFFFAV
14


 


172
AVLHAIYSL
14


 


255
SKLGIVSLL
14


 


294
AVFLPIVVL
14


 


295
VFLPIVVLI
14


 


305
KSILFLPCL
14


 


5
KDITNQEEL
13


 


30
KDTGETSML
13


 


36
SMLKRPVLL
13


 


76
KIAAIIASL
13


 


104
QYFYKIPIL
13


 


110
PILVINKVL
13


 


161
RKQFGLLSF
13


 


176
AIYSLSYPM
13


 


225
VGLAILALL
13


 


256
KLGIVSLLL
13


 


302
LIFKSILFL
13


 


331
NKTEICSQL
13


 


3
SRKDITNQE
12


 


38
LKRPVLLHL
12


 


39
KRPVLLHLH
12


 


53
DEFDCPSEL
12


 


59
SELQHTQEL
12


 


65
QELFPQWHL
12


 


79
AIIASLTFL
12


 


91
LREVIHPLA
12


 


146
KYKKFPHWL
12


 


162
KQFGLLSFF
12


 


188
YRYKLLNWA
12


 


224
IVGLAILAL
12


 


262
LLLGTIHAL
12


 


276
KWIDIKQFV
12


 


300
VVLIFKSIL
12


 


14
WKMKPRRNL
11


 


35
TSMLKRPVL
11


 


73
LPIKIAAII
11


 


78
AAIIASLTF
11


 


83
SLTFLYTLL
11


 


105
YFYKIPILV
11


 


112
LVINKVLPM
11


 


120
MVSITLLAL
11


 


126
LALVYLPGV
11


 


151
PHWLDKWML
11


 


156
KWMLTRKQF
11


 


158
MLTRKQFGL
11


 


160
TRKQFGLLS
11


 


208
WIEHDVWRM
11


 


219
YVSLGIVGL
11


 


222
LGIVGLAIL
11


 


230
LALLAVTSI
11


 


237
SIPSVSDSL
11


 


247
WREFHYIQS
11


 


249
EFHYIQSKL
11


 


254
QSKLGIVSL
11


 


264
LGTIHALIF
11


 


299
IVVLIFKSI
11


 


313
LRKKILKIR
11


 


320
IRHGWEDVT
11

















TABLE XXXII-V2-HLA-B2709







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 5; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













2
WREFHYIQV
19



 


5
FHYIQVNNI
12

















TABLE XXXII-V3-B2709







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 5; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













26
TRFKGLNLI
20



 


1
WREFHYIQI
19


 


2
REFHYIQII
14


 


12
KKSDVPESL
12


 


23
PCLTRFKGL
12


 


25
LTRFKGLNL
12


 


17
PESLWDPCL
11


 


20
LWDPCLTRF
10


 


8
QIIHKKSDV
9

















TABLE XXXII-V4-HLA-B2709







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 9; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score












1
RKQFGLLSL
15


 


5
GLLSLFFAV
14


 


2
KQFGLLSLF
12


 


6
LLSLFFAVL
10


 


9
LFFAVLHAI
10


 


3
QFGLLSLFF
9

















TABLE XXXIII-V1-HLA-B4402







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 3; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













53
DEFDCPSEL
24



 


59
SELQHTQEL
23


 


216
MEIYVSLGI
22


 


65
QELFPQWHL
21


 


294
AVFLPIVVL
21


 


78
AAIIASLTF
18


 


133
GVIAAIVQL
18


 


262
LLLGTIHAL
18


 


269
ALIFAWNKW
18


 


23
EEDDYLHKD
17


 


79
AIIASLTFL
17


 


82
ASLTFLYTL
17


 


156
KWMLTRKQF
17


 


172
AVLHAIYSL
17


 


224
IVGLAILAL
17


 


275
NKWIDIKQF
17


 


5
KDITNQEEL
16


 


36
SMLKRPVLL
16


 


219
YVSLGIVGL
16


 


255
SKLGIVSLL
16


 


295
VFLPIVVLI
16


 


302
LIFKSILFL
16


 


305
KSILFLPCL
16


 


11
EELWKMKPR
15


 


38
LKRPVLLHL
15


 


76
KIAAIIASL
15


 


104
QYFYKIPIL
15


 


110
PILVINKVL
15


 


118
LPMVSITLL
15


 


120
MVSITLLAL
15


 


122
SITLLALVY
15


 


130
LYPGVIAAI
15


 


162
KQFGLLSFF
15


 


180
LSYPMRRSY
15


 


185
RRSYRYKLL
15


 


204
KEDAWIEHD
15


 


248
REFHYIQSK
15


 


254
QSKLGIVSL
15


 


266
TIHALIFAW
15


 


278
IDIKQFVWY
15


 


289
PTFMIAVFL
15


 


301
VLIFKSILF
15


 


316
KILKIRHGW
15


 


1
MESRKDITN
14


 


35
TSMLKRPVL
14


 


72
HLPIKIAAI
14


 


90
LLREVIHPL
14


 


92
REVIHPLAT
14


 


99
ATSHQQYFY
14


 


123
ITLLALVYL
14


 


142
HNGTKYKKF
14


 


149
KFPHWLDKW
14


 


169
FFFAVLHAI
14


 


222
LGIVGLAIL
14


 


225
VGLAILALL
14


 


242
SDSLTWREF
14


 


249
EFHYIQSKL
14


 


288
PPTFMIAVF
14


 


310
LPCLRKKIL
14


 


14
WKMKPRRNL
13


 


30
KDTGETSML
13


 


33
GETSMLKRP
13


 


60
ELQHTQELF
13


 


73
LPIKIAAII
13


 


106
FYKIPILVI
13


 


117
VLPMVSITL
13


 


127
ALVYLPGVI
13


 


139
VQLHNGTKY
13


 


146
KYKKFPHWL
13


 


166
LLSFFFAVL
13


 


182
YPMRRSYRY
13


 


187
SYRYKLLNW
13


 


189
RYKLLNWAY
13


 


200
VQQNKEDAW
13


 


214
WRMEIYVSL
13


 


256
KLGIVSLLL
13


 


291
FMIAVFLPI
13


 


296
FLPIVVLIF
13


 


299
IVVLIFKSI
13


 


309
FLPCLRKKI
13


 


312
CLRKKILKI
13


 


331
NKTEICSQL
13


 


6
DITNQEELW
12


 


10
QEELWKMKP
12


 


22
LEEDDYLHK
12


 


63
HTQELFPQW
12


 


80
IIASLTFLY
12


 


83
SLTFLYTLL
12


 


97
PLATSHQQY
12


 


159
LTRKQFGLL
12


 


161
RKQFGLLSF
12


 


184
MRRSYRYKL
12


 


209
IEHDVWRME
12


 


210
EHDVWRMEI
12


 


221
SLGIVGLAI
12


 


237
SIPSVSDSL
12


 


239
PSVSDSLTW
12


 


244
SLTWREFHY
12


 


251
HYIQSKLGI
12


 


259
IVSLLLGTI
12


 


264
LGTIHALIF
12


 


270
LIFAWNKWI
12


 


277
WIDIKQFVW
12


 


283
FVWYTPPTF
12


 


285
WYTPPTFMI
12


 


324
WEDVTKINK
12


 


19
RRNLEEDDY
11


 


20
RNLEEDDYL
11


 


47
HQTAHADEF
11


 


69
PQWHLPIKI
11


 


87
LYTLLREVI
11


 


101
SHQQYFYKI
11


 


115
NKVLPMVSI
11


 


145
TKYKKFPHW
11


 


151
PHWLDKWML
11


 


158
MLTRKQFGL
11


 


163
QFGLLSFFF
11


 


174
LHAIYSLSY
11


 


206
DAWIEHDVW
11


 


211
HDVWRMEIY
11


 


230
LALLAVTSI
11


 


245
LTWREFHYI
11


 


263
LLGTIHALI
11


 


300
VVLIFKSIL
11

















TABLE XXXIII-V2-HLA-84402







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 5; each start position is


specified, the length of peptide is


9 amino adds, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













3
REFHYIQVN
16



 


5
FHYIQVNNI
10

















TABLE XXXIII-V3-B4402







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 7; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













2
REFHYIQII
23



 


17
PESLWDPCL
22


 


23
PCLTRFKGL
18


 


20
LWDPCLTRF
15


 


12
KKSDVPESL
14


 


13
KSDVPESLW
13


 


26
TRFKGLNLI
13


 


25
LTRFKGLNL
11

















TABLE XXXIII-V4-HLA-B4402







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 9; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













2
KQFGLLSLF
16



 


6
LLSLFFAVL
14


 


9
LFFAVLHAI
14


 


1
RKQFGLLSL
12


 


3
QFGLLSLFF
12

















TABLE XXXIV-V1-B5101







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 3; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













230
LALLAVTSI
27



 


109
IPILVINKV
26


 


293
IAVFLPIVV
25


 


73
LPIKIAAII
24


 


126
LALVYLPGV
24


 


272
FAWNKWIDI
24


 


322
HGWEDVTKI
24


 


233
LAVTSIPSV
23


 


227
LAILALLAV
22


 


287
TPPTFMIAV
22


 


118
LPMVSITLL
21


 


131
LPGVIAAIV
21


 


310
LPCLRKKIL
19


 


206
DAWIEHDVW
18


 


105
YFYKIPILV
17


 


130
YLPGVIAAI
17


 


225
VGLAILALL
17


 


86
FLYTLLREV
16


 


103
QQYFYKIPI
16


 


106
FYKIPILVI
16


 


135
IAAIVQLHN
16


 


169
FFFAVLHAI
16


 


195
WAYQQVQQN
16


 


270
LIFAWNKWI
16


 


291
FMIAVFLPI
16


 


295
VFLPIVVLI
16


 


297
LPIVVLIFK
16


 


312
CLRKKILKI
16


 


69
PQWHLPIKI
15


 


81
IASLTFLYT
15


 


87
LYTLLREVI
15


 


212
DVWRMEIYV
15


 


222
LGIVGLAIL
15


 


259
IVSLLLGTI
15


 


288
PPTFMIAVF
15


 


309
FLPCLRKKI
15


 


57
CPSELQHTQ
14


 


67
LFPQWHLPI
14


 


96
HPLATSHQQ
14


 


101
SHQQYFYKI
14


 


127
ALVYLPGVI
14


 


216
MEIYVSLGI
14


 


217
EIYVSLGIV
14


 


245
LTWREFHYI
14


 


299
IVVLIFKSI
14


 


38
LKRPVLLHL
13


 


53
DEFDCPSEL
13


 


72
HLPIKIAAI
13


 


77
IAAIIASLT
13


 


78
AAIIASLTF
13


 


90
LLREVIHPL
13


 


110
PILVINKVL
13


 


113
VINKVLPMV
13


 


117
VLPMVSITL
13


 


171
FAVLHAIYS
13


 


182
YPMRRSYRY
13


 


221
SLGIVGLAI
13


 


238
IPSVSDSLT
13


 


262
LLLGTIHAL
13


 


263
LLGTIHALI
13


 


268
HALIFAWNK
13


 


302
LIFKSILFL
13

















TABLE XXXIV-V2-HLA-B5101







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 5; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













5
FHYIQVNNI
18



 


2
WREFHYIQV
9

















TABLE XXXIV-V3-HLA-B5101







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 7; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













22
DPCLTRFKG
17



 


26
TRFKGLNLI
15


 


2
REFHYIQII
13


 


1
WREFHYIQI
11


 


16
VPESLWDPC
11


 


23
PCLTRFKGL
11


 


12
KKSDVPESL
10


 


25
LTRFKGLNL
10


 


8
QIIHKKSDV
9


 


15
DVPESLWDP
9


 


4
FHYIQIIHK
8

















TABLE XXXIV-V4-B5101







9mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 9; each start position is


specified, the length of peptide is


9 amino acids, and the end


position for each peptide is the


start position plus eight.









Pos
123456789
score













9
LFFAVLHAI
17



 


6
LLSLFFAVL
12


 


4
FGLLSLFFA
11


 


5
GLLSLFFAV
11


 


1
RKQFGLLSL
8

















TABLE XXXV-V1-HLA-A1







10mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 3; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Pos
1234567890
score













277
WIDIKQFVWY
28



 


121
VSITLLALVY
27


 


210
EHDVWRMEIY
25


 


173
VLHAIYSLSY
23


 


179
SLSYPMRRSY
19


 


243
DSLTWREFHY
19


 


79
AIIASLTFLY
18


 


138
IVQLHNGTKY
18


 


188
YRYKLLNWAY
18


 


21
NLEEDDYLHK
17


 


181
SYPMRRSYRY
17


 


54
EFDCPSELQH
16


 


91
LREVIHPLAT
16


 


169
FFFAVLHAIY
16


 


215
RMEIYVSLGI
16


 


286
YTPPTFMIAV
16


 


18
PRRNLEEDDY
15


 


96
HPLATSHQQY
15


 


98
LATSHQQYFY
15


 


159
LTRKQFGLLS
15


 


241
VSDSLTWREF
15


 


31
DTGETSMLKR
14


 


58
PSELQHTQEL
14


 


323
GWEDVTKINK
14


 


88
YTLLREVIHP
13


 


220
VSLGIVGLAI
13
















TABLE XXXV-V2-HLA-A1







10mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 5; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Pos
1234567890
score












2
WREFHYIQVN
11

















TABLE XXXV-V3-HLA-A1







10mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 7; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Pos
1234567890
score













14
KSDVPESLWD
20



 


21
LWDPCLTRFK
14


 


17
VPESLWDPCL
11


 


2
WREFHYIQII
10


 


19
ESLWDPCLTR
10
















TABLE XXXV-V4-HLA-A1







10mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 9; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Pos
1234567890
score













10
LFFAVLHAIY
15



 


8
LSLFFAVLHA
11


 


3
KQFGLLSLFF
7
















TABLE XXXVI-V1-A0201







10mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 3; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Pos
1234567890
score













232
LLAVTSIPSV
31



 


125
LLALVYLPGV
30


 


261
SLLLGTIHAL
30


 


226
GLAILALLAV
29


 


221
SLGIVGLAIL
28


 


229
ILALLAVTSI
28


 


89
TLLREVIHPL
27


 


223
GIVGLAILAL
27


 


301
VLIFKSILFL
27


 


108
KIPILVINKV
26


 


130
YLPGVIAAIV
26


 


262
LLLGTIHALI
26


 


291
FMIAVFLPIV
26


 


117
VLPMVSITLL
25


 


122
SITLLALVYL
25


 


165
GLLSFFFAVL
25


 


37
MLKRPVLLHL
24


 


81
IASLTFLYTL
23


 


112
LVINKVLPMV
23


 


191
KLLNWAYQQV
23


 


258
GIVSLLLGTI
23


 


75
IKIAAIIASL
22


 


78
AAIIASLTFL
22


 


171
FAVLHAIYSL
22


 


224
IVGLAILALL
22


 


111
ILVINKVLPM
21


 


116
KVLPMVSITL
21


 


119
PMVSITLLAL
21


 


157
WMLTRKQFGL
21


 


293
IAVFLPIVVL
21


 


294
AVFLPIVVLI
21


 


72
HLPIKIAAII
20


 


86
FLYTLLREVI
20


 


120
MVSITLLALV
20


 


129
VYLPGVIAAI
20


 


244
SLTWREFHYI
20


 


253
IQSKLGIVSL
20


 


269
ALIFAWNKWI
20


 


292
MIAVFLPIVV
20


 


309
FLPCLRKKIL
20


 


66
ELFPQWHLPI
19


 


71
WHLPIKIAAI
19


 


90
LLREVIHPLA
19


 


158
MLTRKQFGLL
19


 


218
IYVSLGIVGL
19


 


286
YTPPTFMIAV
19


 


307
ILFLPCLRKK
19


 


43
LLHLHQTAHA
18


 


183
PMRRSYRYKL
18


 


227
LAILALLAVT
18


 


7
ITNQEELWKM
17


 


36
SMLKRPVLLH
17


 


80
IIASLTFLYT
17


 


85
TFLYTLLREV
17


 


127
ALVYLPGVIA
17


 


215
RMEIYVSLGI
17


 


255
SKLGIVSLLL
17


 


105
YFYKIPILVI
16


 


126
LALVYLPGVI
16


 


128
LVYLPGVIAA
16


 


168
SFFFAVLHAI
16


 


228
AILALLAVTS
16


 


236
TSIPSVSDSL
16


 


251
HYIQSKLGIV
16


 


256
KLGIVSLLLG
16


 


299
IVVLIFKSIL
16


 


304
FKSILFLPCL
16


 


329
KINKTEICSQ
16


 


76
KIAAIIASLT
15


 


103
QQYFYKIPIL
15


 


213
VWRMEIYVSL
15


 


216
MEIYVSLGIV
15


 


220
VSLGIVGLAI
15


 


296
FLPIVVLIFK
15


 


302
LIFKSILFLP
15


 


308
LFLPCLRKKI
15

















TABLE XXXVI-V2-HLA-A0201







10mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 5; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Pos
1234567890
score













1
TWREFHYIQV
12



 


4
EFHYIQVNNI
9

















TABLE XXV1-V3-HLA-A0201







10mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 7; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Pos
1234567890
score













25
CLTRFKGLNL
20



 


20
SLWDPCLTRF
18


 


26
LTRFKGLNLI
18


 


8
IQIIHKKSDV
14


 


10
IIHKKSDVPE
12


 


7
YIQIIHKKSD
11


 


12
HKKSDVPESL
11


 


17
VPESLWDPCL
11


 


1
TWREFHYIQI
10


 


2
WREFHYIQII
10


 


9
QIIHKKSDVP
10


 


5
FHYIQIIHKK
9


 


9
SLFFAVLHAI
26


 


6
GLLSLFFAVL
25


 


1
TRKQFGLLSL
16


 


5
FGLLSLFFAV
14


 


7
LLSLFFAVLH
14


 


8
LSLFFAVLHA
12
















TABLE XXXVII-V1-HLA-A0202







10mers: STEAP-1









Pos
1234567890
score










No Results found.
















TABLE XXXVII-V2-HLA-A0202







10mers: STEAP-1









Pos
1234567890
score










No Results found.
















TABLE XXXVII-V3-HLA-A0202







10mers: STEAP-1









Pos
1234567890
score










No Results found.
















TABLE XXXVII-V4-HLA-A0202







10mers: STEAP-1









Pos
1234567890
score










No Results found.

















TABLE XXXVIII-V1-HLA-A0203







10mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 3; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Pos
1234567890
score













70
QWHLPIKIAA
19



 


128
LVYLPGVIAA
19


 


43
LLHLHQTAHA
18


 


71
WHLPIKIAAI
17


 


129
VYLPGVIAAI
17


 


41
PVLLHLHQTA
10


 


69
PQWHLPIKIA
10


 


73
LPIKIAAIIA
10


 


90
LLREVIHPLA
10


 


118
LPMVSITLLA
10


 


127
ALVYLPGVIA
10


 


163
QFGLLSFFFA
10


 


167
LSFFFAVLHA
10


 


187
SYRYKLLNWA
10


 


198
QQVQQNKEDA
10


 


219
YVSLGIVGLA
10


 


222
LGIVGLAILA
10


 


225
VGLAILALLA
10


 


260
VSLLLGTIHA
10


 


264
LGTIHALIFA
10


 


285
WYTPPTFMIA
10


 


42
VLLHLHQTAH
9


 


44
LHLHQTAHAD
9


 


74
PIKIAAIIAS
9


 


91
LREVIHPLAT
9


 


119
PMVSITLLAL
9


 


164
FGLLSFFFAV
9


 


168
SFFFAVLHAI
9


 


188
YRYKLLNWAY
9


 


199
QVQQNKEDAW
9


 


220
VSLGIVGLAI
9


 


223
GIVGLAILAL
9


 


226
GLAILALLAV
9


 


261
SLLLGTIHAL
9


 


265
GTIHALIFAW
9


 


286
YTPPTFMIAV
9
















TABLE XXXVIII-V2-HLA-A0203







10mers: STEAP-1









Pos
123456789
score










No Results found.
















TABLE XXXVIII-V3-HLA-A0203







10mers: STEAP-1









Pos
123456789
score










No Results found.

















TABLE XXXVIII-V4-HLA-A0203







10mers: STEAP-1



Each peptide is a portion of SEQ


ID NO: 9; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each pepfide is the


start position plus nine.









Pos
1234567890
score













4
QFGLLSLFFA
10



 


8
LSLFFAVLHA
10


 


5
FGLLSLFFAV
9


 


9
SLFFAVLHAI
9


 


6
GLLSLFFAVL
8


 


10
LFFAVLHAIY
8
















TABLE XXXIX-V1-HLA-A3







10mers: STEAP-1


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


each start position is


specified, the length of peptide is


10 amino adds, and the end


position for each peptide is the


start position plus nine.









Pos
1234567890
score












137
AIVQLHNGTK
26


 


228
AILALLAVTS
26


 


21
NLEEDDYLHK
24


 


306
SILFLPCLRK
24


 


6
DITNQEELWK
23


 


93
EVIHPLATSH
23


 


138
IVQLHNGTKY
23


 


140
QLHNGTKYKK
23


 


173
VLHAIYSLSY
23


 


307
ILFLPCLRKK
23


 


79
AIIASLTFLY
22


 


116
KVLPMVSITL
22


 


320
IRHGWEDVTK
22


 


153
WLDKWMLTRK
21


 


166
LLSFFFAVLH
21


 


259
IVSLLLGTIH
21


 


296
FLPIVVLIFK
21


 


76
KIAAIIASLT
20


 


127
ALVYLPGVIA
20


 


176
AIYSLSYPMR
20


 


179
SLSYPMRRSY
20


 


263
LLGTIHALIF
20


 


277
WIDIKQFVWY
20


 


86
FLYTLLREVI
19


 


121
VSITLLALVY
19


 


133
GVIAAIVQLH
19


 


165
GLLSFFFAVL
19


 


191
KLLNWAYQQV
19


 


217
EIYVSLGIVG
19


 


234
AVTSIPSVSD
19


 


42
VLLHLHQTAH
18


 


83
SLTFLYTLLR
18


 


130
YLPGVIAAIV
18


 


172
AVLHAIYSLS
18


 


294
AVFLPIVVLI
18


 


319
KIRHGWEDVT
18


 


27
YLHKDTGETS
17


 


37
MLKRPVLLHL
17


 


41
PVLLHLHQTA
17


 


72
HLPIKIAAII
17


 


124
TLLALVYLPG
17


 


212
DVWRMEIYVS
17


 


229
ILALLAVTSI
17


 


231
ALLAVTSIPS
17


 


256
KLGIVSLLLG
17


 


262
LLLGTIHALI
17


 


267
IHALIFAWNK
17


 


269
ALIFAWNKWI
17


 


300
VVLIFKSILF
17


 


312
CLRKKILKIR
17


 


317
ILKIRHGWED
17


 


30
KDTGETSMLK
16


 


66
ELFPQWHLPI
16


 


107
YKIPILVINK
16


 


111
ILVINKVLPM
16


 


128
LVYLPGVIAA
16


 


224
IVGLAILALL
16


 


226
GLAILALLAV
16


 


299
IVVLIFKSIL
16


 


301
VLIFKSILFL
16


 


77
IAAIIASLTF
15


 


89
TLLREVIHPL
15


 


97
PLATSHQQYF
15


 


112
LVINKVLPMV
15


 


180
LSYPMRRSYR
15


 


221
SLGIVGLAIL
15


 


252
YIQSKLGIVS
15


 


261
SLLLGTIHAL
15


 


316
KILKIRHGWE
15


 


43
LLHLHQTAHA
14


 


80
IIASLTFLYT
14


 


90
LLREVIHPLA
14


 


94
VIHPLATSHQ
14


 


110
PILVINKVLP
14


 


113
VINKVLPMVS
14


 


120
MVSITLLALV
14


 


122
SITLLALVYL
14


 


134
VIAAIVQLHN
14


 


147
YKKFPHWLDK
14


 


152
HWLDKWMLTR
14


 


158
MLTRKQFGLL
14


 


186
RSYRYKLLNW
14


 


192
LLNWAYQQVQ
14


 


195
WAYQQVQQNK
14


 


223
GIVGLAILAL
14


 


266
TIHALIFAWN
14


 


292
MIAVFLPIVV
14


 


45
HLHQTAHADE
13


 


60
ELQHTQELFP
13


 


96
HPLATSHQQY
13


 


99
ATSHQQYFYK
13


 


182
YPMRRSYRYK
13


 


199
QVQQNKEDAW
13


 


232
LLAVTSIPSV
13


 


258
GIVSLLLGTI
13


 


279
DIKQFVWYTP
13


 


329
KINKTEICSQ
13


 


8
TNQEELWKMK
12


 


75
IKIAAIIASL
12


 


92
REVIHPLATS
12


 


108
KIPILVINKV
12


 


125
LLALVYLPGV
12


 


139
VQLHNGTKYK
12


 


219
YVSLGIVGLA
12


 


237
SIPSVSDSLT
12


 


240
SVSDSLTWRE
12


 


247
WREFHYIQSK
12


 


283
FVWYTPPTFM
12


 


295
VFLPIVVLIF
12
















TABLE XXXIX-V2-HLA-A3







10mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 5; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Pos
1234567890
score












3
REFHYIQVNN
8


 


1
TWREFHYIQV
7


 


2
WREFHYIQVN
4
















TABLE XXXIX-V3-HLA-A3







10mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 5; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Pos
1234567890
score












25
CLTRFKGLNL
19


 


9
QIIHKKSDVP
17


 


20
SLWDPCLTRF
17


 


5
FHYIQIIHKK
15


 


10
IIHKKSDVPE
14


 


19
ESLWDPCLTR
13


 


21
LWDPCLTRFK
13


 


7
YIQIIHKKSD
11


 


16
DVPESLWDPC
11


 


4
EFHYIQIIHK
10


 


28
RFKGLNLIQS
10


 


8
IQIIHKKSDV
9
















TABLE XXXIX-V4-A3







10mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 9; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Pos
1234567890
score












7
LLSLFFAVLH
22


 


6
GLLSLFFAVL
19


 


3
KQFGLLSLFF
12


 


9
SLFFAVLHAI
12


 


2
RKQFGLLSLF
11


 


10
LFFAVLHAIY
11
















TABLE XL-V1-HLA-A26







10mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 3; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Pos
1234567890
score












277
WIDIKQFVWY
26


 


34
ETSMLKRPVL
25


 


223
GIVGLAILAL
25


 


7
ITNQEELWKM
24


 


31
DTGETSMLKR
23


 


122
SITLLALVYL
23


 


224
IVGLAILALL
23


 


37
MLKRPVLLHL
22


 


79
AIIASLTFLY
22


 


165
GLLSFFFAVL
22


 


295
VFLPIVVLIF
22


 


301
VLIFKSILFL
22


 


117
VLPMVSITLL
21


 


138
IVQLHNGTKY
21


 


210
EHDVWRMEIY
21


 


221
SLGIVGLAIL
21


 


261
SLLLGTIHAL
21


 


89
TLLREVIHPL
20


 


97
PLATSHQQYF
20


 


111
ILVINKVLPM
20


 


116
KVLPMVSITL
20


 


158
MLTRKQFGLL
20


 


212
DVWRMEIYVS
20


 


300
VVLIFKSILF
20


 


93
EVIHPLATSH
19


 


169
FFFAVLHAIY
19


 


173
VLHAIYSLSY
19


 


179
SLSYPMRRSY
19


 


299
IVVLIFKSIL
19


 


66
ELFPQWHLPI
18


 


112
LVINKVLPMV
18


 


263
LLGTIHALIF
18


 


279
DIKQFVWYTP
18


 


283
FVWYTPPTFM
18


 


286
YTPPTFMIAV
18


 


326
DVTKINKTEI
18


 


84
LTFLYTLLRE
17


 


133
GVIAAIVQLH
17


 


141
LHNGTKYKKF
17


 


144
GTKYKKFPHW
17


 


155
DKWMLTRKQF
17


 


160
TRKQFGLLSF
17


 


176
AIYSLSYPMR
17


 


207
AWIEHDVWRM
17


 


243
DSLTWREFHY
17


 


265
GTIHALIFAW
17


 


282
QFVWYTPPTF
17


 


294
AVFLPIVVLI
17


 


302
LIFKSILFLP
17


 


309
FLPCLRKKIL
17


 


6
DITNQEELWK
16


 


12
ELWKMKPRRN
16


 


74
PIKIAAIIAS
16


 


75
IKIAAIIASL
16


 


80
IIASLTFLYT
16


 


108
KIPILVINKV
16


 


217
EIYVSLGIVG
16


 


274
WNKWIDIKQF
16


 


287
TPPTFMIAVF
16


 


289
PTFMIAVFLP
16


 


329
KINKTEICSQ
16


 


21
NLEEDDYLHK
15


 


88
YTLLREVIHP
15


 


120
MVSITLLALV
15


 


125
LLALVYLPGV
15


 


128
LVYLPGVIAA
15


 


149
KFPHWLDKWM
15


 


153
WLDKWMLTRK
15


 


161
RKQFGLLSFF
15


 


172
AVLHAIYSLS
15


 


218
IYVSLGIVGL
15


 


232
LLAVTSIPSV
15


 


235
VTSIPSVSDS
15


 


253
IQSKLGIVSL
15


 


258
GIVSLLLGTI
15


 


266
TIHALIFAWN
15


 


298
PIVVLIFKSI
15


 


307
ILFLPCLRKK
15


 


54
EFDCPSELQH
14


 


60
ELQHTQELFP
14


 


78
AAIIASLTFL
14


 


94
VIHPLATSHQ
14


 


99
ATSHQQYFYK
14


 


119
PMVSITLLAL
14


 


123
ITLLALVYLP
14


 


132
PGVIAAIVQL
14


 


162
KQFGLLSFFF
14


 


191
KLLNWAYQQV
14


 


213
VWRMEIYVSL
14


 


219
YVSLGIVGLA
14


 


226
GLAILALLAV
14


 


229
ILALLAVTSI
14


 


254
QSKLGIVSLL
14


 


63
HTQELFPQWH
13


 


67
LFPQWHLPIK
13


 


103
OQYFYKIPIL
13


 


168
SFFFAVLHAI
13


 


171
FAVLHAIYSL
13


 


184
MRRSYRYKLL
13


 


234
AVTSIPSVSD
13


 


236
TSIPSVSDSL
13


 


237
SIPSVSDSLT
13


 


241
VSDSLTWREF
13


 


293
IAVFLPIVVL
13


 


296
FLPIVVLIFK
13


 


304
FKSILFLPCL
13


 


312
CLRKKILKIR
13


 


76
KIAAIIASLT
12


 


81
IASLTFLYTL
12


 


82
ASLTFLYTLL
12


 


96
HPLATSHQQY
12


 


113
VINKVLPMVS
12


 


121
VSITLLALVY
12


 


124
TLLALVYLPG
12


 


134
YLPGVIAAIV
12


 


181
VIAAIVQLHN
12


 


188
SYPMRRSYRY
12


 


199
YRYKLLNWAY
12


 


240
SVSDSLTWRE
12


 


244
SLTWREFHYI
12


 


248
REFHYIQSKL
12


 


249
EFHYIQSKLG
12


 


256
KLGIVSLLLG
12


 


270
LIFAWNKWID
12


 


303
IFKSILFLPC
12
















TABLE XL-V2-HLA-A26







10mers: STEAP-l


Each peptide is a portion of SEQ


ID NO: 5; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Pos
1234567890
score












4
EFHYIQVNNI
17


 


1
TWREFHYIQV
7
















TABLE XL-V3-HLA-A26







10mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 7; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Pos
1234567890
score












20
SLWDPCLTRF
24


 


16
DVPESLWDPC
19


 


23
DPCLTRFKGL
19


 


4
EFHYIQIIHK
17


 


25
CLTRFKGLNL
17


 


26
LTRFKGLNLI
14


 


28
RFKGLNLIQS
14


 


9
QIIHKKSDVP
12


 


10
IIHKKSDVPE
12


 


7
YIQIIHKKSD
11


 


12
HKKSDVPESL
11
















TABLE XL-V4-A26







10mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 9; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Pos
1234567890
score












6
GLLSLFFAVL
22


 


10
LFFAVLHAIY
19


 


1
TRKQFGLLSL
15


 


2
RKQFGLLSLF
15


 


9
SLFFAVLHAI
15


 


3
KQFGLLSLFF
14


 


4
QFGLLSLFFA
10
















TABLE XLI-V1-HLA-B0702







10mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 3; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Pos
1234567890
score












288
PPTFMIAVFL
23


 


109
IPILVINKVL
22


 


118
LPMVSITLLA
20


 


150
FPHWLDKWML
20


 


287
TPPTFMIAVF
18


 


40
RPVLLHLHQT
17


 


73
LPIKIAAIIA
17


 


34
ETSMLKRPVL
16


 


68
FPQWHLPIKI
16


 


253
IQSKLGIVSL
16


 


223
GIVGLAILAL
15


 


238
IPSVSDSLTW
15


 


78
AAIIASLTFL
14


 


213
VWRMEIYVSL
14


 


255
SKLGIVSLLL
14


 


293
IAVFLPIVVL
14


 


17
KPRRNLEEDD
13


 


35
TSMLKRPVLL
13


 


57
CPSELQHTQE
13


 


75
IKIAAIIASL
13


 


81
IASLTFLYTL
13


 


82
ASLTFLYTLL
13


 


119
PMVSITLLAL
13


 


165
GLLSFFFAVL
13


 


183
PMRRSYRYKL
13


 


218
IYVSLGIVGL
13


 


224
IVGLAILALL
13


 


301
VLIFKSILFL
13


 


304
FKSILFLPCL
13


 


4
RKDITNQEEL
12


 


29
HKDTGETSML
12


 


37
MLKRPVLLHL
12


 


52
ADEFDCPSEL
12


 


116
KVLPMVSITL
12


 


122
SITLLALVYL
12


 


131
LPGVIAAIVQ
12


 


132
PGVIAAIVQL
12


 


184
MRRSYRYKLL
12


 


221
SLGIVGLAIL
12


 


13
LWKMKPRRNL
11


 


19
RRNLEEDDYL
11


 


58
PSELQHTQEL
11


 


66
ELFPQWHLPI
11


 


80
IIASLTFLYT
11


 


89
TLLREVIHPL
11


 


96
HPLATSHQQY
11


 


103
QQYFYKIPIL
11


 


111
ILVINKVLPM
11


 


117
VLPMVSITLL
11


 


145
TKYKKFPHWL
11


 


158
MLTRKQFGLL
11


 


182
YPMRRSYRYK
11


 


220
VSLGIVGLAI
11


 


236
TSIPSVSDSL
11


 


248
REFHYIQSKL
11


 


254
QSKLGIVSLL
11


 


261
SLLLGTIHAL
11


 


290
TFMIAVFLPI
11


 


294
AVFLPIVVLI
11


 


299
IVVLIFKSIL
11


 


330
INKTEICSQL
11
















TABLE XLI-V2-HLA-B0702







10mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 5; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for, each peptide is the


start position plus nine.









Pos
1234567890
score












1
TWREFHYIQV
8


 


4
EFHYIQVNNI
8


 


3
REFHYIQVNN
3
















TABLE XLI-V3-HLA-B0702







10mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 7; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Pos
1234567890
score












17
VPESLWDPCL
21


 


23
DPCLTRFKGL
21


 


25
CLTRFKGLNL
12


 


12
HKKSDVPESL
10
















TABLE XLI-V4-HLA-B0702







10mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 9; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for, each peptide is the


start position plus nine.









Pos
1234567890
score












6
GLLSLFFAVL
13


 


1
TRKQFGLLSL
12


 


3
KQFGLLSLFF
12


 


8
LSLFFAVLHA
9


 


4
QFGLLSLFFA
8


 


5
FGLLSLFFAV
8


 


2
RKQFGLLSLF
7


 


9
SLFFAVLHAI
7
















TABLE XLII-V1-HLA-B08







10mers: STEAP-1









Pos
1234567890
score










No Results found.
















TABLE XLII-V2-HLA-B08







10mers: STEAP-1









Pos
123456789
score










No Results found.
















TABLE XLII-V3-HLA-B08







10mers: STEAP-1









Pos
1234567890
score










No Results found.
















TABLE XLII-V4-HLA-B08







10mers: STEAP-1









Pos
1234567890
score










No Results found.
















TABLE XLIII-V1-HLA-B1510







10mers: STEAP-1









Pos
1234567890
score










No Results found.
















TABLE XLIII-V2-HLA-B1510







10mers: STEAP-1









Pos
123456789
score










No Results found.
















TABLE XLIII-V3-HLA-B1510







10mers: STEAP-1









Pos
1234567890
score






NoResultsfound.

















TABLE XLIII-V4-HLA-B1510







10mers: STEAP-1









Pos
1234567890
score






NoResultsfound.

















TABLE XLIV-V1-HLA-B2705







10mers: STEAP-1









Pos
1234567890
score






NoResultsfound.

















TABLE XLIV-V2-HLA-B2705







10mers: STEAP-1









Pos
1234567890
score






NoResultsfound.

















TABLE XLIV-V3-HLA-B2705







10mers: STEAP-1









Pos
1234567890
score






NoResultsfound.

















TABLE XLIV-V4-HLA-B2705







10mers: STEAP-1









Pos
1234567890
score






NoResultsfound.

















TABLE XLV-V1-HLA-B2709







10mers: STEAP-1









Pos
1234567890
score






NoResultsfound.

















TABLE XLV-V2-HLA-B2709







10mers: STEAP-1









Pos
1234567890
score






NoResultsfound.

















TABLE XLV-V3-HLA-B2709







10mers: STEAP-1









Pos
1234567890
score






NoResultsfound.

















TABLE XLV-V4-HLA-B2709







10mers: STEAP-1









Pos
1234567890
score






NoResultsfound.

















TABLE XLVI-V1-HLA-B4402







STEAP-1


Each peptide is a portion of SEQ


ID NO: 3; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Pos
1234567890
score












59
SELQHTQELF
23


 


248
REFHYIQSKL
23


 


209
IEHDVWRMEI
20


 


261
SLLLGTIHAL
19


 


79
AIIASLTFLY
18


 


148
KKFPHWLDKW
18


 


265
GTIHALIFAW
18


 


71
WHLPIKIAAI
17


 


78
AAIIASLTFL
17


 


109
IPILVINKVL
17


 


116
KVLPMVSITL
17


 


121
VSITLLALVY
17


 


129
VYLPGVIAAI
17


 


223
GIVGLAILAL
17


 


294
AVFLPIVVLI
17


 


301
VLIFKSILFL
17


 


315
KKILKIRHGW
17


 


5
KDITNQEELW
16


 


23
EEDDYLHKDT
16


 


66
ELFPQWHLPI
16


 


75
IKIAAIIASL
16


 


82
ASLTFLYTLL
16


 


89
TLLREVIHPL
16


 


236
TSIPSVSDSL
16


 


269
ALIFAWNKWI
16


 


274
WNKWIDIKQF
16


 


276
KWIDIKQFVW
16


 


287
TPPTFMIAVF
16


 


295
VFLPIVVLIF
16


 


308
LFLPCLRKKI
16


 


324
WEDVTKINKT
16


 


1
MESRKDITNQ
15


 


34
ETSMLKRPVL
15


 


35
TSMLKRPVLL
15


 


117
VLPMVSITLL
15


 


132
PGVIAAIVQL
15


 


168
SFFFAVLHAI
15


 


253
IQSKLGIVSL
15


 


255
SKLGIVSLLL
15


 


293
IAVFLPIVVL
15


 


311
PCLRKKILKI
15


 


22
LEEDDYLHKD
14


 


52
ADEFDCPSEL
14


 


53
DEFDCPSELQ
14


 


81
IASLTFLYTL
14


 


119
PMVSITLLAL
14


 


122
SITLLALVYL
14


 


141
LHNGTKYKKF
14


 


155
DKWMLTRKQF
14


 


162
KQFGLLSFFF
14


 


171
FAVLHAIYSL
14


 


179
SLSYPMRRSY
14


 


216
MEIYVSLGIV
14


 


218
IYVSLGIVGL
14


 


10
QEELWKMKPR
13


 


11
EELWKMKPRR
13


 


29
HKDTGETSML
13


 


37
MLKRPVLLHL
13


 


65
QELFPQWHLP
13


 


103
QQYFYKIPIL
13


 


105
YFYKIPILVI
13


 


158
MLTRKQFGLL
13


 


165
GLLSFFFAVL
13


 


183
PMRRSYRYKL
13


 


184
MRRSYRYKLL
13


 


186
RSYRYKLLNW
13


 


204
KEDAWIEHDV
13


 


205
EDAWIEHDVW
13


 


210
EHDVWRMEIY
13


 


220
VSLGIVGLAI
13


 


224
IVGLAILALL
13


 


238
IPSVSDSLTW
13


 


254
QSKLGIVSLL
13


 


263
LLGTIHALIF
13


 


268
HALIFAWNKW
13


 


277
WIDIKQFVWY
13


 


300
VVLIFKSILF
13


 


309
FLPCLRKKIL
13


 


13
LWKMKPRRNL
12


 


46
LHQTAHADEF
12


 


58
PSELQHTQEL
12


 


62
QHTQELFPQW
12


 


77
IAAIIASLTF
12


 


92
REVIHPLATS
12


 


96
HPLATSHQQY
12


 


138
IVQLHNGTKY
12


 


145
TKYKKFPHWL
12


 


157
WMLTRKQFGL
12


 


160
TRKQFGLLSF
12


 


169
FFFAVLHAIY
12


 


181
SYPMRRSYRY
12


 


188
YRYKLLNWAY
12


 


199
QVQQNKEDAW
12


 


221
SLGIVGLAIL
12


 


241
VSDSLTWREF
12


 


243
DSLTWREFHY
12


 


282
QFVWYTPPTF
12


 


288
PPTFMIAVFL
12


 


304
FKSILFLPCL
12


 


330
INKTEICSQL
11


 


4
RKDITNQEEL
11


 


18
PRRNLEEDDY
11


 


19
RRNLEEDDYL
11


 


33
GETSMLKRPV
11


 


64
TQELFPQWHL
11


 


68
FPQWHLPIKI
11


 


86
FLYTLLREVI
11


 


97
PLATSHQQYF
11


 


126
LALVYLPGVI
11


 


144
GTKYKKFPHW
11


 


161
RKQFGLLSFF
11


 


173
VLHAIYSLSY
11


 


213
VWRMEIYVSL
11


 


244
SLTWREFHYI
11


 


262
LLLGTIHALI
11


 


290
TFMIAVFLPI
11


 


298
PIVVLIFKSI
11


 


299
IVVLIFKSIL
11
















TABLE XLVI-V2-HLA-B4402







10mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 5; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Pos
1234567890
score












3
REFHYIQVNN
13


 


4
EFHYIQVNNI
12
















TABLE XLVI-V3-HLA-B4402







10mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 7; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Pos
1234567890
score












13
KKSDVPESLW
14


 


18
PESLWDPCLT
14


 


23
DPCLTRFKGL
14


 


3
REFHYIQIIH
13


 


20
SLWDPCLTRF
13


 


25
CLTRFKGLNL
12


 


2
WREFHYIQII
11


 


12
HKKSDVPESL
11


 


26
LTRFKGLNLI
11


 


1
TWREFHYIQI
10


 


17
VPESLWDPCL
10


 


4
EFHYIQIIHK
6


 


6
HYIQIIHKKS
6


 


21
LWDPCLTRFK
6
















TABLE XLVI-V4-B4402







10mers: STEAP-1


Each peptide is a portion of SEQ


ID NO: 9; each start position is


specified, the length of peptide is


10 amino acids, and the end


position for each peptide is the


start position plus nine.









Pos
1234567890
score












3
KQFGLLSLFF
15


 


9
SLFFAVLHAI
15


 


6
GLLSLFFAVL
13


 


1
TRKQFGLLSL
12


 


2
RKQFGLLSLF
12


 


10
LFFAVLHAIY
12


 


5
FGLLSLFFAV
5


 


7
LLSLFFAVLH
5
















TABLE XLVII-V1-HLA-B5101







10mers: STEAP-1









Pos
1234567890
score






NoResultsfound.

















TABLE XLVII-V2-HLA-B5101







10mers: STEAP-1









Pos
1234567890
score






NoResultsfound.

















TABLE XLVII-V3-HLA-B5101







10mers: STEAP-1









Pos
1234567890
score






NoResultsfound.

















TABLE XLVII-V4-HLA-B5101







10mers: STEAP-1









Pos
1234567890
score






NoResultsfound.

















TABLE XLVIII-V1-HLA-DRB10101







15mers: STEAP-1


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


each start position is


specified, the length of peptide is 15


amino adds, and the end position for


each peptide is the start position plus


fourteen.









Pos
123456789012345
score












168
SFFFAVLHAIYSLSY
34


 


125
LLALVYLPGVIAAIV
33


 


226
GLAILALLAVTSIPS
32


 


111
ILVINKVLPMVSITL
31


 


128
LVYLPGVIAAIVQLH
30


 


216
MEIYVSLGIVGLAIL
30


 


261
SLLLGTIHALIFAWN
30


 


106
FYKIPILVINKVLPM
28


 


114
INKVLPMVSITLLAL
28


 


229
ILALLAVTSIPSVSD
28


 


70
QWHLPIKIAAIIASL
27


 


161
RKQFGLLSFFFAVLH
27


 


213
VWRMEIYVSLGIVGL
27


 


224
IVGL6JLALLAVTSI
27


 


25
DDYLHKDTGETSMLK
26


 


103
QQYFYKIPILVINKV
26


 


120
MVSITLLALVYLPGV
26


 


157
WMLTRKQFGLLSFFP
26


 


219
YVSLGIVGLAILALL
26


 


227
LAILALLAVTSIPSV
26


 


257
LGIVSLLLGTIHALI
26


 


296
FLPIVVLIFKSILFL
26


 


324
WEDVTKINKTEICSQ
26


 


123
ITLLALVYLPGVIAA
25


 


221
SLGIVGLAILALLAV
25


 


256
KLGIVSLLLGTIHAL
25


 


280
IKQFVWYTPPTFMIA
25


 


298
PIVVLIFKSILFLPC
15


 


307
ILFLPCLRKKILKIR
25


 


71
VVHLPIKIMIIASLT
24


 


81
IASLTFLYTLLREVI
24


 


84
LTFLYTLLREVIHPL
24


 


88
YTLLREVIHPLATSH
24


 


102
HQQYFYKIPILVINK
24


 


115
NKVLPMVSITLLALV
24


 


250
FHYIQSKLGIVSLLL
24


 


258
GIVSLLLGTIHALIF
24


 


282
QFVWYTPPTFMIAVF
24


 


286
YTPPTFMIAVFLPIV
24


 


293
IAVFLPIWLIFKSI
24


 


297
LPIWLIFKSILFLP
24


 


10
QEELWKMKPRRNLEE
23


 


41
PVLLHLHQTAHADEF
23


 


75
IKIMIIASLTFLYT
23


 


117
VLPMVSITLLALVYL
23


 


210
EHDVWRMEIYVSLGI
23


 


247
WREFHYIQSKLGIVS
23


 


281
KQFVWYTPPTFMIAV
23


 


64
TQELFPQWHLPIKIA
22


 


74
PIKIAAIIASLTFLY
22


 


92
REVIHPLATSHQQYF
22


 


108
KIPILVINKVLPMVS
22


 


160
TRKQFGLLSFFFAVL
22


 


218
IYVSLGIVGLAILAL
22


 


223
GIVGLAILALLAVTS
22


 


289
PTFMIAVFLPIVVLI
22


 


290
TFMIAVFLPIVVLIF
22


 


11
EELWKMKPRRNLEED
21


 


56
DCPSELQHTQELFPG
21


 


211
HDVWRMEIYVSLGIV
21


 


232
LLAVTSIPSVSDSLT
21


 


288
PPTFMIAVFLPIVVL
21


 


72
HLPIKIAAIIASLTF
20


 


85
TFLYTLLREVIHPLA
20


 


109
IPILVINKVLPMVSI
20


 


171
FAVLHAIYSLSYPMR
20


 


185
RRSYRYKLLNWAYQQ
20


 


187
SYRYKLLNWAYQQVQ
20


 


244
SLTWREFHYIQSKLG
20


 


253
IQSKLGIVSLLLGTI
20


 


306
SILFLPCLRKKILKI
20


 


315
KKILKIRHGWEDVTK
20


 


68
FPQWHLPIKIAAIIA
19


 


83
SLTFLYTLLREVIHP
19


 


118
LPMVSITLLALVYLP
19


 


136
AAIVQLHNGTKYKKF
19


 


147
YKKFPHWLDKWMLTR
19


 


166
LLSFFFAVLHAIYSL
19


 


181
SYPMRRSYRYKLLNW
19


 


248
REFHYIQSKLGIVSL
19


 


251
HYIQSKLFIVSLLLG
19


 


283
FVWYTPPTFMIAVFL
19


 


302
LIFKSILFLPCLRKK
19


 


126
LALVYLPGVIAAIVQ
18


 


127
ALVYLPGVIAAIVQL
18


 


153
DKWMLTRKQFGLLSF
18


 


205
EDAWIEHDVWRMEIY
18


 


242
SDSLTWREFHYIQSK
18


 


249
EFHYIQSKLGIVSLL
18


 


267
IHALIFAWNKWIDIK
18


 


269
ALIFAWNKWIDIKQF
18


 


291
FMIAVFLPIVVLIFK
18


 


300
VVLIFKSILFLPCLR
18


 


7
ITNQEELWKMKPRRN
17


 


32
TGETSMLKRPVLLHL
17


 


35
TSMLKRPVLLHLHQT
17


 


39
KRPVLLHLHQTAHAD
17


 


40
RPVLLHLHQTAHADE
17


 


67
LFPQWHLPIKIAAII
17


 


91
LREVIHPLATSHQQY
17


 


112
LVINKVLPMVSITLL
17


 


135
IAAIVQLHNGTKYKK
17


 


156
KWMLTRKQFGLLSFF
17


 


164
FGLLSFFFAVLHAIY
17


 


175
HAIYSLSYPMRRSYR
17


 


179
SLSYPMRRSYRYKLL
17


 


194
NWAYQQVQQNKEDAW
17


 


217
EIYVSLGIVGLAILA
17


 


222
LGIVGLAILALLAVT
17


 


239
PSVSDSLTWREFHYI
17


 


254
QSKLGIVSLLLGTIH
17


 


259
IVSLLLGTIHALIFA
17


 


277
WIDIKQFVWYTPPTF
17


 


292
MIAVFLPIVVLIFKS
17


 


294
AVFLPIVVLIFKSIL
17


 


299
IVVLIFKSILFLPCL
17


 


301
VLIFKSILFLPCLRK
17


 


304
FKSILFLPCLRKKIL
17


 


4
RKDITNQEELWKMKP
16


 


24
EDDYLHKDTGETSML
16


 


31
DTGETSMLKRPVLLH
16


 


44
LHLHQTAHADEFDCP
16


 


58
PSELQHTQELFPQWH
16


 


78
AAIIASLTFLYTLLR
16


 


87
LYTLLREVIHPLATS
16


 


95
IHPLATSHQQYFYKI
16


 


107
YKIPILVINKVLPMV
16


 


110
PILVINKVLPMVSIT
16


 


119
PMVSITLLALVYLPG
16


 


122
SITLLALVYLPGVIA
16


 


143
NGTKYKKFPHWLDKW
16


 


148
KKFPHWLDKWMLTRK
16


 


163
QFGLLSFFFAVLHAI
16


 


167
LSFFFAVLHAIYSLS
16


 


170
FFAVLHAIYSLSYPM
16


 


174
LHAIYSLSYPMRRSY
16


 


189
RYKLLNWAYQQVQQN
16


 


192
LLNWAYQQVQQNKED
16


 


215
RMEIYVSLGIVGLAI
16


 


220
VSLGIVGLAILALLA
16


 


230
LALLAVTSIPSVSDS
16


 


260
VSLLLGTIHALIFAW
16


 


264
LGTIHALIFAWNKWI
16


 


272
FAWNKWIDIKQFVWY
16


 


274
WNKWIDIKQFVWYTP
16


 


278
IDIKQFVWYTPPTFM
16


 


314
RKKILKIRHGWEDVT
16


 


321
RHGWEDVTKINKTEI
16
















TABLE XLVIII-V2-HLA-DRB10101







15mers: STEAP-1


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


each start position is


specified, the length of peptide is 15


amino acids, and the end position for


each peptide is the start position plus


fourteen.









Pos
123456789012345
score












4
SLTWREFHYIQVNNI
20


 


2
SDSLTWREFHYIQVN
18
















TABLE XLVIII-V3-HLA-DRB10101







15mers: STEAP-1


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


each start position is


specified, the length of peptide is 15


amino acids, and the end position for


each peptide is the start position plus


fourteen.









Pos
123456789012345
score












9
EFHYIQIIHKKSDVP
26


 


28
DPCLTRFKGLNLIQS
25


 


12
YIQIIHKKSDVPESL
22


 


4
SLTWREFHYIQIIHK
20


 


2
SDSLTWREFHYIQII
18


 


10
FHYIQIIHKKSDVPE
18


 


23
PESLWDPCLTRFKGL
17


 


24
ESLWDPCLTRFKGLN
17


 


7
WREFHYIQIIHKKSD
16


 


20
SDVPESLWDPCLTRF
16


 


15
IIHKKSDVPESLWDP
15


 


26
LWDPCLTRFKGLNLI
12
















TABLE XLVIII-V4-HLA-DRB10101







15mers-STEAP-1


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


each start position is


specified, the length of peptide is 15


amino acids, and the end position for


each peptide is the start position plus


fourteen.









Pos
123456789012345
score












14
SLFFAVLHAIYSLSY
34


 


7
RKQFGLLSLFFAVLH
27


 


3
WMLTRKQFGLLSLFF
26


 


9
QFGLLSLFFAVLHAI
24


 


6
TRKQFGLLSLFFAVL
22


 


10
DKWMLTRKQFGLLSL
18


 


2
KWMLTRKQFGLLSLF
17


 


10
FGLLSLFFAVLHAIY
17


 


12
LLSLFFAVLHAIYSL
17


 


13
LSLFFAVLHAIYSLS
16
















TABLE XLIX-V1-HLA-DRB10301







15mers: STEAP-1


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


each start position is


specified, the length of peptide is 15


amino acids, and the end position for


each peptide is the start position plus


fourteen.









Pos
123456789012345
score












155
DKWMLTRKQFGLLSF
27


 


181
SYPMRRSYRYKLLNW
26


 


297
LPIWLIFKSILFLP
26


 


95
IHPLATSHQQYFYKI
25


 


177
IYSLSYPMRRSYRYK
24


 


307
ILFLPCLRKKILKIR
24


 


19
RRNLEEDDYLHKDTG
22


 


117
VLPMVSITLLALVYL
22


 


163
QFGLLSFFFAVLHAI
22


 


259
IVSLLLGTIHALIFA
22


 


298
PIVVLIFKSILFLPC
22


 


77
IAAIIASLTFLYTLL
21


 


114
INKVLPMVSITLLAL
21


 


115
NKVLPMVSITLLALV
21


 


120
MVSITLLALVYLPGV
21


 


221
SLGIVGLAILALLAV
21


 


222
LGIVGLAILALLAVT
21


 


299
IVVLIFKSILFLPCL
21


 


25
DDYLHKDTGETSMLK
20


 


35
TSMLKRPVLLHLHQT
20


 


87
LYTLLREVIHPLATS
20


 


108
KIPILVINKVLPMVS
20


 


136
AAIVQLHNGTKYKKF
20


 


156
KWMLTRKQFGLLSFF
20


 


206
DAWIEHDVWRMEIYV
20


 


219
YVSLGIVGLAILALL
20


 


293
IAVFLPIVVLIFKSI
20


 


4
RKDITNQEELWKMKP
19


 


13
LWKMKPRRNLEEDDY
19


 


40
RPVLLHLHQTAHADE
19


 


171
FAVLHAIYSLSYPMR
19


 


261
SLLLGTIHALIFAWN
19


 


275
NKWIDIKQFVWYTPP
19


 


306
SILFLPCLRKKILKI
19


 


11
EELWKMKPRRNLEED
18


 


50
AHADEFDCPSELQHT
18


 


167
LSFFFAVLHAIYSLS
18


 


197
YQQVQQNKEDAWIEH
18


 


57
CPSELQHTQELFPQW
17


 


58
PSELQHTQELFPQWH
17


 


84
LTFLYTLLREVIHPL
17


 


132
PGVIAAIVQLHNGTK
17


 


135
IAAIVQLHNGTKYKK
17


 


138
IVQLHNGTKYKKFPH
17


 


190
YKLLNWAYQQVQQNK
17


 


267
IHALIFAWNKWIDIK
17


 


273
AWNKWIDIKQFVWYT
17


 


280
IKQFVWYTPPTFMIA
17


 


317
ILKIRHGWEDVTKIN
17


 


324
WEDVTKINKTEICSQ
17


 


16
MKPRRNLEEDDYLHK
16


 


32
TGETSMLKRPVLLHL
16


 


52
ADEFDCPSELQHTQE
16


 


62
QHTQELFPQWHLPIK
16


 


96
HPLATSHQQYFYKIP
16


 


107
YKIPILVINKVLPMV
16


 


186
RSYRYKLLNWAYQQV
16


 


241
VSDSLTWREFHYIQS
16


 


268
HALIFAWNKWIDIKQ
16


 


34
ETSMLKRPVLLHLHQ
15


 


39
KRPVLLHLHQTAHAD
15


 


153
WLDKWMLTRKQFGLL
15


 


246
TWREFHYIQSKLGIV
15


 


2
ESRKDITNQELLWKM
14


 


148
KKFPHWLDKWMLTRK
14


 


226
GLAILALLAVTSIPS
14


 


232
LLAVTSIPSVSDSLT
14


 


260
VSLLLGTIHALIFAW
14


 


304
FKSILFLPCLRKKIL
14


 


81
IASLTFLYTLLREVI
13


 


88
YTLLREVIHPLATSH
13


 


109
IPILVINKVLPMVSI
13


 


118
LPMVSITLLALVYLP
13


 


122
SITLLALVYLPGVIA
13


 


125
LLALVYLPGVIAAIV
13


 


126
LALVYLPGVIAAIVQ
13


 


131
LPGVIAAIVQLHNGT
13


 


149
KFPHWLDKWMLTRKQ
13


 


170
FFAVLHAIYSLSYPM
13


 


189
RYKLLNWAYQQVQQN
13


 


235
VTSIPSVSDSLTWRE
13


 


237
SIPSVSDSLTWREFH
13


 


254
QSKLGIVSLLLGTIH
13


 


256
KLGIVSLLLGTIHAL
13


 


292
MIAVFLPIVVLIFKS
13
















TABLE XLIX-V2-DRB1-0301







15mers: STEAP-1


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


each start position is


specified, the length of peptide is 15


amino acids, and the end position for


each peptide is the start position plus


fourteen.









Pos
123456789012345
score












1
VSDSLTWREFHYIQV
16


 


2
SDSLTWREFHYIQVN
12
















TABLE XLIX-V3-DRB1030







115mers: STEAP-1


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


each start position is


specified, the length of peptide is 15


amino acids, and the end position for


each peptide is the start position plus


fourteen.









Pos
123456789012345
score












28
DPCLTRFKGLNLIQS
22


 


15
IIHKKSDVPESLWDP
20


 


23
PESLWDPCLTRFKGL
18


 


1
VSDSLTWREFHYIQI
16


 


10
FHYIQIIHKKSOVPE
16


 


22
VPESLWDPCLTRFKG
14


 


2
SDSLTWREFHYIQII
12


 


12
YIQIIHKKSDVPESL
12


 


13
IQIIHKKSDVPESLW
12


 


19
KSDVPESLWDPCLTR
12
















TABLE XLIX-V4-HLA-0301







15mers: STEAP-1


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


each start position is


specified, the length of peptide is 15


amino acids, and the end position for


each peptide is the start position plus


fourteen.









Pos
123456789012345
score












1
DKWMLTRKQFGLLSL
27


 


9
QFGLLSLFFAVLHAI
22


 


2
KWMLTRKQFGLLSLF
20


 


13
LSLFFAVLHAIYSLS
18


 


10
FGLLSLFFAVLHAIY
13
















TABLE XLVIII-V1-HLA-DRB0401







15mers: STEAP-1


Each peptide is a portion of SEQ ID


NO: 3; each start position is


specified, the length of peptide is 15


amino acids, and the end position for


each peptide is the start position plus


fourteen.









Pos
123456789012345
score












84
LTFLYTLLREVIHPL
26


 


88
YTLLREVIHPLATSH
26


 


92
REVIHPLATSHQQYF
26


 


132
PGVIAAIVQLHNGTK
26


 


135
IAAIVQLHNGTKYKK
26


 


190
YKLLNWAYQQVQQNK
26


 


229
ILALLAVTSIPSVSD
26


 


267
IHALIFAWNKWIDIK
26


 


103
QQYFYKIPILVINKV
22


 


144
GTKYKKFPHWLDKWM
22


 


161
RKQFGLLSFFFAVLH
22


 


166
LLSFFFAVLHAIYSL
22


 


167
LSFFFAVLHAIYSLS
22


 


168
SFFFAVLHAIYSLSY
22


 


187
SYRYKLLNWAYQQVQ
22


 


194
NWAYQQVQQNKEDAW
22


 


211
HDVWRMEIYVSLGIV
22


 


216
MEIYVSLGIVGLAIL
22


 


244
SLTWREFHYIQSKLG
22


 


247
WREFHYIQSKLGIVS
22


 


274
WNKWIDIKQFVWYTP
22


 


282
QFVWYTPPTFMIAVF
22


 


288
PPTFMIAVFLPIWL
22


 


306
SILFLPCLRKKILKI
22


 


321
RHGWEDVTKINKTEI
22


 


19
RRNLEEODYLHKDTG
20


 


25
DDYLHKDTGETSMLK
20


 


39
KRPVLLHLHQTAHAD
20


 


40
RPVLLHLHQTAHAOE
20


 


41
PVLLHLHQTAHADEF
20


 


64
TQELFPQWHLPIKIA
20


 


72
HLPIKIAAIIASLTF
20


 


74
PIKIAAIIASLTFLY
20


 


78
AAIIASLTFLYTLLR
20


 


81
IASLTFLYTLLREVI
20


 


87
LYTLLREVIHPLATS
20


 


106
FYKIPILVINKVLPM
20


 


111
ILVINKVLPMVSITL
20


 


114
INKVLPMVSITLLAL
20


 


115
NKVLPMVSITLLALV
20


 


117
VLPMVSITLLALVYL
20


 


120
MVSITLLALVYLPGV
20


 


123
ITLLALVYLPGVIAA
20


 


125
LLALVYLPGVIAAIV
20


 


128
LVYLPGVIAAIVQLH
20


 


163
QFGLLSFFFAVLHAI
20


 


170
FFAVLHAIYSLSYPM
20


 


171
FAVLHAIYSLSYPMR
20


 


181
SYPMRRSYRYKLLNW
20


 


213
VWRMEIYVSLGIVGL
20


 


219
YVSLGIVGLAILALL
20


 


221
SLGIVGLAILALLAV
20


 


222
LGIVGLAILALLAVT
20


 


224
IVGLAILALLAVTSI
20


 


226
GLAILALLAVTSIPS
20


 


227
LAILALLAVTSIPSV
20


 


232
LLAVTSIPSVSDSLT
20


 


235
VTSIPSVSDSLTWRE
20


 


256
KLGIVSLLLDTIHAL
20


 


260
VSLLLGTIHALIFAW
20


 


264
LGTIHALIFAWNKWI
20


 


289
PTFMIAVFLPIVVLI
20


 


290
TFMIAVFLPIVVLIF
20


 


294
AVFLPIVVLIFKSIL
20


 


296
FLPIVVLIFKSILFL
20


 


297
LPIVVLIFKSILFLP
20


 


298
PIVVLIFKSILFLPC
20


 


304
FKSILFLPCLRKKIL
20


 


307
ILFLPCLRKKILKIR
20


 


314
RKKILKIRHGWEDVT
20


 


324
WEDVTKINKTEICSQ
20


 


28
LHKDTGETSMLKRPV
18


 


32
TGETSMLKRPVLLHL
18


 


37
MLKRPVLLHLHQTAH
18


 


54
EFDCPSELQHTQELF
18


 


62
QHTQELFPQWHLPIK
18


 


93
EVIHPLATSHQQYFY
18


 


107
YKIPILVINKVLPMV
18


 


207
AWIEHDVWRMEIYVS
18


 


243
DSLTWREFHYIQSKL
18


 


253
IQSKLGIVSLLLGTI
18


 


258
GIVSLLLGTIHALIF
18


 


320
IRHGWEDVTKINKTE
18


 


11
EELWKMKPRRNLEED
17


 


24
EDDYLHKDTGETSML
17


 


65
QELFPQWHLPIKIAA
16


 


68
FPQWHLPIKIAAIIA
16


 


83
SLTFLYTLLREVIHP
16


 


85
TFLYTLLREVIHPLA
16


 


104
QYFYKIPILVINKVL
16


 


127
ALVYLPGVIAAIVQL
16


 


192
LLNWAYQQVQQNKED
16


 


205
EDAWIEHDVWRMEIY
16


 


249
EFHYIQSKLGIVSLL
16


 


269
ALIFAWNKWIDIKQF
16


 


271
IFAWNKWIDIKQFVW
16


 


283
FVWYTPPTFMIAVFL
16


 


293
IAVFLPIVVLIFKSI
16


 


301
VLIFKSILFLPCLRK
16


 


155
DKWMLTRKQFGLLSF
15


 


4
RKDITNQEELWKMKP
14


 


10
QEELWKMKPRRNLEE
14


 


35
TSMLKRPVLLHLHQT
14


 


43
LLHLHQTAHADEFDC
14


 


77
IAAIIASLTFLYTLL
14


 


95
IHPLATSHQQYFYKI
14


 


108
KIPILVINKVLPMVS
14


 


109
IPILVINKVLPMVSI
14


 


118
LPMVSITLLALVYLP
14


 


122
SITLLALVYLPGVIA
14


 


126
LALVYLPGVIAAIVQ
14


 


131
LPGVIAAIVQLHNGT
14


 


138
IVQLHNGTKYKKFPH
14


 


151
PHWLDKWMLTRKQFG
14


 


164
FGLLSFFFAVLHAIY
14


 


174
LHAIYSLSYPMRRSY
14


 


177
IYSLSYPMRRSYRYK
14


 


189
RYKLLNWAYQQVQQN
14


 


206
DAWIEHDVWRMEIYV
14


 


210
EHDVWRMEIYVSLGI
14


 


217
EIYVSLGIVGLAILA
14


 


230
LALLAVTSIPSVSDS
14


 


238
IPSVSDSLYWREFHY
14


 


254
QSKLGIVSLLLGTIH
14


 


257
LGIVSLLLGTIHALI
14


 


259
IVSLLLGTIHALIFA
14


 


261
SLLLGTIHALIFAWN
14


 


277
WIDIKQFVWYTPPTF
14


 


281
KQFVWYTPPTFMIAV
14


 


292
MIAVFLPIVVLIFKS
14


 


305
KSILFLPCLRKKILK
14


 


317
ILKIRHGWEDVTKIN
14


 


1
MESRKDITNQEELWK
12


 


2
ESRKDITNQEELWKM
12


 


5
KDITNQEELWKMKPR
12


 


6
DITNQEELWKMKPRR
12


 


17
KPRRNLEEDDYLHKD
12


 


21
NLEEDDYLHKDTGET
12


 


27
YLHKDTGETSMLKRP
12


 


38
LKRPVLLHLHQTAHA
12


 


48
QTAHAOEFDCPSELQ
12


 


50
AHADEFDCPSELQHT
12


 


51
HADEFDCPSELQHTQ
12


 


55
FDCPSELQHTQELFP
12


 


56
DCPSELQHTQELFPQ
12


 


67
LFPQWHLPIKIAAII
12


 


69
PQWHLPIKIAAIIAS
12


 


71
WHLPIKIAAIIASLT
12


 


73
LPIKIAAIIASLTFL
12


 


75
IKIAAIIASLTFLYT
12


 


76
KIAAIIASLTFLYTL
12


 


80
IIASLTFLYTLLREV
12


 


89
TLLREVIHPLATSHQ
12


 


100
TSHQQYFYKIPILVI
12


 


105
YFYKIPILVINKVLP
12


 


112
LVINKVLPMVSITLL
12


 


116
KVLPMVSITLLALVY
12


 


129
VYLPGVIAAIVQLHN
12


 


130
YLPGVIAAIVQLHNG
12


 


134
VIMIVQLHNGTKYK
12


 


137
AIVQLHNGTKYKKFP
12


 


148
KKFPHWLDKWMLTRK
12


 


149
KFPHWLDKWMLTRKQ
12


 


152
HWLDKWMLTRKQFGL
12


 


153
WLDKWMLTRKQFGLL
12


 


160
TRKQFGLLSFFFAVL
12


 


165
GLLSFFFAVLHAIYS
12


 


173
VLHAIYSLSYPMRRS
12


 


186
RSYRYKLLNWAYQQV
12


 


191
KLLNWAYQQVQQNKE
12


 


193
LNWAYQQVQQNKEDA
12


 


203
NKEDAWIEHDVWRME
12


 


214
WRMEIYVSLGIVGLA
12


 


218
IYVSLGIVGLAILAL
12


 


223
GIVGLAILALLAVTS
12


 


225
VGLAILALLAVTSIP
12


 


228
AILALLAVTSIPSVS
12


 


234
AVTSIPSVSDSLTWR
12


 


246
TWREFHYIQSKLGIV
12


 


251
HYIQSKLGIVSLLLG
12


 


265
GTIHALIFAWNKWID
12


 


273
AWNKWIDIKQFVWYT
12


 


279
DIKQFVWYTPPTFMI
12


 


291
FMIAVFLPIVVUFK
12


 


295
VFLPIWLIFKSILF
12


 


302
LIFKSILFLPCLRKK
12


 


319
KIRHGWEDVTKINKT
12
















TABLE L-V2-HLA-DR10401







15mers: STEAP-1


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


each start position is


specified, the length of peptide is 15


amino acids, and the end position for


each peptide is the start position plus


fourteen.









Pos
123456789012345
score












4
SLTWREFHYIQVNNI
22


 


3
DSLTWREFHYIQVNN
18
















TABLE L-V3-HLA-DR10401







15mers: STEAP-1


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


each start position is


specified, the length of peptide is 15


amino acids, and the end position for


each peptide is the start position plus


fourteen.









Pos
123456789012345
score












9
EFHYIQIIHKKSDVP
28


 


4
SLTWREFHYIQIIHK
22


 


7
WREFHYIQIIHKKSD
22


 


24
ESLWDPCLTRFKGLN
22


 


28
DPCLTRFKGLNLIQS
20


 


3
DSLTWREFHYIQIIH
18


 


10
FHYIQIIHKKSDVPE
14
















TABLE L-V4-DR10401







15mers: STEAP-1


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


each start position is


specified, the length of peptide is 15


amino acids, and the end position for


each peptide is the start position plus


fourteen.









Pos
123456789012345
score












7
RKQFGLLSLFFAVLH
22


 


13
LSLFFAVLHAIYSLS
22


 


14
SLFFAVLHAIYSLSY
22


 


9
QFGLLSLFFAVLHAI
20


 


12
LLSLFFAVLHAIYSL
20


 


1
DKWMLTRKQFGLLSL
15


 


10
FGLLSLFFAVLHAIY
14


 


4
MLTRKQFGLLSLFFA
12


 


6
TRKQFGLLSLFFAVL
12


 


11
GLLSLFFAVLHAIYS
12
















TABLE LI-V1-DRB1101







15mers: STEAP-1


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


each start position is


specified, the length of peptide is 15


amino acids, and the end position for


each peptide is the start position plus


fourteen.









Pos
123456789012345
score












108
KIPILVINKVLPMVS
28


 


88
YTLLREVIHPLATSH
27


 


167
LSFFFAVLHAIYSLS
26


 


68
FPQWHLPIKIMIIA
24


 


154
LDKWMLTRKQFGLLS
24


 


321
RHGWEDVTKINKTEI
24


 


168
SFFFAVLHAIYSLSY
23


 


247
WREFHYIQSKLGIVS
23


 


161
RFQFGLLSFFFAVLH
22


 


324
WEDVTKINKTEICSQ
22


 


7
ITNQEELWKMKPRRN
21


 


84
LTFLYTLLREVIHPL
21


 


297
LPIVVLIFKSILFLP
21


 


307
ILFLPCLRKKILKIR
21


 


22
LEEDDYLHKDTGETS
20


 


39
KRPVLLHLHQTAHAD
20


 


122
SITLLALVYLPGVIA
20


 


314
RKKILKIRHGWEDVT
20


 


11
EELWKMKPRRNLEED
19


 


85
TFLYTLLREVIHPLA
19


 


111
ILVINKVLPMVSITL
19


 


125
LLALVYLPGVIAAIV
19


 


144
GTKYKKFPHWLDKWM
19


 


210
EHDVWRMEIYVSLGI
19


 


254
QSKLGIVSLLLGTIH
19


 


283
FVWYTPPTFMIAVFL
19


 


293
IAVFLPIVVLIFKSI
19


 


306
SILFLPCLRKKILKI
19


 


226
GLAILALLAVTSIPS
18


 


229
ILALLAVTSIPSVSD
18


 


31
DTGETSMLKRPVLLH
17


 


103
QQYFYKIPILVINKV
17


 


194
NWAYQQVQQNKEDAW
17


 


274
WNKWIDIKQFVWYTP
17


 


185
RRSYRYKLLNWAYQQ
16


 


187
SYRYKLLNWAYQQVQ
16


 


216
MEIYVSLGIVGLAIL
16


 


244
SLTWREFHYIQSKLG
16


 


268
HALIFAWNKWIDIKQ
16


 


301
VLIFKSILFLPCLRK
16


 


311
PCLRKKILKIRHGWE
16


 


10
QEELWKMKPRRNLEE
15


 


32
TGETSMLKRPVLLHL
15


 


37
MLKRPVLLHLHQTAH
15


 


64
TQELFPQWHLPIKIA
15


 


81
IASLTFLYTLLREVI
15


 


106
FYKIPILVINKVLPM
15


 


138
IVQLHNGTKYKKFPH
15


 


177
IYSLSYPMRRSYRYK
15


 


181
SYPMRRSYRYKLLNW
15


 


203
NKEDAWIEHDVWRME
15


 


260
VSLLLGTIHALIFAW
15


 


43
LLHLHQTAHADEFDC
14


 


55
FDCPSELQHTQELFP
14


 


114
INKVLPMVSITLLAL
14


 


120
MVSITLLALVYLPGV
14


 


132
PGVIAAIVQLHNGTK
14


 


140
QLHNGTKYKKFPHWL
14


 


148
KKFPHWLDKWMLTRK
14


 


153
WLDKWMLTRKQFGLL
14


 


178
YSLSYPMRRSYRYKL
14


 


196
AYQQVQQNKEDAWIE
14


 


207
AWIEHDVWRMEIYVS
14


 


215
RMEIYVSLGIVGLAI
14


 


232
LLAVTSIPSVSDSLT
14


 


277
WIDIKQFVWYTPPTF
14


 


287
TPPTFMIAVFLPIVV
14


 


294
AVFLPIVVLIFKSIL
14


 


295
VFLPIVVLIFKSILF
14


 


300
VVLIFKSILFLPCLR
14


 


304
FKSILFLPCLRKKIL
14


 


67
LFPQWHLPIKIAAII
13


 


71
WHLPIKIPAIIASLT
13


 


74
PIKIAAIIASLTFLY
13


 


115
NKVLPMVSITLLALV
13


 


128
LVYLPGVIAAIVQLH
13


 


166
LLSFFFAVLHAIYSL
13


 


170
FFAVLHAIYSLSYPM
13


 


212
DVWRMEIYVSLGIVG
13


 


217
EIYVSLGIVGLAILA
13


 


219
YVSLGIVGLAILALL
13


 


223
GIVGLAILALLAVTS
13


 


235
VTSIPSVSDSLTWRE
13


 


250
FHYIQSKLGIVSLLL
13


 


257
LGIVSLLLGTIHALI
13


 


261
SLLLGTIHALIFAWN
13


 


289
PTFMIAVFLPIVVLI
13


 


296
FLPIVVLIFKSILFL
13


 


312
CLRKKILKIRHGWED
13
















TABLE LI-V2-HLA-DRB1-1101







15mers: STEAP-1


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


each start posiUon is


specified, the length of peptide is 15


amino acids, and the end position for


each peptide is the start position plus


fourteen.









Pos
123456789012345
score












4
SLTWREFHYIQVNNI
10


 


3
DSLTWREFHYIQVNN
9


 


2
SDSLTWREFHYIQVN
8
















TABLE LI-V3-DRB11101







15mers: STEAP-1


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


each start position is


specified, the length of peptide is 15


amino acids, and the end position for


each peptide is the start position plus


fourteen.









Pos
123456789012345
score












9
EFHYIQIIHKKSDVP
31


 


25
SLWDPCLTRFKGLNL
21


 


10
FHYIQIIHKKSDVPE
20


 


7
WREFHYIQIIHKKSD
16
















TABLE LI-V4-DRB1-1101







15mers: STEAP-1


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


each start position is


specified, the length of peptide is 15


amino acids, and the end position for


each peptide is the start position plus


fourteen.









Pos
123456789012345
score












13
LSLFFAVLHAIYSLS
26


 


14
SLFFAVLHAIYSLSY
23


 


7
RKQFGLLSLFFAVLH
22


 


9
QFGLLSLFFAVLHAI
13
















TABLE LII







Exon compositions of 8P1D4 v.1









Exon number
Start
End












1
1
34


2
35
149


3
150
662


4
663
827


5
828
1176
















TABLE LIII





Nucleotide sequence of transcript variant 8P1D4 v.2 (SEQ ID NO: 81)
















ggggcccgca cctctgggca gcagcggcag ccgagactca cggtcaagct aaggcgaaga
60


 


gtgggtggct gaagccatac tattttatag aattaatgga aagcagaaaa gacatcacaa
120


 


accaagaaga actttggaaa atgaagccta ggagaaattt agaagaagac gattatttgc
180


 


ataaggacac gggagagacc agcatgctaa aaagacctgt gcttttgcat ttgcaccaaa
240


 


cagcccatgc tgatgaattt gactgccctt cagaacttca gcacacacag gaactctttc
300


 


cacagtggca cttgccaatt aaaatagctg ctattatagc atctctgact tttctttaca
360


 


ctcttctgag ggaagtaatt caccccttag caacttccca tcaacaatat ttttataaaa
420


 


ttccaatcct ggtcatcaac aaagtcttgc caatggtttc catcactctc ttggcattgg
480


 


tttacctgcc aggtgtgata gcagcaattg tccaacttca taatggaacc aagtataaga
540


 


agtttccaca ttggttggat aagtggatgt taacaagaaa gcagtttggg cttctcagtt
600


 


tcttttttgc tgtactgcat gcaatttata gtctgtctta cccaatgagg cgatcctaca
660


 


gatacaagtt gctaaactgg gcatatcaac aggtccaaca aaataaagaa gatgcctgga
720


 


ttgagcatga tgtttggaaa atggagattt atgtgtctct gggaattgtg ggattggcaa
780


 


tactggctct gttggctgtg acatctaetc catctgtgag tgactctttg acatggagag
840


 


aatttcacta tattcaggta aataatatat aaaataaccc taagaggtaa atcttctttt
900


 


tgtgtttatg atatagaata ttgactttac ccataaaaaa taacaaatgt ttttcaaggg
960


 


cagcaaagat cttatacttg ttccaattaa taatgtgctc tcctgttgtt ttccctattg
1020


 


Cttctaatta ggacaagtgt ttcctagaca aaataaaagg cattaaaata ttctttgttc
1080


 


tttttttttt tgtttgtttg ttttttgttg gtttgtttgt ttttttgaga tgaagtctcg
1140


 


ctctgttgcc catgctggag tacagtggca gatctcggct cactgcaacc ctgcgcctcc
1200


 


tgggttcagg cgattctctt cctcagcctc cctgagtagc tgggattaca ggcacccatc
1260


 


accatgtcca gctaattttt tatttttagt tagagacagg gttttcccat gttggccagg
1320


 


ctggtctcga tctcctgacc tcaaatgatc cgcccacctc ggcctcccaa agtgctggga
1380


 


tgacagttgt gagccaccac actcagcctg ctctttctaa tatttgaaac ttgttagaca
1440


 


atttgctacc catctaatgt atatettagg aatccaatat ggcatggttt attatttctt
1500


 


aaaaaaaata ttcttttacc tgtcacctga atttagtaat gccttttatg ttacacaact
1560


 


tagcactttc cagaaacaaa aactctctcc ttgaaataat agagttttta tctaccaaag
1620


 


atatgctagt gtctcatttc aaaggctgct ttttccagct tacatettat atacttactc
1680


 


acttgaagtt tctaaatatc cttgtaattt taaaactatc tcagatttac tgaggtttat
1740


 


cttctggtgg tagattatcc etaagaagag tgatgtgcca gaatcactct gggatccttg
1800


 


tctgacaaga ttcaaaggac taaatttaat tcagtcatga acactgccaa ttaccgttta
1860


 


tgggtagaca tctetggaaa tttccacaag gtcagacatt cgcaactatc ccttctacat
1920


 


gtccacacgt atactccaac actttattag gcatctgatt agtttggaaa gtatgcctcc
1980


 


atctgaatta gtccagtgtg gcttagagtt ggtacaacat tctcacagaa tttcctaatt
2040


 


ttgtaggttc agcctgataa ccactggagt tctttggtcc tcattaaata gctttcttca
2100


 


cacattgctc tgcctgttac acatatgatg aacactgctt tttagacttc attaggaatt
2160


 


taggactgca tcttgacaac tgagcctatt ctactatatg tacaatacct agcccataat
2220


 


aggtatacaa tacacatttg gtaaaactaa ttttcaacca atgacatgta tttttcaact
2280


 


agtaacctag aaatgtttca cttaaaatct gagaactggt tacactacaa gttaccttgg
2340


 


agattcatat atgaaaacgc aaacttagct atttgattgt attcactggg acttaagaat
2400


 


gcgcctgaat aattgtgagt tcgatttgtt ctggcaggct aatgaccatt tccagtaaag
2460


 


tgaatagagg tcagaagtcg tataaaagag gtgttgtcag aacaccgttg agattacata
2520


 


ggtgaacaac tatttteaag caactetatt tgtgtagtga caaagcatcc caatgcaggc
2580


 


tgaaatgttt catcacatct ctggatctct ctattttgtg cagacattga aaaaattgtt
2640


 


catattattt ccatgttatc agaatatttg attttttaaa aacataggcc aagttcattc
2700


 


acttcattat tcatttatca aaatcagagt gaatcacatt agtcgccttc acaactgata
2760


 


aagatcactg aagtcaaatt gatttttgct ataatcttca atctacctat atttaattga
2820


 


gaatctaaaa tgtacaaatc attgtgttga ttctgcagtg atcctgctat aagtaagact
2880


 


cagtccctga ttttaggtat cctgtgaaaa gcagaattaa gacaaataca caagagacaa
2940


 


agcacaaaaa ataaatatca taaggggatg aacaaaatgg tggagaaaga gtagacaaag
3000


 


tttttgatca cctgccttca aagaaaggct gtgaattttg ttcacttaga cagcttggag
3060


 


acaagaaatt acccaaaagt aaggtgagga ggataggcaa aaagagcaga aagatgtgaa
3120


 


tggacattgt tgagaaatgt gataggaaaa caatcataga taaaggattt ccaagcaaca
3180


 


gagcatatcc agatgaggta ggatgggata aactcttatt gaaccaatct tcaccaattt
3240


 


tgtttttctt ttgcagagca agctaggaat tgtttccctt ctactgggca caatacacgc
3300


 


attgattttt gcctggaata agtggataga tataaaacaa tttgtatggt atacacctcc
3360


 


aacttttatg atagctgttt tccttccaat tgttgtcctg atatttaaaa gcatactatt
3420


 


cctgccatgc ttgaggaaga agatactgaa gattagacat ggttgggaag acgtcaccaa
3480


 


aattaacaaa actgagatat gttcccagtt gtagaattac tgtttacaca catttttgtt
3540


 


caatattgat atattttatc accaacattt caagtttgta tttgttaata aaatgattat
3600


 


tcaaggaaaa aaaaaaaaaa aaaaaaa
3627
















TABLE LIV





Nucleotide sequence alignment of 8P1D4v.1 (SEQ ID NO: 82) and 8P1D4 v.2


(SEQ ID NO: 83)















Score = 1584 bits (824), Expect = 0.0Identities = 826/827 (99%) Strand = Plus/Plus


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


Score = 714 bits (371), Expect = 0.0Identities = 371/371 (100%) Strand = Plus/Plus


 








 








 








 








 








 








 






















TABLE LV





Peptide sequences of protein coded by 8P1D4 v.2 (SEQ ID NO: 84)
















MESRKDITNQ EELWKMKPRR NLEEDDYLHK DTGETSMLKR PVLLHLHQTA HADEFDCPSE
60


 


LQHTQELFPQ WHLPIKIAAI IASLTFLYTL LREVIHPLAT SHQQYFYKIP ILVINKVLPM
120


 


VSITLLALVY LPGVIAAIVQ LHNGTKYKKF PHWLDKWMLT RKQFGLLSFF FAVLHAIYSL
180


 


SYPMRRSYRY KLLNWAYQQV QONKEDAWIE HDVWRMEIYV SLGIVGLAIL ALLAVTSIPS
240


 


VSDSLTWREF HYIQVNNI
258
















TABLE LVI





Amino acid sequence alignment of 8P1D4v.1 (SEQ ID NO: 85)


and 8P1D4 v.2 (SEQ ID NO: 86)















Score = 521 bits (1342), Expect = e−147Identities = 254/254 (100%),


Positives = 254/254 (100%)


 










8P1D4v.1:
1
MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSE
60




MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSE



8P1D4v.2:
1
MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSE
60


 


8P1D4v.1:
61
LQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPM
120




LOHTOELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPM



8P1D4v.2:
61
LQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPM
120


 


8P1D4v.1:
121
VSITLLALVYLPGVIAAIVQLHNGTKYKKPPHWLDKWMLTRKQFGLLSFFFAVLHAIYSL
180




VSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSL



8P1D4v.2:
121
VSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSL
180


 


8P1D4v.1:
181
SYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPS
240




SYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPS



8P1D4v.2:
181
SYPMRRSYRYKILNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPS
240


 


8P1D4v.1:
241
VSDSLTWREFHYIO
254




VSDSLTWREFHYIQ



8P1D4v.2:
241
VSDSLTWREFHYIQ
254
















TABLE LVII





Nucleotide sequence of transcript variant 8P1D4 v.3 (SEQ ID NO: 87)
















ggggcccgca cctctgggca gcagcggcag ccgagactca cggtcaagct aaggcgaaga
60


 


gtgggtggct gaagccatac tattttatag aattaatgga aagcagaaaa gacatcacaa
120


 


accaagaaga actttggaaa atgaagccta ggagaaattt agaagaagac gattatttgc
180


 


ataaggacac gggagagacc agcatgctaa aaagacctgt gcttttgcat ttgcaccaaa
240


 


cagcccatgc tgatgaattt gactgccctt cagaacttca gcacacacag gaactctttc
300


 


cacagtggca cttgccaatt aaaatagctg ctattatagc atctctgact tttctttaca
360


 


ctcttctgag ggaagtaatt caccctttag caacttccca tcaacaatat ttttataaaa
420


 


ttccaatcct ggtcatcaac aaagtcttgc caatggtttc catcactctc ttggcattgg
480


 


tttacctgcc aggtgtgata gcagcaattg tccaacttca taatggaacc aagtataaga
540


 


agtttccaca ttggttggat aagtggatgt taacaagaaa gcagtttggg cttctcagtt
600


 


tcttttttgc tgtactgcat gcaatttata gtctgtctta cccaatgagg cgatcctaca
660


 


gatacaagtt gctaaactgg gcatatcaac aggtccaaca aaataaagaa gatgcctgga
720


 


ttgagcatga tgtttggaga atggagattt atgtgtctct gggaattgtg ggattggcaa
780


 


tactggctct gttggctgtg acatctattc catctgtgag tgactctttg acatggagag
840


 


aatttcacta tattcagatt atccataaga agagtgatgt gccagaatca ctctgggatc
900


 


cttgtctgac aagattcaaa ggactaaatt taattcagtc atgaacactg ccaattaccg
960


 


tttatgggta gacatctttg gaaatttcca caagagcaag ctaggaattg tttcccttct
1120


 


actgggcaca atacacgcat tgatttttgc ctggaataag tggatagata taaaacaatt
1080


 


tgtatggtat acacctccaa cttttatgat agctgttttc cttccaattg ttgtcctgat
1140


 


atttaaaagc atactattcc tgccatgctt gaggaagaag atactgaaga ttagacatgg
1200


 


ttgggaagac gtcaccaaaa ttaacaaaac tgagatatgt tcccagttgt agaattactg
1260


 


tttacacaca tttttgttca atattgatat attttatcac caacatttca tgttaataaa
1320


 


tgttaataaa atgattattc aaggaaaaaa aaaaaaaaaa aaaaa
1365
















TABLE LVIII





Nucleotide sequence alignment of 8P1D4v.2 (SEQ ID NO: 88) and 8P1D4


v.3 (SEQ ID NO: 89)















Score = 1642 bits (854), Expect = 0.0Identities = 856/857 (99%) Strand = Plus/Plus


 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 


Score = 267 bits (139), Expect = 2e−68Identities = 139/139 (100%) Strand = Plus/Plus


 








 








 








 


Score = 717 bits (373), Expect = 0.0Identities = 373/373 (100%) Strand = Plus/Plus


 








 








 








 








 








 








 






















TABLE LIX





Peptide sequences of protein coded by 8P1D4 v.3 (SEQ ID NO: 90)
















MESRKDITNQ EELWKMKPRR NLEEDDYLHK DTGETSMLKR PVLLHLHQTA HADEFDCPSE
60


 


LQHTQELFPQ WHLPIKIAAI IASLTFLYTL LREVIHPLAT SHQQYFYKIP ILVINKVLPM
120


 


VSITLLALVY LPGVIAAIVQ LHNGTKYKKF PMWLDKWMLT RKQFGLLSFF FAVLHAIYSL
180


 


SYPMRRSYRY KLLNWAYQQV QQNKEDAWIE HDVWRNEIYV SLGIVGLAIL ALLAVTSIPS
240


 


VSDSLTWREF HYIQIIHKKS DVPESLWDPC LTRFKGLNLI QS
282
















TABLE LX





Amino acid sequence alignment of 8P1D4 v.2 (SEQ ID NO: 91)


and 8P1D4 v.3 (SEQ ID NO: 92)















Score = 522 bits (1345) , Expect = e−147Identities = 254/255 (99%),


Positives = 255/255 (99%)


 










8P1D4v.2:
1
MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEPDCPSE
60




MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEPDCPSE



8P1D4v.3:
1
MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEPDCPSE
60


 


8P1D4v.2:
61
LQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPM
120




LQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPM



8P1D4v.3:
61
LQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPM
120


 


8P1D4v.2:
121
VSITLLALVYLPGVIAAIVQLHNGRKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSL
180




VSITLLALVYLPGVIAAIVQLMNGTKYKKFPHWLDKWMLTRKOFGLLSFFFAVLHAIYSL



8P1D4v.3:
121
VSITLLALVYLPGVIAAIVOLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSL
180


 


8P1D4v.2:
181
SYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPS
240




SYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPS



8P1D4v.3:
181
SYPMRRSYRYKLLNWAYQOVQONKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPS
240


 


8P1D4v.2:
241
VSDSLTWREFHYIQV
255




VSDSLTWREFHYIQ+



8P1D4v.3:
241
VSDSLTWREFHYIQI
255
















TABLE LXI





SEARCH Peptides
















STEAP 1 Variant 1:



nonamers, decamers and 15mers: aa 1-339 (SEQ ID NO: 71)



MESRKDITNQ EELWKMKPRR NLEEDDYLHK DTGETSMLKR PVLLHLHQTA HADEFDCPSE
60


 


LQHTQELFPQ WULPIKIAAI IASLTFLYTL LREVIHPLAT SHQQYFYKIP ILVINKVLPM
120


 


VSITLLALVY LPGVIAAIVQ LHNGTKYKKF PHWLDKWMLT RKQFGLLSFF FAVLHAIYSL
180


 


SYPMRRSYRY KLLNWAYQQV QQNKEDAWIE HDVWRMEIYV SLGIVGLAIL ALLAVTSIPS
240


 


VLIFKSILFL PCLRKKILKI RHGWEDVTKI NKTEICSQL
339


 


Variant 2:



9-mers aa 247-258 (SEQ ID NO: 72)



WREFHYIQVNNI



 


10-mers aa 246258 (SEQ ID NO: 73)



TWREFHYIQVNNI



 


15-mers aa 241258 (SEQ ID NO: 74)



VSDSLTWREFHYIQVNNI



 


Variant 3:



9-mers aa 247- (SEQ ID NO: 75)



WREFHYIQIIHKKSDVPESLWDPCLTRFKGLNLIQS



 


10-mers aa 246- (SEQ ID NO: 76)



TWREFHYIQIIHKKSDVPESLWDPCLTRFKGLNLIQS



 


15-mers aa 241- (SEQ ID NO: 77)



VSDSLTWREFHYIQIIHKKSDVPESLWDPCLTRFKGLNLIQS



 


Variant 4:



9-mers aa 160-176 (SEQ ID NO: 78)



RKQFGLLSLFFAVLHAI



 


10-mers aa 159-177 (SEQ ID NO: 79)



TRKQFGLLSLFFAVLHAIY



 


15-mers aa 154-182 (SEQ ID NO: 80)



DKWMLTRKQFGLLSLFFAVLHAIYSLSYP

## Claims

1. An isolated heteropolymer comprising a peptide consisting of amino acids 307-315 of SEQ ID NO:3 wherein L308 is substituted with M, V, I, S, A, T, F, C, or G, or wherein K315 is substituted with Y, R, H, F or A, and a peptide consisting of amino acids 118-126 of SEQ ID NO:3, wherein L126 is substituted with M, F, W, Y, A, I or V.

2. A composition comprising the heteropolymer of claim 1.

3. The composition of claim 2, further comprising a peptide selected from the group consisting of amino acids 86-98, 262-270 and 302-310 of SEQ ID NO:3.

4. The composition of claim 2, further comprising a peptide that is a Pan-DR-binding epitope.

5. The composition of claim 2, further comprising an HTL peptide selected from the group consisting of SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47.