# Patent

## Title
Nucleic acid and corresponding protein named 158P1D7 useful in the treatment and detection of bladder and other cancers

## Abstract

A novel gene (designated 158P1D7) and its encoded protein are described. While 158P1D7 exhibits tissue specific expression in normal adult tissue, it is aberrantly expressed in multiple cancers including set forth in Table 1. Consequently, 158P1D7 provides a diagnostic and/or therapeutic target for cancers. The 158P1D7 gene or fragment thereof, or its encoded protein or a fragment thereof, can be used to elicit an immune response.

## Background
This application is a continuation-in-part of U.S. patent application Ser. No. 10/280,340 filed 25 Oct. 2002 and a continuation-in-part of U.S. patent application Ser. No. 10/277,292 filed 21 Oct. 2002, both of which are continuations of U.S. patent application Ser. No. 09/935,430 filed Aug. 22, 2001, which claimed the priority benefit of U.S. Provisional Patent Application 60/227,098, filed Aug. 22, 2000, and 60/282,739, filed Apr. 10, 2001. This application also claims priority benefit of U.S. Provisional Patent Application 60/446,633, filed Feb. 10, 2003. The entire contents of all of these applications are fully incorporated herein by reference.
SUBMISSION ON COMPACT DISC
The contents of the following submission on compact discs are incorporated herein by reference in its entirety: A compact disc copy of the Sequence Listing (COPY 1) (file name: 511582005020, date recorded: Jul. 27, 2004, size: 288,768 bytes): and a duplicate compact disc copy of the Sequence Listing (COPY 2) (file name: 511582005020, date recorded: Jul. 27, 2004, size: 288,768 bytes).


FIELD OF THE INVENTION
The invention described herein relates to novel nucleic acid sequences and thier encoded proteins, referred to as 158P1D7 and variants thereof, and to diagnostic and therapeutic methods and compositions useful in the management of various cancers that express 158P1D7 and variants thereof.
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.
Of all new cases of cancer in the United States, bladder cancer represents approximately 5 percent in men (fifth most common neoplasm) and 3 percent in women (eighth most common neoplasm). The incidence is increasing slowly, concurrent with an increasing older population. In 1998, there was an estimated 54,500 cases, including 39,500 in men and 15,000 in women. The age-adjusted incidence in the United States is 32 per 100,000 for men and 8 per 100,000 in women. The historic male/female ratio of 3:1 may be decreasing related to smoking patterns in women. There were an estimated 11,000 deaths from bladder cancer in 1998 (7,800 in men and 3,900 in women). Bladder cancer incidence and mortality strongly increase with age and will be an increasing problem as the population becomes more elderly.
Bladder cancers comprise a heterogeneous group of diseases. The main determinants of disease control and survival are histology and extent of disease. The main codes for these factors include pathology classification, the International Classification of Diseases-Oncology (ICDO), and staging classification of extent of disease, the TNM classification. (Table XXI). For a general discussion of bladder and other urogenital cancers, see, e.g., Volgelzang, et al., Eds. Comprehensive Textbook of Genitourinary Oncology, (Williams & Wilkins, Baltimore 1996), in particular pages 295-556.
Three primary types of tumors have been reported in the bladder. The most common type of bladder cancer is Transitional cell carcinoma (TCC); this accounts for about 90% of all bladder cancers. The second form of bladder cancer is squamous cell carcinoma, which accounts for about 8% of all bladder cancers where schistosomiasis is not endemic, and approximately 75% of bladder carcinomas where schistosomiasis is endemic. Squamous cell carcinomas tend to invade deeper layers of the bladder. The third type of bladder cancer is adenocarcinoma, which account for 1%-2% of bladder cancers; these are primarily invasive forms of cancer.
Bladder cancer is commonly detected and diagnosed using cytoscopy and urine cytology. However these methods demonstrate poor sensitivity. Relatively more reliable methods of detection currently used in the clinic include the bladder tumor antigen (BTA) stat test, NMP22 protein assay, telomerase expression and hyaluronic acid and hyaluronidase (HA-HAase) urine test. The advantage of using such markers in the diagnosis of bladder cancer is their relative high sensitivity in earlier tumor stages compared to standard cytology.
For example, the BTA stat test has 60-80% sensitivity and 50-70% specificity for bladder cancer, while the HA-HAase urine test shows 90-92% sensitivity and 80-84% specificity for bladder cancer (J Urol 2001 165:1067). In general, sensitivity for stage Ta tumors was 81% for nuclear matrix protein (NMP22), 70% for telomerase, 32% for bladder tumor antigen (BTA) and 26% for cytology (J Urol 2001 166:470; J Urol 1999, 161:810). Although the telomeric repeat assay which measures telomerase activity is relatively sensitive, instability of telomerase in urine presently renders this detection method unreliable.
Most bladder cancers recur in the bladder. Generally, 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.
Intravesical bacilli Calmette-Guerin (BCG) is a common and efficacious immunotherapeutic agent used in the treatment of bladder cancer. BCG is also used as a prophylactic agent to prevent recurrence of bladder cancer. However, 30% of patients fail to respond to BCG therapy and go on to develop invasive and metastatic disease (Catalona et al. J Urol 1987, 137:220-224). BCG-related side effects have been frequently observed such as drug-induced cystitis, risk of bacterial infection, and hematuria, amongst others. Other alternative immunotherapies have been used for the treatment of bladder cancer, such as KLH (Flamm et al. Urologe 1994; 33:138-143) interferons (Bazarbashi et al. J Surg Oncol. 2000; 74:181-4), and MAGE-3 peptide loaded dendritic cells (Nishiyama et al. Clin Cancer Res 2001; 7:23-31). All these approaches are still experimental (Zlotta et al. Eur Urol 2000; 37 Suppl 3:10-15). There continues to be a significant need for diagnostic and treatment modalities that are beneficial for bladder cancer patients. Furthermore, from a worldwide standpoint, several cancers stand out as the leading killers. In particular, carcinomas of the lung, prostate, breast, colon, pancreas, and ovary are 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, 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.
Prostate cancer is the fourth most prevalent cancer in men worldwide. 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. 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 patents 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.
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 lunch and bronchial cancers.
An estimated 182,800 new invasive cases of breast cancer were expected to have occurred among women in the United States during 2000. Additionally, about 1,400 new cases of breast cancer were expected to be diagnosed in men in 2000. After increasing about 4% per year in the 1980s, breast cancer incidence rates in women have leveled off in the 1990s to about 110.6 cases per 100,000.
In the U.S. alone, there were an estimated 41,200 deaths (40,800 women, 400 men) in 2000 due to breast cancer. Breast cancer ranks second among cancer deaths in women. According to the most recent data, mortality rates declined significantly during 1992-1996 with the largest decreases in younger women, both white and black. These decreases were probably the result of earlier detection and improved treatment.
Taking into account the medical circumstances and the patient's preferences, treatment of breast cancer may involve lumpectomy (local removal of the tumor) and removal of the lymph nodes under the arm; mastectomy (surgical removal of the breast) and removal of the lymph nodes under the arm; radiation therapy; chemotherapy; or hormone therapy. Often, two or more methods are used in combination. Numerous studies have shown that, for early stage disease, long-term survival rates after lumpectomy plus radiotherapy are similar to survival rates after modified radical mastectomy. Significant advances in reconstruction techniques provide several options for breast reconstruction after mastectomy. Recently, such reconstruction has been done at the same time as the mastectomy.
Local excision of ductal carcinoma in situ (DCIS) with adequate amounts of surrounding normal breast tissue may prevent the local recurrence of the DCIS. Radiation to the breast and/or tamoxifen may reduce the chance of DCIS occurring in the remaining breast tissue. This is important because DCIS, if left untreated, may develop into invasive breast cancer. Nevertheless, there are serious side effects or sequelae to these treatments. There is, therefore, a need for efficacious breast cancer treatments.
There were an estimated 23,100 new cases of ovarian cancer in the United States in 2000. It accounts for 4% of all cancers among women and ranks second among gynecologic cancers. During 1992-1996, ovarian cancer incidence rates were significantly declining. Consequent to ovarian cancer, there were an estimated 14,000 deaths in 2000. Ovarian cancer causes more deaths than any other cancer of the female reproductive system.
Surgery, radiation therapy, and chemotherapy are treatment options for ovarian cancer. Surgery usually includes the removal of one or both ovaries, the fallopian tubes (salpingo-oophorectomy), and the uterus (hysterectomy). In some very early tumors, only the involved ovary will be removed, especially in young women who wish to have children. In advanced disease, an attempt is made to remove all intra-abdominal disease to enhance the effect of chemotherapy. There continues to be an important need for effective treatment options for ovarian cancer.
There were an estimated 28,300 new cases of pancreatic cancer in the United States in 2000. Over the past 20 years, rates of pancreatic cancer have declined in men. Rates among women have remained approximately constant but may be beginning to decline. Pancreatic cancer caused an estimated 28,200 deaths in 2000 in the United States. Over the past 20 years, there has been a slight but significant decrease in mortality rates among men (about −0.9% per year) while rates have increased slightly among women.
Surgery, radiation therapy, and chemotherapy are treatment options for pancreatic cancer. These treatment options can extend survival and/or relieve symptoms in many patients but are not likely to produce a cure for most. There is a significant need for additional therapeutic and diagnostic options for pancreatic cancer.
SUMMARY OF THE INVENTION
The present invention relates to a novel nucleic acid sequence and its encoded polypeptide, designated 158P1D7. As used herein, “158P1D7” may refer to the novel polynucleotides or polypeptides or variants thereof or both of the disclosed invention.
Nucleic acids encoding 158P1D7 are over-expressed in the cancer(s) listed in Table I. Northern blot expression analysis of 158P1D7 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 158P1D7 are provided. The tissue-related profile of 158P1D7 in normal adult tissues, combined with the over-expression observed in bladder tumors, shows that 158P1D7 is aberrantly over-expressed in at least some cancers. Thus, 158P1D7 nucleic acids and polypeptides serve as a useful diagnostic agent (or indicator) and/or therapeutic target for cancers of the tissues, such as those listed in Table I.
The invention provides polynucleotides corresponding or complementary to all or part of the 158P1D7 nucleic acids, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding 158P1D7-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 about 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100 or more than 100 contiguous amino acids of a 158P1D7-related protein, as well as the peptides/proteins themselves; DNA, RNA, DNA/RNA hybrids, and related molecules (such as PNAs), polynucleotides or oligonucleotides complementary or having at least a 90% homology to 158P1D7 nucleic acid sequences or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the 158P1D7 genes, mRNAs, or to 158P1D7-encoding polynucleotides. Also provided are means for isolating cDNAs and the gene(s) encoding 158P1D7. Recombinant DNA molecules containing 158P1D7 polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 158P1D7 gene products are also provided. The invention further provides antibodies that bind to 158P1D7 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. The invention also comprises T cell clones that recognize an epitope of 158P1D7 in the context of a particular HLA molecule.
The invention further provides methods for detecting the presence, amount, and status of 158P1D7 polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 158P1D7 polynucleotides and polypeptides. A typical embodiment of this invention provides methods for monitoring 158P1D7 polynucleotides and polypeptides in a tissue or hematology sample having or suspected of having some form of growth dysregulation such as cancer.
Note that to determine the starting position of any peptide set forth in Tables V-XVIII and XXII to XLIX (collectively HLA Peptide Tables) respective to its parental protein, e.g., variant 1, variant 2, etc., reference is made to three factors: the particular variant, the length of the peptide in an HLA Peptide Table, and the Search Peptides in Table VII. Generally, a unique Search Peptide is used to obtain HLA peptides of a particular for a particular variant. The position of each Search Peptide relative to its respective parent molecule is listed in Table 55. 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 XLIX to obtain the actual position of the HLA peptides in their parental molecule. For example, if a particular Search Peptide begins at position 150 of its parental molecule, one must add 150-1, i.e., 149 to each HLA peptide amino acid position to calculate the position of that amino acid in the parent molecule.
The invention further provides various immunogenic or therapeutic compositions and strategies for treating cancers that express 158P1D7 such as bladder cancers, including therapies aimed at inhibiting the transcription, translation, processing or function of 158P1D7 as well as cancer vaccines.
The invention further provides a method of generating a mammalian immune response directed to a protein of FIG. 2, where the method comprises exposing cells of the mammal's immune system to a portion of a) a 158P1D7-related protein and/or b) a nucleotide sequence that encodes said protein, whereby an immune response is generated to said protein. The 158P1D7-related protein can comprise at least one T cell or at least one B cell epitope; and, upon contacting the epitope with a mammalian immune system T cell or B cell respectively, the T cell or B cell is activated. The immune system cell is a B cell, a cytotoxic T cell (CTL), and/or a helper T cell (HTL). When the immune system cell is a B cell, the activated B cell generates antibodies that specifically bind to the 158P1D7-related protein. When the immune system cell is a T cell that is a cytotoxic T cell (CTL), the activated CTL kills an autologous cell that expresses the 158P1D7-related protein. When the immune system cell is a T cell that is a helper T cell (HTL), the activated HTL secretes cytokines that facilitate the cytotoxic activity of a cytotoxic T cell (CTL) or the antibody-producing activity of a B cell.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. 158P1D7 SSH nucleic acid sequence. The 158P1D7 SSH sequence contains 231 bp.
FIG. 2. A) The cDNA and amino acid sequence of 158P1D7 variant 1 (also called “158P1D7 v.1” or “158P1D7 variant 1”) is shown in FIG. 2A. The start methionine is underlined. The open reading frame extends from nucleic acid 23-2548 including the stop codon.
B) The cDNA and amino acid sequence of 158P1D7 variant 2 (also called “158P1D7 v.2”) is shown in FIG. 2B. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 23-2548 including the stop codon.
C) The cDNA and amino acid sequence of 158P1D7 variant 3 (also called “158P1D7 v.3”) is shown in FIG. 2C. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 23-2221 including the stop codon.
D) The cDNA and amino acid sequence of 158P1D7 variant 4 (also called “158P1D7 v.4′) is shown in FIG. 2D. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 23-1210 including the stop codon.
E) The cDNA and amino acid sequence of 158P1D7 variant 5 (also called “158P1D7 v.5”) is shown in FIG. 2E. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 480-3005 including the stop codon.
F) The cDNA and amino acid sequence of 158P1D7 variant 6 (also called “158P1D7 v.6”) is shown in FIG. 2F. The codon for the start methionine is underlined. The open reading frame extends from nucleic acid 23-1612 including the stop codon.
FIG. 3.
A) The amino acid sequence of 158P1D7 v.1 is shown in FIG. 3A; it has 841 amino acids.
B) The amino acid sequence of 158P1D7 v.3 is shown in FIG. 3B; it has 732 amino acids.
C) The amino acid sequence of 158P1D7 v.4 is shown in FIG. 3C; it has 395 amino acids.
D) The amino acid sequence of 158P1D7 v.6 is shown in FIG. 3D; it has 529 amino acids.
As used herein, a reference to 158P1D7 includes all variants thereof, including those shown in FIGS. 2, 3, 10, 11, and 12 unless the context clearly indicates otherwise.
FIG. 4. Alignment BLAST homology of 158P1D7 v.1 amino acid to hypothetical protein FLJ22774.
FIG. 5. FIG. 5a: Amino acid sequence alignment of 158P1D7 with human protein. FIG. 5b: Amino acid sequence alignment of 158P1D7 with human protein similar to IGFALS.
FIG. 6. Expression of 158P1D7 by RT-PCR. First strand cDNA was prepared from vital pool 1 (VP1: liver, lung and kidney), vital pool 2 (VP2, pancreas, colon and stomach), prostate xenograft pool (LAPC-4AD, LAPC-4AI, LAPC-9AD, LAPC-9AI), prostate cancer pool, bladder cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, breast cancer pool, and metastasis pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P1D7, was performed at 30 cycles of amplification. Strong expression of 158P1D7 is observed in bladder cancer pool and breast cancer pool. Lower levels of expression are observed in VP1, VP2, xenograft pool, prostate cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, and metastasis pool.
FIG. 7. Expression of 158P1D7 in normal human tissues. Two multiple tissue northern blots, with 2 μg of mRNA/lane, were probed with the 158P1D7 fragment. Size standards in kilobases (kb) are indicated on the side. The results show expression of 158P1D7 in prostate, liver, placenta, heart and, to lower levels, in small intestine and colon.
FIG. 8. Expression of 158P1D7 in bladder cancer patient specimens. FIG. 8A. RNA was extracted from the bladder cancer cell lines (CL), normal bladder (N), bladder tumors (T) and matched normal adjacent tissue (NAT) isolated from bladder cancer patients. Northern blots with 10 μg of total RNA/lane were probed with the 158P1D7 fragment. Size standards in kilobases (kb) are indicated on the side. The results show expression of 158P1D7 in 1 of 3 bladder cancer cell lines. In patient specimens, 158P1D7 expression is detected in 4 of 6 tumors tested. FIG. 8B. In another study, 158P1D7 expression is detected in all patient tumors tested (8B). The expression observed in normal adjacent tissues (isolated from diseased tissues) but not in normal tissue, isolated from healthy donors, may indicate that these tissues are not fully normal and that 158P1D7 may be expressed in early stage tumors.
FIG. 9. Expression of 158P1D7 in lung cancer patient specimens. RNA was extracted from lung cancer cell lines (CL), lung tumors (T), and their normal adjacent tissues (NAT) isolated from lung cancer patients. Northern blot with 10 μg of total RNA/lane was probed with the 158P1D7 fragment. Size standards in kilobases (kb) are indicated on the side. The results show expression of 158P1D7 in 1 of 3 lung cancer cell lines and in all 3 lung tumors tested, but not in normal lung tissues.
FIG. 10. Expression of 158P1D7 in breast cancer patient specimens. RNA was extracted from breast cancer cell lines (CL), normal breast (N), and breast tumors (T) isolated from breast cancer patients. Northern blot with 10 μg of total RNA/lane was probed with the 158P1D7 fragment: Size standards in kilobases (kb) are indicated on the side. The results show expression of 158P1D7 in 2 of 3 breast cancer cell lines and in 2 breast tumors, but not in normal breast tissue.
FIG. 11. FIGS. 11(a)-(d): Hydrophilicity amino acid profile of 158P1D7 v.1, v.3, v.4, and v.6 determined by computer algorithm sequence analysis using the method of Hopp and Woods (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828) accessed on the Protscale website located on the World Wide Web at (URL: expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
FIG. 12. FIGS. 12(a)-(d): Hydropathicity amino acid profile of 158P1D7 v.1, v.3, v.4, and v.6 determined by computer algorithm sequence analysis using the method of Kyte and Doolite (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132) accessed on the ProtScale website located on the World Wide Web at (URL: expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
FIG. 13. FIGS. 13(a)-(d): Percent accessible residues amino acid profile of 158P1D7 v.1, v.3, v.4, and v.6 determined by computer algorithm sequence analysis using the method of Janin (Janin J., 1979 Nature 277:491-492) accessed on the ProtScale website located on the World Wide Web at (URL: expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
FIG. 14. FIGS. 14(a)-(d): Average flexibility amino acid profile of 158P1D7 v.1, v.3, v.4, and v.6 determined by computer algorithm sequence analysis using the method of Bhaskaran and Ponnuswamy (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255) accessed on the ProtScale website located on the World Wide Web at (URL: expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
FIG. 15. FIGS. 15(a)-(d): Beta-turn amino acid profile of 158P1D7 v.1, v.3, v.4, and v.6 determined by computer algorithm sequence analysis using the method of Deleage and Roux (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294) accessed on the ProtScale website located on the World Wide Web at (URL: expasy.ch/cgi-bin/protscale.pl) through the ExPasy molecular biology server.
FIG. 16. FIGS. 16(A)-(D): Secondary structure and transmembrane domains prediction for 158P1D7 protein variants. The secondary structures of 158P1D7 protein variants 1 (SEQ ID NO: 104), v.3 (SEQ ID NO: 105), v.4 (SEQ ID NO: 106), and v.6 (SEQ ID NO: 107), respectively, were predicted using the HNN—Hierarchical Neural Network method (NPS@: Network Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]:147-150 Combet C., Blanchet C., Geourjon C. and Deleage G., http://pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsann.html), accessed from the ExPasy molecular biology server located on the World Wide Web at (.expasy.ch/tools/). This method predicts the presence and location of alpha helices, extended strands, and random coils from the primary protein sequence. The percent of the protein variant in a given secondary structure is also listed. FIGS. 16E, 16G, 16I, and 16K: Schematic representation of the probability of existence of transmembrane regions of 158P1D7 protein variants 1, 3, 4, and 6, 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. 16F, 16H, 16J, and 16L: Schematic representation of the probability of the existence of transmembrane regions of 158P1D7 protein variants 1, 3, 4, and 6, respectively, based on the TMHMM algorithm of Sonnhammer, von Heijne, and Krogh (Erik L. L. Sonnhammer, Gunnar von Heijne, and Anders Krogh: A hidden Markov model for predicting transmembrane helices in protein sequences. In Proc. of Sixth Int. Conf. on Intelligent Systems for Molecular Biology, p 175-182 Ed J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, Calif.: AAAI Press, 1998). The TMpred and TMHMM algorithms are accessed from the ExPasy molecular biology server located on the World Wide Web at (.expasy.ch/tools/). Protein variants 1 and 3 are predicted to contain 1 transmembrane region and protein variants 3 and 4 are not predicted to have transmembrane regions. All variants contain a hydrophobic stretch at their amino terminus that may encode a signal peptide.
FIG. 17. Schematic alignment of SNP variants of 158P1D7. Schematic alignment of SNP variants of 158P1D7. Variant 158P1D7 v.2 is a variant with single nucleotide differences at 1546. Though this SNP variant is shown on transcript variant 158P1D7 v.1, it could also occur in any other transcript variants that contains the base pairs. Numbers correspond to those of 158P1D7 v.1. Black box shows sequence similar to 158P1D7 v.1. SNP is indicated above the box.
FIG. 18. Schematic alignment of protein variants of 158P1D7. Schematic alignment of protein variants of 158P1D7. Protein variants correspond to nucleotide variants. Nucleotide variant 158P1D7 v.2 and v.5 code for the same protein as v.1. Nucleotide variants 158P1D7 v.3 and v.4 are transcript variants of v.1, as shown in FIG. 12. Variant v.6 is a single nucleotide different from v.4 but codes for a protein that differs in the C-terminal portion from the protein coded by v.4. Black boxes represent sequence similar to v.1. Hatched box represents amino acid sequence not present in v.1. Numbers underneath the box correspond to 158P1D7 v.1.
FIG. 19. Exon compositions of transcript variants of 158P1D7. Variant 158P1D7 v.3, v.4, v.5 and v.6 are transcript variants of 158P1D7 v.1. Variant 158P1D7 v.3 spliced 2069-2395 out of variant 158P1D7 v.1 and variant v.4 spliced out 1162-2096 out of v.1. Variant v.5 added another exon and 2 bp to the 5′ end and extended 288 bp to the 3′ end of variant v.1. Variant v.6 spliced at the same site as v.4 but spliced out an extra ‘g’ at the boundary. Numbers in “( )” underneath the boxes correspond to those of 158P1D7 v.1. Lengths of introns and exons are not proportional.
FIG. 20. 158P1D7 Expression in Melanoma Cancer. RNA was extracted from normal skin cell line Detroit-551, and from the melanoma cancer cell line A375. Northern blots with 10 ug of total RNA were probed with the 158P1D7 DNA probe. Size standards in kilobases are on the side. Results show expression of 158P1D7 in the melanoma cancer cell line but not in the normal cell line.
FIG. 21. 158P1D7 Expression in cervical cancer patient specimens. First strand cDNA was prepared from normal cervix, cervical cancer cell line Hela, and a panel of cervical cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P1D7, was performed at 26 and 30 cycles of amplification. Results show expression of 158P1D7 in 5 out of 14 tumor specimens tested but not in normal cervix nor in the cell line.
FIG. 22. Detection of 158P1D7 protein in recombinant cells with monoclonal antibodies. Cell lysates from the indicated cell lines were separated by SDS-PAGE and then transferred to nitrocellulose for Western blotting. The blots were probed with 5 ug/ml of the indicated anti-158P1D7 monoclonal antibodies (MAbs) in PBS+0.2% Tween 20+1% non-fat milk, washed, and then incubated with goat anti-mouse IgG-HRP secondary Ab. Immunoreactive bands were then visualized by enhanced chemoluminescence and exposure to autoradiographic film. Arrows indicate the  ̃95 KD and 90 kD 158P1D7 protein doublet band which suggest 158P1D7 is post-translationally modified to generate 2 different molecular weight species. These results demonstrate expression of 158P1D7 protein in recombinant cells and specific detection of the protein with monoclonal antibodies.
FIG. 23. Surface staining of 158P1D7-expressing 293T and UMUC cells with anti-158P1D7 monoclonal antibodies. Transiently transfected 293T cells expressing 158P1D7 and stable 158P1D7-expressing UMUC bladder cancer cells were analyzed for surface expression of 158P1D7 with monoclonal antibodies (MAbs) by flow cytometry. Transfected 293T control vector and 158P1D7 vector cells and stable UMUC-neo and UMUC-158P1D7 cells were stained with 10 ug/ml and 1 ug/ml, respectively, of the indicated MAbs. Surface bound MAbs were detected by incubation with goat anti-mouse IgG-PE secondary Ab and then subjected to FACS analysis. 158P1D7-expressing 293T and UMUC cells exhibited an increase in relative fluorescence compared to control cells demonstrating surface expression and detection of 158P1D7 protein by each of the MAbs.
FIG. 24. Surface staining of endogenous 158P1D7-expressing LAPC9 prostate cancer and UGB1 bladder cancer xenograft cells with MAb M15-68(2)22.1.1. LAPC9 and UGB1 xenograft cells were subjected to surface staining with either control mouse IgG antibody or MAb M15-68(2).1.1 at 1 ug/ml. Surface bound MAbs were detected by incubation with goat anti-mouse IgG-PE secondary Ab and then subjected to FACS analysis. Both LAPC9 and UGB1 cells exhibited an increase in relative fluorescence with the anti-158P1D7 MAb demonstrating surface expression and detection of 158P1D7 protein.
FIG. 25. Monoclonal antibody-mediated internalization of endogenous surface 158P1D7 in NCI-H146 small cell lung cancer cells. NCI-H146 cells were stained with 5 ug/ml of the indicated MAbs at 4° C. for 1.5 hours, washed, and then either left at 4° C. or moved to 37° C. for 10 and 30 minutes. Residual surface bound MAb was then detected with anti-mouse IgG-PE secondary antibody. The decrease in the mean fluorescence intensity (MF) of cells moved to 37° C. compared to cells left at 4° C. demonstrates internalization of surface bound 158P1D7/MAb complexes.
FIG. 26. Binding of the 158P1D7 extracellular domain to human umbilical vein endothelial cells. The recombinant extracellular domain (ECD) of 158P1D7 (amino acids 16-608) was iodinated to high specific activity using the iodogen (1,3,4,5-tetrachloro-3a,6a-diphenylglycoluril) method. Human umbilical vein endothelial cells (HUVEC) at 90% confluency in 6 well plates was incubated with 1 nM of 125I-158P1D7 ECD in the presence (non-specific binding) or absence (Total binding) of 50 fold excess unlabeled ECD for 2 hours at either 4° C. or 37° C. Cells were washed, solubilized in 0.5M NaOH, and subjected to gamma counting. The data shows specific binding of 158P1D7 ECD to HUVEC cells suggesting the presence of an 158P1D7 receptor on HUVEC cells. FIG. 26A. Shows that the 158P1D7 ECD bound directly to the surface of HUVEC cells as detected by the 158P1D7 specific MAb. FIG. 26B. Shows specific binding of 158P1D7 ECD to HUVEC cells suggesting the presence of an 158P1D7 receptor on HUVEC cells.
FIG. 27. 158P1D7 enhances the growth of bladder cancer in mice. Male ICR-SCID mice, 5-6 weeks old (Charles River Laboratory, Wilmington, Mass.) were used and maintained in a strictly controlled environment in accordance with the NIH Guide for the Care and Use of Laboratory Animals. 158P1D7 transfected UM-UC-3 cells and parental cells were injected into the subcutaneous space of SCID mice. Each mouse received 4×106 cells suspended in 50% (v/v) of Matrigel. Tumor size was monitored through caliper measurements twice a week. The longest dimension (L) and the dimension perpendicular to it (W) were taken to calculate tumor volume according to the formula W2×L/2. The Mann-Whitney U test was used to evaluate differences of tumor growth. All tests were two sided with {acute over (α)}=0.05.
FIG. 28. Internalization of M15-68(2).31.1.1 in NCI-H146 cells. Endogenous-158P1D7 expressing NCI-H146 cells were incubated with 5 ug/ml of MAb M15-68(2).31.1.1 at 4° C. for 1 hour, washed, and then incubated with goat anti-mouse IgG-PE secondary antibody and washed. Cells were then either left at 4° C. or moved to 37° C. for 30 minutes. Cells were then subjected to fluorescent and brightfield microscopy. Cells that remained at 4° C. exhibited a halo of fluorescence on the cells demonstrative of surface staining. Cells moved to 37° C. exhibited a loss of the halo of surface fluorescence and the generation of punctate internal fluorescence indicative of internalization of the 158P1D7/MAb complexes.
FIG. 29. Effect of 158P1D7 RNAi on cell survival. As control, 3T3 cells, a cell line with no detectable expression of 158P1D7 mRNA, was also treated with the panel of siRNAs (including oligo 158P1D7.b) and no phenotype was observed. This result reflects the fact that the specific protein knockdown in the LNCaP and PC3 cells is not a function of general toxicity, since the 3T3 cells did not respond to the 158P1D7.b oligo. The differential response of the three cell lines to the Eg5 control is a reflection of differences in levels of cell transfection and responsiveness of the cell lines to oligo treatment.
FIG. 30. 158P1D7 MAb Retards the Growth of Human Bladder Cancer Xenografts in Mice. Male ICR-SCID mice, 5-6 weeks old (Charles River Laboratory, Wilmington, Mass.) were used and were maintained in a strictly-controlled environment in accordance with the NIH Guide for the Care and Use of Laboratory Animals. UG-BL, a patient bladder cancer, was used to establish xenograft models. Stock tumors regularly maintained in SCID mice were sterilely dissected, minced, and digested using Pronase (Calbiochem, San Diego, Calif.). Cell suspensions generated were incubated overnight at 37° C. to obtain a homogeneous single-cell suspension. Each mouse received 2.5×106 cells at the subcutaneous site of right flank. A Murine monoclonal antibody to 158P1D7 was tested at a dose of 500 μg/mouse in the study. PBS was used as control. MAbs were dosed intra-peritoneally twice a week for a total of 12 doses, starting on the same day of tumor cell injection. Tumor size was monitored through caliper measurements twice a week. The longest dimension (L) and the dimension perpendicular to it (W) were taken to calculate tumor volume according to the formula: W2×L/2. The results show that Anti-158P1D7 mAbs are capable of inhibiting the growth of human bladder carcinoma in mice.
FIG. 31. 158P1D7 MAbs Retard Growth of Human Prostate Cancer Xenografts in Mice. Male ICR-SCID mice, 5-6 weeks old (Charles River Laboratory, Wilmington, Mass.) were used and were maintained in a strictly-controlled environment in accordance with the NIH Guide for the Care and Use of Laboratory Animals. LAPC-9AD, an androgen-dependent human prostate cancer, was used to establish xenograft models. Stock tumors were regularly maintained in SCID mice. At the day of implantation, stock tumors were harvested and trimmed of necrotic tissues and minced to 1 mm3 pieces. Each mouse received 4 pieces of tissues at the subcutaneous site of right flank. A Murine monoclonal antibody to 158P1D7 was tested at a dose of 500 μg/mouse and 500 μg/mouse respectively. PBS and anti-KLH monoclonal antibody were used as controls. The study cohort consisted of 4 groups with 6 mice in each group. MAbs were dosed intra-peritoneally twice a week for a total of 8 doses. Treatment was started when tumor volume reached 45 mm3. Tumor size was monitored through caliper measurements twice a week. The longest dimension (L) and the dimension perpendicular to it (W) were taken to calculate tumor volume according to the formula: W2×L/2. The Student's t test and the Mann-Whitney U test, where applicable, were used to evaluate differences of tumor growth. All tests were two-sided with α=0.05.
FIG. 32. Effect of 158P1D7 on Proliferation of Rat1 cells. cells were grown overnight in 0.5% FBS and then compared to cells treated with 10% FBS. The cells were evaluated for proliferation at 18-96 hr post-treatment by a 3H-thymidine incorporation assay and for cell cycle analysis by a BrdU incorporation/propidium iodide staining assay. The results show that the Rat-1 cells expressing the 158P1D7 antigen grew effectively in low serum concentrations (0.1%) compared to the Rat-1-Neo cells.
FIG. 33. 158P1D7 Enhances Entry Into the S Phase. Cells were labeled with 10 □M BrdU, washed, trypsinized and fixed in 0.4% paraformaldehyde and 70% ethanol. Anti-BrdU-FITC (Pharmigen) was added to the cells, the cells were washed and then incubated with 10 □g/ml propidium iodide for 20 min prior to washing and analysis for fluorescence at 488 nm. The results show that there was increased labeling of cells in S-phase (DNA synthesis phase of the cell cycle) in 3T3 cells that expressed the 158P1D7 antigen relative to control cells.
FIG. 34. FIG. 34A. The cDNA (SEQ ID NO: 108) and amino acid sequence (SEQ ID NO: 109) of M15/X68 (2)18 VH clone#1. FIG. 34B. The cDNA (SEQ ID NO: 110) and amino acid sequence (SEQ ID NO: 111) of M15/X68 (2)18 VL clone #2.
FIG. 35. FIG. 35A. The amino acid sequence (SEQ ID NO: 112) of M15/X68 (2)18 VH clone #1. FIG. 35B. The amino acid sequence (SEQ ID NO: 113) of M15/X68 (2)18 VL clone #2.
FIG. 36. Detection of 158P1D7 protein by immunohistochemistry in various cancer patient specimens. Tissue was obtained from patients with bladder transitional cell carcinoma, breast ductal carcinoma and lung carcinoma. The results showed expression of 158P1D7 in the tumor cells of the cancer patients' tissue panel (A) bladder transitional cell carcinoma, invasive Grade III (B) bladder transitional cell carcinoma, papillary Grade II. (C) breast infiltrating ductal carcinoma, moderately differentiated, (D) breast infiltrating ductal carcinoma, moderate to poorly differentiated, (E) lung squamous cell carcinoma, (F) lung adenocarcinoma, well differentiated. The expression of 158P1D7 in bladder transitional cell carcinoma tissues was detected mostly around the cell membrane indicating that 158P1D7 is membrane associated.



DETAILED DESCRIPTION OF THE INVENTION
Outline of Sections
I.) Definitions
II.) 158P1D7 Polynucleotides

    
    
        II.A.) Uses of 158P1D7 Polynucleotides
        
            II.A.1.) Monitoring of Genetic Abnormalities
            II.A.2.) Antisense Embodiments
            II.A.3.) Primers and Primer Pairs
            II.A.4.) Isolaton of 158P1D7-Encoding Nucleic Acid Molecules
            II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems
        
        
    
    


III.) 158P1D7-related Proteins

    
    
        III.A.) Motif-bearing Protein Embodiments
        III.B.) Expression of 158P1D7-related Proteins
        III.C.) Modifications of 158P1D7-related Proteins
        III.D.) Uses of 158P1D7-related Proteins
    
    


IV.) 158P1D7 Antibodies
V.) 158P1D7 Cellular Immune Responses
VI.) 158P1D7 Transgenic Animals
VII.) Methods for the Detection of 158P1D7
VIII.) Methods for Monitoring the Status of 158P1D7-related Genes and Their Products
IX.) Identification of Molecules That Interact With 158P1D7
X.) Therapeutic Methods and Compositions

    
    
        X.A.) Anti-Cancer Vaccines
        X.B.) 158P1D7 as a Target for Antibody-Based Therapy
        X.C.) 158P1D7 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 158P1D7.
XII.) Inhibition of 158P1D7 Protein Function

    
    
        XII.A.) Inhibition of 158P1D7 With Intracellular Antibodies
        XII.B.) Inhibition of 158P1D7 with Recombinant Proteins
        XII.C.) Inhibition of 158P1D7 Transcription or Translation
        XII.D.) General Considerations for Therapeutic Strategies
    
    


XIII.) Identification, Characterization and Use of Modulators of 158P1D7
XIV.) RNAi and Therapeutic use of small interfering RNA (siRNAs)
XV.) KITS
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 “invasive bladder cancer” means bladder cancers that have extended into the bladder muscle wall, and are meant to include stage stage T2-T4 and disease under the TNM (tumor, node, metastasis) system. In general, these patients have substantially less favorable outcomes compared to patients having non-invasive cancer. Following cystectomy, 50% or more of the patients with invasive cancer will develop metastasis (Whittmore. Semin Urol 1983; 1:4-10).
“Altering the native glycosylation paftem” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence 158P1D7 (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 158P1D7. 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 158P1D7-related protein). For example an analog of the 158P1D7 protein can be specifically bound by an antibody or T cell that specifically binds to 158P1D7 protein.
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-158P1D7 antibodies bind 158P1D7 proteins, or a fragment thereof, and 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-158P1D7 antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-158P1D7 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 one or more than one codon having a usage frequency of less than about 20%, more preferably less than about 30% or 40%. A sequence may be “completely optimized” to contain no codon having a usage frequency of less than about 20%, more preferably less than about 30% or 40%. 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 one or more than one function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Examples of cytotoxic agents include, but are not limited to maytansinoids, yttrium, bismuth, ricin, ricin A-chain, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, sapaonaria officinalis inhibitor, and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu. Antibodies may also be conjugated to an anti-cancer pro-drug activating enzyme capable of converting the pro-drug to its active form.
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, or present, 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 nucleic acids other than those of 158P1D7 or that encode polypeptides other than 158P1D7 gene product or fragments thereof. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated 158P1D7 polynucleotide. A protein is said to be “isolated,” for example, when physical, mechanical and/or chemical methods are employed to remove the 158P1D7 protein from cellular constituents that are normally associated, or present, with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated 158P1D7 protein. Alternatively, an isolated protein can be prepared by synthetic or 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 bladder cancer” and “metastatic disease” mean bladder cancers that have spread to regional lymph nodes or to distant sites, and are meant to stage T×N×M+ under the TNM system. The most common site for bladder cancer metastasis is lymph node. Other common sites for metastasis include lung, bone and liver.
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 an 158P1D7-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 mammals, such as humans.
The term “polynucleotide” means a polymeric form of nucleotides of at least 3, 4, 5, 6, 7, 8, 9, or 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 is often used interchangeably with “oligonucleotide”, although “oligonucleotide” may be used to refer to the subset of polynucleotides less than about 50 nucleotides in length. A polynucleotide can comprise a nucleotide sequence disclosed herein wherein thymidine (T) (as shown for example in 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”, thus “peptide” may be used to refer to the subset of polypeptides less than about 50 amino acids in length.
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.
A “recombinant” DNA or RNA molecule is a DNA or RNA molecule that has been subjected to molecular manipulation in vitro.
“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.
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 add residues in the corresponding position(s) of a specifically described protein (e.g. the 158P1D7 protein shown in FIG. 2 or FIG. 3). An analog is an example of a variant protein.
The 158P1D7-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 158P1D7 proteins or fragments thereof, as well as fusion proteins of a 158P1D7 protein and a heterologous polypeptide are also included. Such 158P1D7 proteins are collectively referred to as the 158P1D7-related proteins, the proteins of the invention, or 158P1D7. The term “158P1D7-related protein refers to a polypeptide fragment or an 158P1D7 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 about 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100 or more than 100 amino acids.
II.) 158P1D7 Polynucleotides
One aspect of the invention provides polynucleotides corresponding or complementary to all or part of an 158P1D7 gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding an 158P1D7-related protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to an 158P1D7 gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides that hybridize to an 158P1D7 gene, mRNA, or to an 158P1D7 encoding polynucleotide (collectively, “158P1D7 polynucleotides”). In all instances when referred to in this section, T can also be U in FIG. 2.
Embodiments of a 158P1D7 polynucleotide include: a 158P1D7 polynucleotide having the sequence shown in FIG. 2, the nucleotide sequence of 158P1D7 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 158P1D7 nucleotides comprise, without limitation:

    
    
        (a) a polynucleotide comprising or consisting of the sequence as shown in FIG. 2, wherein T can also be U;
        (b) a polynucleotide comprising or consisting of the sequence as shown in FIG. 2, from nucleotide residue number 23 through nucleotide residue number 2548, wherein T can also be U;
        (c) a polynucleotide that encodes a 158P1D7-related protein whose sequence is encoded by the cDNAs contained in the plasmid designated p158P1D7-Turbo/3PX deposited with American Type Culture Collection as Accession No. PTA-3662 on 22 Aug. 2001 (sent via Federal Express on 20 Aug. 2001);
        (d) a polynucleotide that encodes an 158P1D7-related protein that is at least 90% homologous to the entire amino acid sequence shown in FIG. 2;
        (e) a polynucleotide that encodes an 158P1D7-related protein that is at least 90% identical to the entire amino acid sequence shown in FIG. 2;
        (f) a polynucleotide that encodes at least one peptide set forth in Tables V-XVIII;
        (g) a polynucleotide that encodes a peptide region of at least 5 amino acids of FIG. 3 in any whole number increment up to 841 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of FIG. 11;
        (h) a polynucleotide that encodes a peptide region of at least 5 amino acids of FIG. 3 in any whole number increment up to 841 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of FIG. 12;
        (i) a polynucleotide that encodes a peptide region of at least 5 amino acids of FIG. 3 in any whole number increment up to 841 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of FIG. 13;
        (j) a polynucleotide that encodes a peptide region of at least 5 amino acids of FIG. 3 in any whole number increment up to 841 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile on FIG. 14;
        (k) a polynucleotide that encodes a peptide region of at least 5 amino acids of FIG. 3 in any whole number increment up to 841 that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of FIG. 15;
        (l) a polynucleotide that is fully complementary to a polynucleotide of any one of (a)-(k);
        (m) a polynucleotide that selectively hybridizes under stringent conditions to a polynucleotide of (a)-(l);
        (n) a peptide that is encoded by any of (a)-(k); and,
        (o) a polynucleotide of any of (a)-(m) or peptide of (n) 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 158P1D7 polynucleotides that encode specific portions of the 158P1D7 mRNA sequence (and those which are complementary to such sequences) such as those that encode the protein and fragments thereof, for example of 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, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825 or 841 contiguous 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 158P1D7 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the 158P1D7 protein shown in FIG. 2, or FIG. 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the 158P1D7 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 30 to about amino acid 40 of the 158P1D7 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 40 to about amino acid 50 of the 158P1D7 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 50 to about amino acid 60 of the 158P1D7 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 60 to about amino acid 70 of the 158P1D7 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 70 to about amino acid 80 of the 158P1D7 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 80 to about amino acid 90 of the 158P1D7 protein shown in FIG. 2 or FIG. 3, polynucleotides encoding about amino acid 90 to about amino acid 100 of the 158P1D7 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 158P1D7 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 the 158P1D7 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 158P1D7 protein shown in FIG. 2 or FIG. 3 can be generated by a variety of techniques well known in the art. These polynucleotide fragments can include any portion of the 158P1D7 sequence as shown in FIG. 2 or FIG. 3.
Additional illustrative embodiments of the invention disclosed herein include 158P1D7 polynucleotide fragments encoding one or more of the biological motifs contained within the 158P1D7 protein sequence, including one or more of the motif-bearing subsequences of the 158P1D7 protein set forth in Tables V-XVIII. In another embodiment, typical polynucleotide fragments of the invention encode one or more of the regions of 158P1D7 that exhibit homology to a known molecule. In another embodiment of the invention, typical polynucleotide fragments can encode one or more of the 158P1D7 N-glycosylation sites, cAMP and cGMP-dependent protein kinase phosphorylation sites, casein kinase II phosphorylation sites or N-myristoylation site and amidation sites.
II.A.) Uses of 158P1D7 Polynucleotides
II.A.1.) Monitoring of Genetic Abnormalities
The polynucleotides of the preceding paragraphs have a number of different specific uses. The human 158P1D7 gene maps to the chromosomal location set forth in Example 3. For example, because the 158P1D7 gene maps to this chromosome, polynucleotides that encode different regions of the 158P1D7 protein 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 158P1D7 protein provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the chromosomal region that encodes 158P1D7 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 158P1D7 was shown to be highly expressed in bladder and other cancers, 158P1D7 polynucleotides are used in methods assessing the status of 158P1D7 gene products in normal versus cancerous tissues. Typically, polynucleotides that encode specific regions of the 158P1D7 protein 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 158P1D7 gene, such as such 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 158P1D7. 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 158P1D7 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., 158P1D7. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The 158P1D7 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 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 158P1D7 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 158P1D7 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 the 158P1D7 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 158P1D7 mRNA and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, 158P1D7 antisense oligonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to 158P1D7 mRNA. Optionally, 158P1D7 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 158P1D7. Alternatively, the antisense molecules are modified to employ ribozymes in the inhibition of 158P1D7 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 this nucleotides of the invention include primers and primer pairs, which allow the specific amplification of polynucleotides of the invention or of any specific parts thereof, and probes that selectively or specifically hybridize to nucleic acid molecules of the invention or to any part thereof. Primers may also be used as probes and 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 158P1D7 polynucleotide in a sample and as a means for detecting a cell expressing a 158P1D7 protein.
Examples of such probes include polypeptides comprising all or part of the human 158P1D7 cDNA sequence shown in FIG. 2. Examples of primer pairs capable of specifically amplifying 158P1D7 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 158P1D7 mRNA. Preferred probes of the invention are polynucleotides of more than about 9, about 12, about 15, about 18, about 20, about 23, about 25, about 30, about 35, about 40, about 45, and about 50 consecutive nucleotides found in 158P1D7 nucleic acids disclosed herein.
The 158P1D7 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 158P1D7 gene(s), mRNA(s), or fragments thereof; as reagents for the diagnosis and/or prognosis of bladder cancer and other cancers; as coding sequences capable of directing the expression of 158P1D7 polypeptides; as tools for modulating or inhibiting the expression of the 158P1D7 gene(s) and/or translation of the 158P1D7 transcript(s); and as therapeutic agents.
II.A.4.) Isolation of 158P1D7-Encoding Nucleic Acid Molecules
The 158P1D7 cDNA sequences described herein enable the isolation of other polynucleotides encoding 158P1D7 gene product(s), as well as the isolation of polynucleotides encoding 158P1D7 gene product homologs, alternatively spliced isoforms, allelic variants, and mutant forms of the 158P1D7 gene product as well as polynucleotides that encode analogs of 158P1D7-related proteins. Various molecular cloning methods that can be employed to isolate full length cDNAs encoding an 158P1D7 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 158P1D7 gene cDNAs can be identified by probing with a labeled 158P1D7 cDNA or a fragment thereof. For example, in one embodiment, the 158P1D7 cDNA (FIG. 2) or a portion thereof can be synthesized and used as a probe to retrieve overlapping and full-length cDNAs corresponding to a 158P1D7 gene. The 158P1D7 gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with 158P1D7 DNA probes or primers.
The present invention includes the use of any probe as described herein to identify and isolate a 158P1D7 or 158P1D7 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.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems
The invention also provides recombinant DNA or RNA molecules containing an 158P1D7 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 158P1D7 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 bladder cancer cell lines such as SCaBER, UM-UC3, HT1376, RT4, T24, TCC-SUP, J82 and SW780, other transfectable or transducible bladder cancer cell lines, 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 158P1D7 or a fragment, analog or homolog thereof can be used to generate 158P1D7 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 158P1D7 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, 158P1D7 can be expressed in several bladder cancer and non-bladder cell lines, including for example SCaBER, UM-UC3, HT1376, RT4, T24, TCC-SUP, J82 and SW780. The host-vector systems of the invention are useful for the production of a 158P1D7 protein or fragment thereof. Such host-vector systems can be employed to study the functional properties of 158P1D7 and 158P1D7 mutations or analogs.
Recombinant human 158P1D7 protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a 158P1D7-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding 158P1D7 or fragment, analog or homolog thereof, the 158P1D7 or related protein is expressed in the 293T cells, and the recombinant 158P1D7 protein is isolated using standard purification methods (e.g., affinity purification using anti-158P1D7 antibodies). In another embodiment, a 158P1D7 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 158P1D7 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 the 158P1D7 coding sequence can be used for the generation of a secreted form of recombinant 158P1D7 protein.
As discussed herein, redundancy in the genetic code permits variation in 158P1D7 gene sequences. In particular, it is known in the art that specific host species often have specific codon preferences, and thus one can adapt the disclosed sequence as preferred for a desired host. For example, preferred analog codon sequences typically have rare codons (i.e., codons having a usage frequency of less than about 20% in known sequences of the desired host) replaced with higher frequency codons. Codon preferences for a specific species are calculated, for example, by utilizing codon usage tables available on the INTERNET such as at URL URL: dna.affrc.go.jp/ ̃nakamura/codon.html.
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.) 158P1D7-related Proteins
Another aspect of the present invention provides 158P1D7-related proteins. Specific embodiments of 158P1D7 proteins comprise a polypeptide having all or part of the amino acid sequence of human 158P1D7 as shown in FIG. 2 or FIG. 3. Alternatively, embodiments of 158P1D7 proteins comprise variant, homolog or analog polypeptides that have alterations in the amino acid sequence of 158P1D7 shown in FIG. 2 or FIG. 3.
In general, naturally occurring allelic variants of human 158P1D7 share a high degree of structural identity and homology (e.g., 90% or more homology). Typically, allelic variants of the 158P1D7 protein contain conservative amino acid substitutions within the 158P1D7 sequences described herein or contain a substitution of an amino acid from a corresponding position in a homologue of 158P1D7. One class of 158P1D7 allelic variants are proteins that share a high degree of homology with at least a small region of a particular 158P1D7 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 or more 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 158P1D7 proteins such as polypeptides having amino acid insertions, deletions and substitutions. 158P1D7 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 158P1D7 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, 158P1D7 variants, analogs or homologs, have the distinguishing attribute of having at least one epitope that is “cross reactive” with a 158P1D7 protein having the amino acid sequence of FIG. 2. As used in this sentence, “cross reactive” means that an antibody or T cell that specifically binds to an 158P1D7 variant also specifically binds to the 158P1D7 protein having the amino acid sequence of FIG. 2. A polypeptide ceases to be a variant of the protein shown in FIG. 2 when it no longer contains any epitope capable of being recognized by an antibody or T cell that specifically binds to the 158P1D7 protein. Those skilled in the art understand that antibodies that recognize proteins bind to epitopes of varying size, and a grouping of the order of about four or five amino acids, contiguous or not, is regarded as a typical number of amino acids in a minimal epitope. See, e.g., Nair et al., J. Immunol 2000 165 (12): 6949-6955; Hebbes et al., Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Immunol (1985) 135(4):2598-608.
Another class of 158P1D7-related protein variants share 70%, 75%, 80%, 85% or 90% or more similarity with the amino acid sequence of FIG. 2 or a fragment thereof. Another specific class of 158P1D7 protein variants or analogs comprise one or more of the 158P1D7 biological motifs described herein or presently known in the art. Thus, encompassed by the present invention are analogs of 158P1D7 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 the 158P1D7 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 the 158P1D7 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 the 158P1D7 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 10 to about amino acid 20 of the 158P1D7 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 20 to about amino acid 30 of the 158P1D7 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 30 to about amino acid 40 of the 158P1D7 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 40 to about amino acid 50 of the 158P1D7 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 50 to about amino acid 60 of the 158P1D7 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 60 to about amino acid 70 of the 158P1D7 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 70 to about amino acid 80 of the 158P1D7 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 80 to about amino acid 90 of the 158P1D7 protein shown in FIG. 2 or FIG. 3, polypeptides consisting of about amino acid 90 to about amino acid 100 of the 158P1D7 protein shown in FIG. 2 or FIG. 3, etc. throughout the entirety of the 158P1D7 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 the 158P1D7 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.
158P1D7-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 158P1D7-related protein. In one embodiment, nucleic acid molecules provide a means to generate defined fragments of the 158P1D7 protein (or variants, homologs or analogs thereof).
III.A.) Motif-bearing Protein Embodiments
Additional illustrative embodiments of the invention disclosed herein include 158P1D7 polypeptides comprising the amino acid residues of one or more of the biological motifs contained within the 158P1D7 polypeptide sequence set forth in FIG. 2 or FIG. 3. Various motifs are known in the art, and a protein can be evaluated for the presence of such motifs by a number of publicly available Internet sites (see, e.g., URL addresses: pfam.wustl.edu/; searchlauncher.bcm.tmc.edu/seq-search/struc-predict.html; psort.ims.u-tokyo.ac.jp/; URL: cbs.dtu.dk/; ebi.ac.uk/interpro/scan.html; expasy.ch/tools/scnpsitl.html; EpimatrixTM and EpimerTM, Brown University, brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS, bimas.dcrt.nih.gov/.).
Motif bearing subsequences of the 158P1D7 protein are set forth and identified in Table XIX.
Table XX sets forth several frequently occurring motifs based on pfam searches (see URL address pfam.wustl.edu/). The columns of Table XX list (1) motif name abbreviation, (2) percent identity found amongst the different member of the motif family, (3) motif name or description and (4) most common function; location information is included if the motif is relevant for location.
Polypeptides comprising one or more of the 158P1D7 motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 158P1D7 motifs discussed above are associated with growth dysregulation and because 158P1D7 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-XVIII. CTL epitopes can be determined using specific algorithms to identify peptides within an 158P1D7 protein that are capable of optimally binding to specified HLA alleles (e.g., Table IV; EpimatrixTM and EpimerTM, Brown University, URL: brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS, URL: bimas.dcrt.nih.gov/.) Moreover, processes for identifying peptides that have sufficient binding affinity for HLA molecules and which are correlated with being immunogenic epitopes, are well known in the art, and are carried out without undue experimentation. In addition, processes for identifying peptides that are immunogenic epitopes, are well known in the art, and are carried out without undue experimentation either in vitro or in vivo.
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 9733602 to Chesnut et al.; Sette, Immunogenetics 1999 50(34): 201-212; Sette et al., J. Immunol. 2001 166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20; Kondo et al., Immunogenetics 1997 45(4): 249-258; Sidney et al., J. Immunol. 1996 157(8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75 (1994)); Kast et al., 1994 152(8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61(3): 266-278; Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625-1633; Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al., J. Immunol. 1991 147(8): 2663-2669; Alexander et al., Immunity 1994 1(9): 751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92.
Related embodiments of the inventions include polypeptides comprising combinations of the different motifs set forth in Table XIX, and/or, one or more of the predicted CTL epitopes of Table V through Table XVIII, and/or, one or more of the T cell binding motifs known in the art. Preferred embodiments contain no insertions, deletions or substitutions either within the motifs or the intervening sequences of the polypeptides. In addition, embodiments which include a number of either N-terminal and/or C-terminal amino acid residues on either side of these motifs may be desirable (to, for example, include a greater portion of the polypeptide architecture in which the motif is located). Typically the number of N-terminal and/or C-terminal amino acid residues on either side of a motif is between about 1 to about 100 amino acid residues, preferably 5 to about 50 amino acid residues.
158P1D7-related proteins are embodied in many forms, preferably in isolated form. A purified 158P1D7 protein molecule will be substantially free of other proteins or molecules that impair the binding of 158P1D7 to antibody, T cell or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a 158P1D7-related proteins include purified 158P1D7-related proteins and functional, soluble 158P1D7-related proteins. In one embodiment, a functional, soluble 158P1D7 protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.
The invention also provides 158P1D7 proteins comprising biologically active fragments of the 158P1D7 amino acid sequence shown in FIG. 2 or FIG. 3. Such proteins exhibit properties of the 158P1D7 protein, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the 158P1D7 protein; to be bound by such antibodies; to elicit the activation of HTL or CTL; and/or, to be recognized by HTL or CTL.
158P1D7-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 on the basis of immunogenicity. Fragments that contain such structures are particularly useful in generating subunit-specific anti-158P1D7 antibodies, or T cells or in identifying cellular factors that bind to 158P1D7.
CTL epitopes can be determined using specific algorithms to identify peptides within an 158P1D7 protein that are capable of optimally binding to specified HLA alleles (e.g., by using the SYFPEITHI site at World Wide Web URL syfpeithi.bmi-heidelberg.com/; the listings in Table IV(AY(E); EpimatixTM and EpimerTM, Brown University, URL (URL: brown.edu/Research/TB-HIV_Lab/epimatrx/epimabix.html); and BIMAS, URL: bimas.dcrt.nih.gov/). Illustrating this, peptide epitopes from 158P1D7 that are presented in the context of human MHC class I molecules HLA-A1, A2, A3, A11, A24, B7 and B35 were predicted (Tables V-XVIII). Specifically, the complete amino acid sequence of the 158P1D7 protein was entered into the HLA Peptide Motif Search algorithm found in the Bioinformatics and Molecular Analysis Section (BIMAS) web site listed above. 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 158P1D7 predicted binding peptides are shown in Tables V-XVIII herein. In Tables V-XVIII, the top 50 ranking 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. The binding score corresponds to the estimated half time of dissociation of complexes containing the peptide at 37° C. at pH 6.5. Peptides with the highest binding score are predicted to be the most tightly bound to HLA Class I on the cell surface for the greatest period of time and thus represent the best immunogenic targets for T-cell recognition.
Actual binding of peptides to an HLA allele can be evaluated by stabilization of HLA expression on the antigen-processing defective cell line T2 (see, e.g., Xue et al., Prostate 30:73-8 (1997) and Peshwa et al., Prostate 36:129-38 (1998)). Immunogenicity of specific peptides can be evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes (CTL) in the presence of antigen presenting cells such as dendritic cells.
It is to be appreciated that every epitope predicted by the BIMAS site, EpimerTM and EpimatrixTM sites, or specified by the HLA class I or class II motifs available in the art or which become part of the art such as set forth in Table IV (or determined using World Wide Web site URL syfpeithi.bmi-heidelberg.com/) are to be “applied” to the 158P1D7 protein. As used in this context “applied” means that the 158P1D7 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 the 158P1D7 of 8, 9, 10, or 11 amino acid residues that bears an HLA Class I motif, or a subsequence of 9 or more amino acid residues that bear an HLA Class II motif are within the scope of the invention.
III.B.) Expression of 158P1D7-related Proteins
In an embodiment described in the examples that follow, 158P1D7 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 158P1D7 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 158P1D7 protein in transfected cells. The secreted HIS-tagged 158P1D7 in the culture media can be purified, e.g., using a nickel column using standard techniques.
III.C.) Modifications of 158P1D7-related Proteins
Modifications of 158P1D7-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 158P1D7 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of the 158P1D7. Another type of covalent modification of the 158P1D7 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 158P1D7 comprises linking the 158P1D7 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 158P1D7-related proteins of the present invention can also be modified to form a chimeric molecule comprising 158P1D7 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 the 158P1D7 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 158P1D7. A chimeric molecule can comprise a fusion of a 158P1D7-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 the 158P1D7. In an alternative embodiment, the chimeric molecule can comprise a fusion of a 158P1D7-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 158P1D7 polypeptide in place of at least one variable region within an Ig molecule. In a preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgG1 molecule. For the production of immunoglobulin fusions see, e.g., U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
III.D.) Uses of 158P1D7-related Proteins
The proteins of the invention have a number of different uses. As 158P1D7 is highly expressed in bladder and other cancers, 158P1D7-related proteins are used in methods that assess the status of 158P1D7 gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of the 158P1D7 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 158P1D7-related proteins comprising the amino acid residues of one or more of the biological motifs contained within the 158P1D7 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, 158P1D7-related proteins that contain the amino acid residues of one or more of the biological motifs in the 158P1D7 protein are used to screen for factors that interact with that region of 158P1D7.
158P1D7 protein fragments/subsequences are particularly useful in generating and characterizing domain-specific antibodies (e.g., antibodies recognizing an extracellular or intracellular epitope of an 158P1D7 protein), for identifying agents or cellular factors that bind to 158P1D7 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 158P1D7 genes, or by analogs, homologs or fragments thereof, have a variety of uses, including but not limited to generating antibodies and in methods for identifying ligands and other agents and cellular constituents that bind to an 158P1D7 gene product. Antibodies raised against an 158P1D7 protein or fragment thereof are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of 158P1D7 protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers. 158P1D7-related nucleic acids or proteins are also used in generating HTL or CTL responses.
Various immunological assays useful for the detection of 158P1D7 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 158P1D7-expressing cells (e.g., in radioscintigraphic imaging methods). 158P1D7 proteins are also particularly useful in generating cancer vaccines, as further described herein.
IV.) 158P1D7 Antibodies
Another aspect of the invention provides antibodies that bind to 158P1D7-related proteins. Preferred antibodies specifically bind to a 158P1D7-related protein and do not bind (or bind weakly) to peptides or proteins that are not 158P1D7-related proteins. For example, antibodies bind 158P1D7 can bind 158P1D7-related proteins such as the homologs or analogs thereof.
158P1D7 antibodies of the invention are particularly useful in bladder cancer 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 158P1D7 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 158P1D7 is involved, such as advanced or metastatic bladder cancers.
The invention also provides various immunological assays useful for the detection and quantification of 158P1D7 and mutant 158P1D7-related proteins. Such assays can comprise one or more 158P1D7 antibodies capable of recognizing and binding a 158P1D7-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 histocompatbility complex (MHC) binding assays.
In addition, immunological imaging methods capable of detecting bladder cancer and other cancers expressing 158P1D7 are also provided by the invention, including but not limited to radioscintigraphic imaging methods using labeled 158P1D7 antibodies. Such assays are clinically useful in the detection, monitoring, and prognosis of 158P1D7 expressing cancers such as bladder cancer.
158P1D7 antibodies are also used in methods for purifying a 158P1D7-related protein and for isolating 158P1D7 homologues and related molecules. For example, a method of purifying a 158P1D7-related protein comprises incubating an 158P1D7 antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a 158P1D7-related protein under conditions that permit the 158P1D7 antibody to bind to the 158P1D7-related protein; washing the solid matrix to eliminate impurities; and eluting the 158P1D7-related protein from the coupled antibody. Other uses of the 158P1D7 antibodies of the invention include generating anti-idiotypic antibodies that mimic the 158P1D7 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 158P1D7-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 158P1D7 can also be used, such as a 158P1D7GST-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 158P1D7-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 158P1D7-related protein or 158P1D7 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 add sequence of 158P1D7 as shown in FIG. 2 or FIG. 3 can be analyzed to select specific regions of the 158P1D7 protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of the 158P1D7 amino acid sequence are used to identify hydrophilic regions in the 158P1D7 structure (see, e.g., the Example entitled “Antigenicity profiles”). Regions of the 158P1D7 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, Hopp and Woods, Kyte-Doolittle, Janin, Bhaskaran and Ponnuswamy, Deleage and Roux, Garnier-Robson, Eisenberg, Karplus-Schultz, or Jameson-Wolf analysis. Thus, each region identified by any of these programs or methods is within the scope of the present invention. Methods for the generation of 158P1D7 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 158P1D7 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.
158P1D7 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 158P1D7-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.
One embodiment of the invention is a mouse hybridoma that produces murine monoclonal antibodies designated X68(2)18 (a.k.a. M15-68(2)18.1.1) deposited with American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, Va. 20108 on 6 Feb. 2004 and assigned Accession No. ______.
The antibodies or fragments of the invention can also be produced, by recombinant means. Regions that bind specifically to the desired regions of the 158P1D7 protein can also be produced in the context of chimeric or complementarity determining region (CDR) grafted antibodies of multiple species origin. Humanized or human 158P1D7 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. Nat. 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 158P1D7 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 158P1D7 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 158P1D7 antibodies with an 158P1D7-related protein can be established by a number of well known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses using, as appropriate, 158P1D7-related proteins, 158P1D7-expressing cells or extracts thereof. A 158P1D7 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 158P1D7 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.) 158P1D7 Cellular Immune Responses
The mechanism by which T cells recognize antigens has been delineated. Efficacious peptide epitope vaccine compositions of the invention induce a therapeutic or prophylactic immune responses in very broad segments of the world-wide population. For an understanding of the value and efficacy of compositions of the invention that induce cellular immune responses, a brief review of immunology-related technology is provided.
A complex of an HLA molecule and a peptidic antigen acts as the ligand recognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985; Townsend, A. and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R. N., Annu. Rev. Immunol. 11:403, 1993). Through the study of single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues that correspond to motifs required for specific binding to HLA antigen molecules have been identified and are set forth in Table IV (see also, e.g., Southwood, et al., J. Immunol. 160:3363, 1998; Rammensee, et al., Immunogenetics 41:178, 1995; Rammensee et al., SYFPEITHI, access via World Wide Web at URL syfpeithi.bmi-heidelberg.com/; Sette, A. and Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et al., Cell 74:929-937, 1993; Kondo et al., J. Immunol. 155:4307-4312, 1995; Sidney et al., J. Immunol 157:3480-3490, 1996; Sidney et al., Human Immunol. 45:79-93, 1996; Sette, A. and Sidney, J. Immunogenetics 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 51 Cr-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.) 158P1D7 Transgenic Animals
Nucleic acids that encode a 158P1D7-related protein can also be used to generate either transgenic animals or “knock out” animals which, in turn, are useful in the development and screening of therapeutically useful reagents. In accordance with established techniques, cDNA encoding 158P1D7 can be used to clone genomic DNA that encodes 158P1D7. The cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode 158P1D7. 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 158P1D7 transgene incorporation with tissue-specific enhancers.
Transgenic animals that include a copy of a transgene encoding 158P1D7 can be used to examine the effect of increased expression of DNA that encodes 158P1D7. 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 158P1D7 can be used to construct a 158P1D7 “knock out” animal that has a defective or altered gene encoding 158P1D7 as a result of homologous recombination between the endogenous gene encoding 158P1D7 and altered genomic DNA encoding 158P1D7 introduced into an embryonic cell of the animal. For example, cDNA that encodes 158P1D7 can be used to clone genomic DNA encoding 158P1D7 in accordance with established techniques. A portion of the genomic DNA encoding 158P1D7 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 the 158P1D7 polypeptide.
VII.) Methods for the Detection of 158P1D7
Another aspect of the present invention relates to methods for detecting 158P1D7 polynucleotides and polypeptides and 158P1D7-related proteins, as well as methods for identifying a cell that expresses 158P1D7. The expression profile of 158P1D7 makes it a diagnostic marker for metastasized disease. Accordingly, the status of 158P1D7 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 158P1D7 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 158P1D7 polynucleotides in a biological sample, such as urine, serum, bone, prostatic fluid, tissues, semen, cell preparations, and the like. Detectable 158P1D7 polynucleotides include, for example, a 158P1D7 gene or fragment thereof, 158P1D7 mRNA, alternative splice variant 158P1D7 mRNAs, and recombinant DNA or RNA molecules that contain a 158P1D7 polynucleotide. A number of methods for amplifying and/or detecting the presence of 158P1D7 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 an 158P1D7 mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using an 158P1D7 polynucleotides as sense and antisense primers to amplify 158P1D7 cDNAs therein; and detecting the presence of the amplified 158P1D7 cDNA. Optionally, the sequence of the amplified 158P1D7 cDNA can be determined.
In another embodiment, a method of detecting a 158P1D7 gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using 158P1D7 polynucleotides as sense and antisense primers; and detecting the presence of the amplified 158P1D7 gene. Any number of appropriate sense and antisense probe combinations can be designed from the nucleotide sequence provided for the 158P1D7 (FIG. 2) and used for this purpose.
The invention also provides assays for detecting the presence of an 158P1D7 protein in a tissue or other biological sample such as urine, serum, semen, bone, prostate, cell preparations, and the like. Methods for detecting a 158P1D7-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 158P1D7-related protein in a biological sample comprises first contacting the sample with a 158P1D7 antibody, a 158P1D7-reactive fragment thereof, or a recombinant protein containing an antigen binding region of a 158P1D7 antibody; and then detecting the binding of 158P1D7-related protein in the sample.
Methods for identifying a cell that expresses 158P1D7 are also within the scope of the invention. In one embodiment, an assay for identifying a cell that expresses a 158P1D7 gene comprises detecting the presence of 158P1D7 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 158P1D7 riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 158P1D7, 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 158P1D7 gene comprises detecting the presence of 158P1D7-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 158P1D7-related proteins and cells that express 158P1D7-related proteins.
158P1D7 expression analysis is also useful as a tool for identifying and evaluating agents that modulate 158P1D7 gene expression. For example, 158P1D7 expression is significantly upregulated in bladder cancer, and is expressed in cancers of the tissues listed in Table I. Identification of a molecule or biological agent that inhibits 158P1D7 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 158P1D7 expression by RT-PCR, nucleic acid hybridization or antibody binding.
VIII.) Methods for Monitoring the Status of 158P1D7-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): 437A38 (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 158P1D7 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 158P1D7 in a biological sample of interest can be compared, for example, to the status of 158P1D7 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 158P1D7 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 158P1D7 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 158P1D7 expressing cells) as well as the level, and biological activity of expressed gene products (such as 158P1D7 mRNA, polynucleotides and polypeptides). Typically, an alteration in the status of 158P1D7 comprises a change in the location of 158P1D7 and/or 158P1D7 expressing cells and/or an increase in 158P1D7 mRNA and/or protein expression.
158P1D7 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 the 158P1D7 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 158P1D7 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 the 158P1D7 gene), Northern analysis and/or PCR analysis of 158P1D7 mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of 158P1D7 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 158P1D7 proteins and/or associations of 158P1D7 proteins with polypeptide binding partners). Detectable 158P1D7 polynucleotides include, for example, a 158P1D7 gene or fragment thereof, 158P1D7 mRNA, alternative splice variants, 158P1D7 mRNAs, and recombinant DNA or RNA molecules containing a 158P1D7 polynucleotide.
The expression profile of 158P1D7 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 158P1D7 provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The invention provides methods and assays for determining 158P1D7 status and diagnosing cancers that express 158P1D7, such as cancers of the issues listed in Table I. For example, because 158P1D7 mRNA is so highly expressed in bladder and other cancers relative to normal bladder tissue, assays that evaluate the levels of 158P1D7 mRNA transcripts or proteins in a biological sample can be used to diagnose a disease associated with 158P1D7 dysregulation, and can provide prognostic information useful in defining appropriate therapeutic options.
The expression status of 158P1D7 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 158P1D7 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 158P1D7 in a biological sample can be examined by a number of well-known procedures in the art. For example, the status of 158P1D7 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 158P1D7 expressing cells (e.g. those that express 158P1D7 mRNAs or proteins). This examination can provide evidence of dysregulated cellular growth, for example, when 158P1D7-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 158P1D7 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 bladder) to a different area of the body (such as a lymph node). By example, evidence of dysregulated cellular growth is important 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 158P1D7 gene products by determining the status of 158P1D7 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 158P1D7 gene products in a corresponding normal sample. The presence of aberrant 158P1D7 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 158P1D7 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 158P1D7 mRNA can, for example, be evaluated in tissue samples including but not limited to those listed in Table I. The presence of significant 158P1D7 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 158P1D7 mRNA or express it at lower levels.
In a related embodiment, 158P1D7 status is determined at the protein level rather than at the nucleic acid level. For example, such a method comprises determining the level of 158P1D7 protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 158P1D7 expressed in a corresponding normal sample. In one embodiment, the presence of 158P1D7 protein is evaluated, for example, using immunohistochemical methods. 158P1D7 antibodies or binding partners capable of detecting 158P1D7 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 158P1D7 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 158P1D7 may be indicative of the presence or promotion of a tumor. Such assays therefore have diagnostic and predictive value where a mutation in 158P1D7 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 158P1D7 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 the 158P1D7 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 DBCCR1, PAX6 and APC genes have been detected in bladder cancers leading to aberrant expression of the genes (Esteller et al., Cancer Res 2001; 61:3225-3229) 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 which 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 158P1D7. Gene amplification is measured in a sample directly, for example, by conventional Southern blotting or Northern blotting to quantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies are employed that recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn are labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
Biopsied tissue or peripheral blood can be conveniently assayed for the presence of cancer cells using for example, Northern, dot blot or RT-PCR analysis to detect 158P1D7 expression. The presence of RT-PCR amplifiable 158P1D7 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.
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 158P1D7 mRNA or 158P1D7 protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of 158P1D7 mRNA expression correlates to the degree of susceptibility. In a specific embodiment, the presence of 158P1D7 in bladder or other tissue is examined, with the presence of 158P1D7 in the sample providing an indication of bladder cancer susceptibility (or the emergence or existence of a bladder tumor). Similarly, one can evaluate the integrity 158P1D7 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 158P1D7 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 158P1D7 mRNA or 158P1D7 protein expressed by tumor cells, comparing the level so determined to the level of 158P1D7 mRNA or 158P1D7 protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of 158P1D7 mRNA or 158P1D7 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 158P1D7 is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors. Another embodiment is the evaluation of the integrity of 158P1D7 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 158P1D7 mRNA or 158P1D7 protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of 158P1D7 mRNA or 158P1D7 protein expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of 158P1D7 mRNA or 158P1D7 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 158P1D7 expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer. Also, one can evaluate the integrity 158P1D7 nucleotide and amino add 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 158P1D7 gene and 158P1D7 gene products (or perturbations in 158P1D7 gene and 158P1D7 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. PSCA, H-ras and p53 expression 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 158P1D7 gene and 158P1D7 gene products (or perturbations in 158P1D7 gene and 158P1D7 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 158P1D7 gene and 158P1D7 gene products (or perturbations in 158P1D7 gene and 158P1D7 gene products) and another factor associated with malignancy entails detecting the overexpression of 158P1D7 mRNA or protein in a tissue sample, detecting the overexpression of BLCA-4A mRNA or protein in a tissue sample (or PSCA expression), and observing a coincidence of 158P1D7 mRNA or protein and BLCA-4 mRNA or protein overexpression (or PSCA expression) (Amara et al., 2001, Cancer Res 61:4660-4665; Konety et al., Clin Cancer Res, 2000, 6(7):2618-2625). In a specific embodiment, the expression of 158P1D7 and BLCA-4 mRNA in bladder tissue is examined, where the coincidence of 158P1D7 and BLCA-4 mRNA overexpression in the sample indicates the existence of bladder cancer, bladder cancer susceptibility or the emergence or status of a bladder tumor.
Methods for detecting and quantifying the expression of 158P1D7 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 158P1D7 mRNA include in situ hybridization using labeled 158P1D7 riboprobes, Northern blot and related techniques using 158P1D7 polynucleotide probes, RT-PCR analysis using primers specific for 158P1D7, 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 158P1D7 mRNA expression. Any number of primers capable of amplifying 158P1D7 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 158P1D7 protein can be used in an immunohistochemical assay of biopsied tissue.
IX.) Identification of Molecules that Interact with 158P1D7
The 158P1D7 protein and nucleic acid sequences disclosed herein allow a skilled artisan to identify proteins, small molecules and other agents that interact with 158P1D7, as well as pathways activated by 158P1D7 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 158P1D7 protein sequences. In such methods, peptides that bind to 158P1D7 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 158P1D7 protein.
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 158P1D7 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 158P1D7 are used to identify protein-protein interactions mediated by 158P1D7. Such interactions can be examined using immunoprecipitation techniques (see, e.g., Hamilton B J, et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51). 158P1D7 protein can be immunoprecipitated from 158P1D7-expressing cell lines using anti-158P1D7 antibodies. Alternatively, antibodies against His-tag can be used in a cell line engineered to express fusions of 158P1D7 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 158P1D7 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 158P1D7'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 158P1D7 related ion channel, protein pump, or cell communication functions 158P1D7 are identified and used to treat patients that have a cancer that expresses 158P1D7 (see, e.g., Hille, B., Ionic Channels of Excitable Membranes 2nd Ed., Sinauer Assoc., Sunderland, Mass., 1992). Moreover, ligands that regulate 158P1D7 function can be identified based on their ability to bind 158P1D7 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 158P1D7 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 158P1D7.
An embodiment of this invention comprises a method of screening for a molecule that interacts with an 158P1D7 amino acid sequence shown in FIG. 2 or FIG. 3, comprising the steps of contacting a population of molecules with the 158P1D7 amino acid sequence, allowing the population of molecules and the 158P1D7 amino acid sequence to interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the 158P1D7 amino acid sequence, and then separating molecules that do not interact with the 158P1D7 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 158P1D7 amino acid sequence. The identified molecule can be used to modulate a function performed by 158P1D7. In a preferred embodiment, the 158P1D7 amino acid sequence is contacted with a library of peptides.
X.) Therapeutic Methods and Compositions
The identification of 158P1D7 as a protein that is normally expressed in a restricted set of tissues, but which is also expressed in bladder and other cancers, opens a number of therapeutic approaches to the treatment of such cancers. As contemplated herein, 158P1D7 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 the 158P1D7 protein are useful for patients suffering from a cancer that expresses 158P1D7. These therapeutic approaches generally fall into two classes. One class comprises various methods for inhibiting the binding or association of the 158P1D7 protein with its binding partner or with other proteins. Another class comprises a variety of methods for inhibiting the transcription of the 158P1D7 gene or translation of 158P1D7 mRNA.
X.A.) Anti-Cancer Vaccines
The invention provides cancer vaccines comprising a 158P1D7-related protein or 158P1D7-related nucleic acid. In view of the expression of 158P1D7, cancer vaccines prevent and/or treat 158P1D7-expressing cancers with minimal or no effects on non-target tissues. The use of a tumor antigen in a vaccine that generates humoral and/or cell-mediated immune responses as anti-cancer therapy is well known in the art (see, e.g., 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 158P1D7-related protein, or a 158P1D7-encoding nucleic acid molecule and recombinant vectors capable of expressing and presenting the 158P1D7 immunogen (which typically comprises a number of antibody or T cell epitopes). Skilled artisans understand that a wide variety of vaccine systems for delivery of immunoreactive epitopes are known in the art (see, e.g., Heryln et al., Ann Med 1999 Feb. 31(11):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 the 158P1D7 protein shown in FIG. 2 or analog or homolog thereof) so that the mammal generates an immune response that is specific for that epitope (e.g. generates antibodies that specifically recognize that epitope). In a preferred method, the 158P1D7 immunogen contains a biological motif, see e.g., Tables V-XVIII, or a peptide of a size range from 158P1D7 indicated in FIG. 11, FIG. 12, FIG. 13, FIG. 14, and FIG. 15.
The entire 158P1D7 protein, immunogenic regions or epitopes thereof can be combined and delivered by various means. Such vaccine compositions can include, for example, lipopeptides (e.g., Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) (“PLG”) microspheres (see, e.g., Eldridge, et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine 12:299-306, 1994; Jones et al., Vaccine 13:675-681, 1995), peptide compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin Exp Immunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J. P., J. Immunol. Methods 196:17-32, 1996), peptides formulated as multivalent peptides; peptides for use in ballistic delivery systems, typically crystallized peptides, viral delivery vectors (Perkus, M. E. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 379, 1996; Chakrabarti, S. et al., Nature 320:535,1986; Hu, S. L. et al., Nature 320:537,1986; Kieny, M.-P. et al., AIDS Bio/Technology 4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al., Virology 175:535, 1990), particles of viral or synthetic origin (e.g., Kofler, N. et al., J. Immunol. Methods. 192:25, 1996; Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. et al., Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R., and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al., Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol. 148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked or particle absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993; Robinson, H. L., Hunt, L. A., and Webster, R. G., Vaccine 11:957, 1993; Shiver, J. W. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 423, 1996; Cease, K. B., and Berzofsky, J. A., Annu. Rev. Immunol. 12:923, 1994 and Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993). Toxin-targeted delivery technologies, also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Mass.) may also be used.
In patients with 158P1D7-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 158P1D7 protein that bind corresponding HLA alleles (see e.g., Table IV; EpimerTM and EpimatrixTM, Brown University (URL brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and, BIMAS, (URL bimas.dcrt.nih.gov/; SYFPEITHI at URL syfpeithi.bmi-heidelberg.com/). In a preferred embodiment, the 158P1D7 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 or a peptide of 8, 9, 10 or 11 amino acids specified by an HLA Class I motif/supermotif (e.g., Table IV (A), Table IV (D), or Table IV (E)) and/or a peptide of at least 9 amino acids that comprises an HLA Class II motif/supermotif (e.g., Table IV (B) or Table IV (C)). As is appreciated in the art, the HLA Class I binding groove is essentially closed ended so that peptides of only a particular size range can fit into the groove and be bound, generally HLA Class I epitopes are 8, 9, 10, or 11 amino acids long. In contrast, the HLA Class II binding groove is essentially open ended; therefore a peptide of about 9 or more amino acids can be bound by an HLA Class II molecule. Due to the binding groove differences between HLA Class I and II, HLA Class I motifs are length specific, i.e., position two of a Class I motif is the second amino acid in an amino to carboxyl direction of the peptide. The amino acid positions in a Class II motif are relative only to each other, not the overall peptide, i.e., additional amino acids can be attached to the amino and/or carboxyl termini of a motif-bearing sequence. HLA Class II epitopes are often 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long, or longer than 25 amino acids.
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. the 158P1D7 protein) so that an immune response is generated. A typical embodiment consists of a method for generating an immune response to 158P1D7 in a host, by contacting the host with a sufficient amount of at least one 158P1D7 B cell or cytotoxic T-cell epitope or analog thereof; and at least one periodic interval thereafter re-contacting the host with the 158P1D7 B cell or cytotoxic T-cell epitope or analog thereof. A specific embodiment consists of a method of generating an immune response against a 158P1D7-related protein or a man-made multiepitopic peptide comprising: administering 158P1D7 immunogen (e.g. the 158P1D7 protein or a peptide fragment thereof, an 158P1D7 fusion protein or analog etc.) in a vaccine preparation to a human or another mammal. Typically, such vaccine preparations further contain a suitable adjuvant (see, e.g., U.S. Pat. No. 6,146,635) or a universal helper epitope such as a PADRETM peptide (Epimmune Inc., San Diego, Calif.; see, e.g., Alexander et al., J. Immunol. 2000 164(3); 164 (3): 1625-1633; Alexander et al., Immunity 1994 1(9): 751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92). An alternative method comprises generating an immune response in an individual against a 158P1D7 immunogen by: administering in vivo to muscle or skin of the individual's body a DNA molecule that comprises a DNA sequence that encodes an 158P1D7 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.
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 158P1D7. Constructs comprising DNA encoding a 158P1D7-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 158P1D7 protein/immunogen. Alternatively, a vaccine comprises a 158P1D7-related protein. Expression of the 158P1D7-related protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells that bear 158P1D7 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 URL: genweb.com). Nucleic acid-based delivery is described, for instance, in Wolff et. al., Science 247:1465 (1990) as well as U.S. Pat. Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720. Examples of DNA-based delivery technologies include “naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., U.S. Pat. No. 5,922,687).
For therapeutic or prophylactic immunization purposes, proteins of the invention can be expressed via viral or bacterial vectors. Various viral gene delivery systems that can be used in the practice of the invention include, but are not limited to, vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus, and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol. 8:658-663; Tsang et al. J. Natl. Cancer Inst. 87:982-990 (1995)). Non-viral delivery systems can also be employed by introducing naked DNA encoding a 158P1D7-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 158P1D7-related nucleic acid molecule. In one embodiment, the full-length human 158P1D7 cDNA is employed. In another embodiment, 158P1D7 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 158P1D7 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 bladder cancer, autologous dendritic cells pulsed with peptides of the MAGE-3 antigen are being used in a Phase I clinical trial to stimulate bladder cancer patients' immune systems (Nishiyama et al., 2001, Clin Cancer Res, 7(1):23-31). Thus, dendritic cells can be used to present 158P1D7 peptides to T cells in the context of MHC class I or II molecules. In one embodiment, autologous dendritic cells are pulsed with 158P1D7 peptides capable of binding to MHC class I and/or class II molecules. In another embodiment, dendritic cells are pulsed with the complete 158P1D7 protein. Yet another embodiment involves engineering the overexpression of the 158P1D7 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 158P1D7 can also be engineered to express immune modulators, such as GM-CSF, and used as immunizing agents.
X.B.) 158P1D7 as a Target for Antibody-based Therapy
158P1D7 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 158P1D7 is expressed by cancer cells of various lineages relative to corresponding normal cells, systemic administration of 158P1D7-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 158P1D7 are useful to treat 158P1D7-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.
158P1D7 antibodies can be introduced into a patient such that the antibody binds to 158P1D7 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 158P1D7, 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 the 158P1D7 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. 158P1D7), 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-158P1D7 antibody) that binds to a marker (e.g. 158P1D7) 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 158P1D7, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 158P1D7 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-158P1D7 antibodies can be done in accordance with various approaches that have been successfully employed in the treatment of other types of cancer, including but not limited to colon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari et al., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al., 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al., 1994, Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res. 55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin. Immunol. 11:117-127). Some therapeutic approaches involve conjugation of naked antibody to a toxin, such as the conjugation of Y91 or I131 to anti-CD20 antibodies (e.g., ZevalinTM, IDEC Pharmaceuticals Corp. or BexxarTM, Coulter Pharmaceuticals), while others involve co-administration of antibodies and other therapeutic agents, such as HerceptinTM (trastuzumab) with paclitaxel (Genentech, Inc.). To treat bladder cancer, for example, 158P1D7 antibodies can be administered in conjunction with radiation, chemotherapy or hormone ablation.
Although 158P1D7 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 158P1D7 expression, preferably using immunohistochemical assessments of tumor tissue, quantitative 158P1D7 imaging, or other techniques that reliably indicate the presence and degree of 158P1D7 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-158P1D7 monoclonal antibodies that treat bladder and other cancers include those that initiate a potent immune response against the tumor or those that are directly cytotoxic. In this regard, anti-158P1D7 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-158P1D7 mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express 158P1D7. 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-158P1D7 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 158P1D7 antigen with high affinity but exhibit low or no antigenicity in the patient.
Therapeutic methods of the invention contemplate the administration of single anti-158P1D7 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-158P1D7 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-158P1D7 mAbs are administered in their naked” or unconjugated form, or can have a therapeutic agent(s) conjugated to them.
Anti-158P1D7 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-158P1D7 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.
Based on clinical experience with the Herceptin 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-158P1D7 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 158P1D7 expression in the patient, the extent of circulating shed 158P1D7 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 158P1D7 in a given sample (e.g. the levels of circulating 158P1D7 antigen and/or 158P1D7 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-158P1D7 antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a 158P1D7-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-158P1D7 antibodies that mimic an epitope on a 158P1D7-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.) 158P1D7 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 158P1D7 antigen, or derives at least some therapeutic benefit when the antigen was tumor-associated.
In some embodiments, it may be desirable to combine the class I peptide components with components that induce or facilitate neutralizing antibody and or helper T cell responses directed to the target antigen. A preferred embodiment of such a composition comprises class I and class II epitopes in accordance with the invention. An alternative embodiment of such a composition comprises a class I and/or class II epitope in accordance with the invention, along with a cross reactive HTL epitope such as PADRETM (Epimmune, San Diego, Calif.) molecule (described e.g., in U.S. Pat. No. 5,736,142).
A vaccine of the invention can also include antigen-presenting cells (APC), such as dendritic cells (DC), as a vehicle to present peptides of the invention. Vaccine compositions can be created in vitro, following dendritic cell mobilization and harvesting, whereby loading of dendritic cells occurs in vitro. For example, dendritic cells are transfected, e.g., with a minigene in accordance with the invention, or are pulsed with peptides. The dendritic cell can then be administered to a patient to elicit immune responses in vivo. Vaccine compositions, either DNA- or peptide-based, can also be administered in vivo in combination with dendritc cell mobilization whereby loading of dendritic cells occurs in vivo.
Preferably, the following principles are utilized when selecting an array of epitopes for inclusion in a polyepitopic composition for use in a vaccine, or for selecting discrete epitopes to be included in a vaccine and/or to be encoded by nucleic acids such as a minigene. It is preferred that each of the following principles be balanced in order to make the selection. The multiple epitopes to be incorporated in a given vaccine composition may be, but need not be, contiguous in sequence in the native antigen from which the epitopes are derived.
1.) Epitopes are selected which, upon administration, mimic immune responses that have been observed to be correlated with tumor clearance. For HLA Class I this includes 34 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 TMs 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 158P1D7, the PADRE® universal helper T cell epitope (or multiple HTL epitopes from 158P1D7), and an endoplasmic reticulum-translocating signal sequence can be engineered. A vaccine may also comprise epitopes that are derived from other TAAs.
The immunogenicity of a multi-epitopic minigene can be confirmed in transgenic mice to evaluate the magnitude of CTL induction responses against the epitopes tested. Further, the immunogenicity of DNA-encoded epitopes in vivo can be correlated with the in vitro responses of specific CTL lines against target cells transfected with the DNA plasmid. Thus, these experiments can show that the minigene serves to both: 1.) generate a CTL response and 2.) that the induced CTLs recognized cells expressing the encoded epitopes.
For example, to create a DNA sequence encoding the selected epitopes (minigene) for expression in human cells, the amino acid sequences of the epitopes may be reverse translated. A human codon usage table can be used to guide the codon choice for each amino acid. These epitope-encoding DNA sequences may be directly adjoined, so that when translated, a continuous polypeptide sequence is created. To optimize expression and/or immunogenicity, additional elements can be incorporated into the minigene design. Examples of amino acid sequences that can be reverse translated and included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes, antibody epitopes, a ubiquitination signal sequence, and/or an endoplasmic reticulum targeting signal. In addition, HLA presentation of CTL and HTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL or HTL epitopes; these larger peptides comprising the epitope(s) are within the scope of the invention.
The minigene sequence may be converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) may be synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides can be joined, for example, using T4 DNA ligase. This synthetic minigene, encoding the epitope polypeptide, can then be cloned into a desired expression vector.
Standard regulatory sequences well known to those of skill in the art are preferably included in the vector to ensure expression in the target cells. Several vector elements are desirable: a promoter with a down-stream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g. ampicillin or kanamycin resistance). Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.
Additional vector modifications may be desired to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the minigene. The inclusion of mRNA stabilization sequences and sequences for replication in mammalian cells may also be considered for increasing minigene expression.
Once an expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is transformed into an appropriate E. coli strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as all other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell bank.
In addition, immunostimulatory sequences (ISSs or CpGs) appear to play a role in the immunogenicity of DNA vaccines. These sequences may be included in the vector, outside the minigene coding sequence, if desired to enhance immunogenicity.
In some embodiments, a bi-cistronic expression vector which allows production of both the minigene-encoded epitopes and a second protein (included to enhance or decrease immunogenicity) can be used. Examples of proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, or for HTL responses, pan-DR binding proteins (PADRETM, Epimmune, San Diego, Calif.). Helper (HTL) epitopes can be joined to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows direction of the HTL epitopes to a cell compartment different than that of the CTL epitopes. If required, this could facilitate more efficient entry of HTL epitopes into the HLA class II pathway, thereby improving HTL induction. In contrast to HTL or CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-β) may be beneficial in certain diseases.
Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in E. coli, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and grown to saturation in shaker flasks or a bioreactor according to well-known techniques. Plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion-exchange resins supplied by QIAGEN, Inc. (Valencia, Calif.). If required, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.
Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). This approach, known as “naked DNA,” is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of minigene DNA vaccines, an alternative method for formulating purified plasmid DNA may be desirable. A variety of methods have been described, and new techniques may become available. Cationic lipids, glycolipids, and fusogenic liposomes can also be used in the formulation (see, e.g., as described by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987). In addition, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds (PINC) could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.
Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes. For example, the plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays. The transfection method used will be dependent on the final formulation. Electroporation can be used for “naked” DNA, whereas cationic lipids allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS). These cells are then chromium-51 (51Cr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by 51Cr release, indicates both production of, and HLA presentation of, minigene-encoded CTL epitopes. Expression of HTL epitopes may be evaluated in an analogous manner using assays to assess HTL activity.
In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations. Transgenic mice expressing appropriate human HLA proteins are immunized with the DNA product. The dose and route of administration are formulation dependent (e.g., IM for DNA in PBS, intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-one days after immunization, splenocytes are harvested and restimulated for one week in the presence of peptides encoding each epitope being tested. Thereafter, for CTL effector cells, assays are conducted for cytolysis of peptide-loaded, 51Cr-labeled target cells using standard techniques. Lysis of target cells that were sensitized by HLA loaded with peptide epitopes, corresponding to minigene-encoded epitopes, demonstrates DNA vaccine function for in vivo induction of CTLs. Immunogenicity of HTL epitopes is confirmed in transgenic mice in an analogous manner.
Alternatively, the nucleic acids can be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253. Using this technique, particles comprised solely of DNA are administered. In a further alternative embodiment, DNA can be adhered to particles, such as gold particles.
Minigenes can also be delivered using other bacterial or viral delivery systems well known in the art, e.g., an expression construct encoding epitopes of the invention can be incorporated into a viral vector such as vaccinia.
X.C.2. Combinations of CTL Peptides with Helper Peptides
Vaccine compositions comprising CTL peptides of the invention can be modified, e.g., analoged, to provide desired attributes, such as improved serum half life, broadened population coverage or enhanced immunogenicity.
For instance, the ability of a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence which contains at least one epitope that is capable of inducing a T helper cell response. Although a CTL peptide can be directly linked to a T helper peptide, often CTL epitope/HTL epitope conjugates are linked by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. The spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer need not be comprised of the same residues and thus may be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more usually three to six residues and sometimes 10 or more residues. The CTL peptide epitope can be linked to the T helper peptide epitope either directly or via a spacer either at the amino or carboxy terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the T helper peptide may be acylated.
In certain embodiments, the T helper peptide is one that is recognized by T helper cells present in a majority of a genetically diverse population. This can be accomplished by selecting peptides that bind to many, most, or all of the HLA class II molecules. Examples of such amino acid bind many HLA Class II molecules include sequences from antigens such as tetanus toxoid at positions 830-843 (QYIKANSKFIGITE; SEQ ID NO: 24), Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: 25), and Streptococcus 18 kD protein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO: 26). Other examples include peptides bearing a DR 1-4-7 supermotif, or either of the DR3 motifs.
Alternatively, it is possible to prepare synthetic peptides capable of stimulating T helper lymphocytes, in a loosely HLA-restricted fashion, using amino acid sequences not found in nature (see, e.g., PCT publication WO 95/07707). These synthetic compounds called Pan-DR-binding epitopes (e.g., PADRETM, Epimmune, Inc., San Diego, Calif.) are designed to most preferably bind most HLA-DR (human HLA class II) molecules. For instance, a pan-DR-binding epitope peptide having the formula: aKXVAAWTLKAAa (SEQ ID NO: 27), 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 specifically prime 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 158P1D7. 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 158P1D7.
X.D. Adoptive Immunotherapy
Antigenic 158P1D7-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 dendritc 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 158P1D7. 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 158P1D7. 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 158P1D7-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 158P1D7, a vaccine comprising 158P1D7-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 effectively stimulate a cytotoxic T cell response; compositions which stimulate helper T cell responses can also be given in accordance with this embodiment of the invention.
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 □g and the higher value is about 10,000; 20,000; 30,000; or 50,000 □g. Dosage values for a human typically range from about 500 □g to about 50,000 □g 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 patent'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 □g and the higher value is about 10,000; 20,000; 30,000; or 50,000 □g. Dosage values for a human typically range from about 500 □g to about 50,000 □g 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, welting 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 the peptide composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable carrier, preferably an aqueous carrier, and is administered in a volume of fluid 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).
Proteins(s) of the invention, and/or nucleic acids encoding the protein(s), can also be administered via liposomes, which may also serve to: 1) target the proteins(s) to a particular tissue, such as lymphoid tissue; 2) to target selectively to diseases cells; or, 3) to increase the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes either filled or decorated with a desired peptide of the invention can be directed to the site of lymphoid cells, where the liposomes then deliver the peptide compositions. Liposomes for use in accordance with the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.
For targeting cells of the immune system, a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.
For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the invention, and more preferably at a concentration of 25%-75%.
For aerosol administration, immunogenic peptides are preferably supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptides are about 0.01%-20% by weight, preferably about 1%-10%. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from about 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute about 0.1%-20% by weight of the composition, preferably about 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery.
XI.) Diagnostic and Prognostic Embodiments of 158P1D7.
As disclosed herein, 158P1D7 polynucleotides, polypeptides, reactive cytotoxic T cells (CTL), reactive helper T cells (HTL) and anti-polypeptide antibodies are used in well known diagnostic, prognostic and therapeutic assays that examine conditions associated with dysregulated cell growth such as cancer, in particular the cancers listed in Table I (see, e.g., both its specific pattern of tissue expression as well as its overexpression in certain cancers as described for example in Example 4).
158P1D7 can be used in a manner analogous to, or as complementary to, the bladder associated antigen combination, mucins and CEA, represented in a diagnostic kit called ImmunoCytTM. ImmunoCyt a is a commercially available assay to identify and monitor the presence of bladder cancer (see Fradet et al., 1997, Can J Urol, 4 (3):400-405). A variety of other diagnostic markers are also used in similar contexts including p53 and H-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 the 158P1D7 polynucleotides and polypeptides (as well as the 158P1D7 polynucleotide probes and anti-158P1D7 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 158P1D7 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 158P1D7 polynucleotides described herein can be utilized to detect 158P1D7 overexpression or the metastasis of bladder 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 158P1D7 polypeptides described herein can be utilized to generate antibodies for use in detecting 158P1D7 overexpression or the metastasis of bladder 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 bladder 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 158P1D7 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 158P1D7-expressing cells (lymph node) is found to contain 158P1D7-expressing cells such as the 158P1D7 expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis.
Alternatively 158P1D7 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 158P1D7 or express 158P1D7 at a different level are found to express 158P1D7 or have an increased expression of 158P1D7 (see, e.g., the 158P1D7 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 158P1D7) such as ImmunoCytTM, PSCA etc. (see, e.g., Fradet et al., 1997, Can J Urol, 4(3):400405; Amara et al., 2001, Cancer Res 61:46604665). Just as PSA polynucleotide fragments and polynucleotide variants are employed by skilled artisans for use in methods of monitoring PSA, 158P1D7 polynucleotide fragments and polynucleotide variants are used in an analogous manner. In particular, typical PSA polynucleotides used in methods of monitoring PSA are probes or primers which consist of fragments of the PSA cDNA sequence. Illustrating this, primers used to PCR amplify a PSA polynucleotide must include less than the whole PSA sequence to function in the polymerase chain reaction. In the context of such PCR reactions, skilled artisans generally create a variety of different polynucleotide fragments that can be used as primers in order to amplify different portions of a polynucleotide of interest or to optimize amplification reactions (see, e.g., Caetano-Anolles, G. Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., Methods Mol. Biol. 98: 121-154 (1998)). An additional illustration of the use of such fragments is provided in Example 4, where a 158P1D7 polynucleotide fragment is used as a probe to show the expression of 158P1D7 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. the 158P1D7 polynucleotide shown in FIG. 2) 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. 158P1D7 polypeptide fragments and polypeptide analogs or variants can also be used in an analogous manner. This practice of using polypeptide fragments or polypeptide variants to generate antibodies (such as anti-PSA antibodies or T cells) is typical in the art with a wide variety of systems such as fusion proteins being used by practitioners (see, e.g., Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubel et al. eds., 1995). In this context, each epitope(s) functions to provide the architecture with which an antibody or T cell is reactive. Typically, skilled artisans create a variety of different polypeptide fragments that can be used in order to generate immune responses specific for different portions of a polypeptide of interest (see, e.g., U.S. Pat. No. 5,840,501 and U.S. Pat. No. 5,939,533). For example it may be preferable to utilize a polypeptide comprising one of the 158P1D7 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. the 158P1D7 polypeptide shown in FIG. 2).
As shown herein, the 158P1D7 polynucleotides and polypeptides (as well as the 158P1D7 polynucleotide probes and anti-158P1D7 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 158P1D7 gene products, in order to evaluate the presence or onset of a disease condition described herein, such as bladder cancer, are used to identify patients for preventive measures or further monitoring, as has been done so successfully with PSA for monitoring prostate cancer. Materials such as 158P1D7 polynucleotides and polypeptides (as well as the 158P1D7 polynucleotide probes and anti-158P1D7 antibodies used to identify the presence of these molecules) satisfy a need in the art for molecules having similar or complementary characteristics to PSA in situations of bladder cancer. Finally, in addition to their use in diagnostic assays, the 158P1D7 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 158P1D7 gene maps (see Example 3 below). Moreover, in addition to their use in diagnostic assays, the 158P1D7-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, 158P1D7-related proteins or polynucleotides of the invention can be used to treat a pathologic condition characterized by the over-expression of 158P1D7. 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 the 158P1D7 antigen. Antibodies or other molecules that react with 158P1D7 can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.
XII.) Inhibition of 158P1D7 Protein Function
The invention includes various methods and compositions for inhibiting the binding of 158P1D7 to its binding partner or its association with other protein(s) as well as methods for inhibiting 158P1D7 function.
XII.A.) Inhibition of 158P1D7 with Intracellular Antibodies
In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to 158P1D7 are introduced into 158P1D7 expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti-158P1D7 antibody is expressed intracellularly, binds to 158P1D7 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 precisely target the intrabody to the desired intracellular compartment. For example, intrabodies targeted to the endoplasmic reticulum (ER) are engineered to incorporate a leader peptide and, optionally, a C-terminal ER retention signal, such as the KDEL amino acid motif. Intrabodies intended to exert activity in the nucleus are engineered to include a nuclear localization signal. Lipid moieties are joined to intrabodies in order to tether the intrabody to the cytosolic side of the plasma membrane. Intrabodies can also be targeted to exert function in the cytosol. For example, cytosolic intrabodies are used to sequester factors within the cytosol, thereby preventing them from being transported to their natural cellular destination.
In one embodiment, intrabodies are used to capture 158P1D7 in the nucleus, thereby preventing its activity within the nucleus. Nuclear targeting signals are engineered into such 158P1D7 intrabodies in order to achieve the desired targeting. Such 158P1D7 intrabodies are designed to bind specifically to a particular 158P1D7 domain. In another embodiment, cytosolic intrabodies that specifically bind to the 158P1D7 protein are used to prevent 158P1D7 from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus (e.g., preventing 158P1D7 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 bladder, for example, the PSCA promoter and/or promoter/enhancer can be utilized (See, for example, U.S. Pat. No. 5,919,652 issued 6 Jul. 1999 and Lin et al. PNAS, USA 92(3):679-683 (1995)).
XII.B.) Inhibition of 158P1D7 with Recombinant Proteins
In another approach, recombinant molecules bind to 158P1D7 and thereby inhibit 158P1D7 function. For example, these recombinant molecules prevent or inhibit 158P1D7 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 158P1D7 specific antibody molecule. In a particular embodiment, the 158P1D7 binding domain of a 158P1D7 binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two 158P1D7 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 158P1D7, whereby the dimeric fusion protein specifically binds to 158P1D7 and blocks 158P1D7 interaction with a binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking technologies.
XII.C.) Inhibition of 158P1D7 Transcription or Translation
The present invention also comprises various methods and compositions for inhibiting the transcription of the 158P1D7 gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of 158P1D7 mRNA into protein.
In one approach, a method of inhibiting the transcription of the 158P1D7 gene comprises contacting the 158P1D7 gene with a 158P1D7 antisense polynucleotide. In another approach, a method of inhibiting 158P1D7 mRNA translation comprises contacting the 158P1D7 mRNA with an antisense polynucleotide. In another approach, a 158P1D7 specific ribozyme is used to cleave the 158P1D7 message, thereby inhibiting translation. Such antisense and ribozyme based methods can also be directed to the regulatory regions of the 158P1D7 gene, such as the 158P1D7 promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a 158P1D7 gene transcription factor are used to inhibit 158P1D7 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 158P1D7 by interfering with 158P1D7 transcriptional activation are also useful to treat cancers expressing 158P1D7. Similarly, factors that interfere with 158P1D7 processing are useful to treat cancers that express 158P1D7. 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 158P1D7 (i.e., antisense, ribozyme, polynucleotides encoding intrabodies and other 158P1D7 inhibitory molecules). A number of gene therapy approaches are known in the art. Recombinant vectors encoding 158P1D7 antisense polynucleotides, ribozymes, factors capable of interfering with 158P1D7 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 158P1D7 to a binding partner, etc.
In vivo, the effect of a 158P1D7 therapeutic composition can be evaluated in a suitable animal model. For example, xenogenic bladder cancer models can be used, wherein human bladder cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SCID mice (Shibayama et al., 1991, J Urol., 146(4):1136-7; Beecken et al., 2000, Urology, 56(3):521-526). Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.
In vivo assays that evaluate the promotion of apoptosis are useful in evaluating therapeutic compositions. In one embodiment, xenografts from tumor bearing mice treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.
The therapeutic compositions used in the practice of the foregoing methods can be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include any material that when combined with the therapeutic composition retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16th Edition, A. Osal, Ed., 1980).
Therapeutic formulations can be solubilized and administered via any route capable of delivering the therapeutic composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumor, intradermal, intraorgan, orthotopic, and the like. A preferred formulation for intravenous injection comprises the therapeutic composition in a solution of preserved bacteriostatic water, sterile unpreserved water, and/or diluted in polyvinylchloride or polyethylene bags containing 0.9% sterile Sodium Chloride for Injection, USP. Therapeutic protein preparations can be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted in bacteriostatic water (containing for example, benzyl alcohol preservative) or in sterile water prior to injection.
Dosages and administration protocols for the treatment of cancers using the foregoing methods will vary with the method and the target cancer, and will generally depend on a number of other factors appreciated in the art.
XIII.) Identification, Characterization and Use of Modulators of 158P1D7
Methods to Identify and Use Modulators
In one embodiment, screening is performed to identify modulators that induce or suppress a particular expression profile, suppress or induce specific pathways, preferably generating the associated phenotype thereby. In another embodiment, having identified differentially expressed genes important in a particular state; screens are performed to identify modulators that alter expression of individual genes, either increase or decrease. In another embodiment, screening is performed to identify modulators that alter a biological function of the expression product of a differentially expressed gene. Again, having identified the importance of a gene in a particular state, screens are performed to identify agents that bind and/or modulate the biological activity of the gene product.
In addition, screens are done for genes that are induced in response to a candidate agent. After identifying a modulator (one that suppresses a cancer expression pattern leading to a normal expression pattern, or a modulator of a cancer gene that leads to expression of the gene as in normal tissue) a screen is performed to identify genes that are specifically modulated in response to the agent. Comparing expression profiles between normal tissue and agent-treated cancer tissue reveals genes that are not expressed in normal tissue or cancer tissue, but are expressed in agent treated tissue, and vice versa. These agent-specific sequences are identified and used by methods described herein for cancer genes or proteins. In particular these sequences and the proteins they encode are used in marking or identifying agent-treated cells. In addition, antibodies are raised against the agent-induced proteins and used to target novel therapeutics to the treated cancer tissue sample.
Modulator-related Identification and Screening Assays:
Gene Expression-related Assays
Proteins, nucleic acids, and antibodies of the invention are used in screening assays. The cancer-associated proteins, antibodies, nucleic acids, modified proteins and cells containing these sequences are used in screening assays, such as evaluating the effect of drug candidates on a “gene expression profile,” expression profile of polypeptides or alteration of biological function. In one embodiment, the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent (e.g., Davis, G F, et al., J Biol Screen 7:69 (2002); Zlokarnik, et al., Science 279:84-8 (1998); Heid, Genome Res 6:986-94, 1996).
The cancer proteins, antibodies, nucleic acids, modified proteins and cells containing the native or modified cancer proteins or genes are used in screening assays. That is, the present invention comprises methods for screening for compositions which modulate the cancer phenotype or a physiological function of a cancer protein of the invention. This is done on a gene itself or by evaluating the effect of drug candidates on a “gene expression profile” or biological function. In one embodiment, expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring after treatment with a candidate agent, see Zlokarnik, supra.
A variety of assays are executed directed to the genes and proteins of the invention. Assays are run on an individual nucleic acid or protein level. That is, having identified a particular gene as up regulated in cancer, test compounds are screened for the ability to modulate gene expression or for binding to the cancer protein of the invention. “Modulation” in this context includes an increase or a decrease in gene expression. The preferred amount of modulation will depend on the original change of the gene expression in normal versus tissue undergoing cancer, with changes of at least 10%, preferably 50%, more preferably 100-300%, and in some embodiments 300-1000% or greater. Thus, if a gene exhibits a 4-fold increase in cancer tissue compared to normal tissue, a decrease of about four-fold is often desired; similarly, a 10-fold decrease in cancer tissue compared to normal tissue a target value of a 10-fold increase in expression by the test compound is often desired. Modulators that exacerbate the type of gene expression seen in cancer are also useful, e.g., as an upregulated target in further analyses.
The amount of gene expression is monitored using nucleic acid probes and the quantification of gene expression levels, or, alternatively, a gene product itself is monitored, e.g., through the use of antibodies to the cancer protein and standard immunoassays. Proteomics and separation techniques also allow for quantification of expression.
Expression Monitoring to Identify Compounds that Modify Gene Expression
In one embodiment, gene expression monitoring, i.e., an expression profile, is monitored simultaneously for a number of entities. Such profiles will typically involve one or more of the genes of FIG. 2. In this embodiment, e.g., cancer nucleic acid probes are attached to biochips to detect and quantify cancer sequences in a particular cell. Alternatively, PCR can be used. Thus, a series, e.g., wells of a microtiter plate, can be used with dispensed primers in desired wells. A PCR reaction can then be performed and analyzed for each well.
Expression monitoring is performed to identify compounds that modify the expression of one or more cancer-associated sequences, e.g., a polynucleotide sequence set out in FIG. 2. Generally, a test modulator is added to the cells prior to analysis. Moreover, screens are also provided to identify agents that modulate cancer, modulate cancer proteins of the invention, bind to a cancer protein of the invention, or interfere with the binding of a cancer protein of the invention and an antibody or other binding partner.
In one embodiment, high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such “combinatorial chemical libraries” are then screened in one or more assays to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional “lead compounds,” as compounds for screening, or as therapeutics.
In certain embodiments, combinatorial libraries of potential modulators are screened for an ability to bind to a cancer polypeptide or to modulate activity. Conventionally, new chemical entities with useful properties are generated by identifying a chemical compound (called a “lead compound”) with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis.
As noted above, gene expression monitoring is conveniently used to test candidate modulators (e.g., protein, nucleic acid or small molecule). After the candidate agent has been added and the cells allowed to incubate for a period, the sample containing a target sequence to be analyzed is, e.g., added to a biochip.
If required, the target sequence is prepared using known techniques. For example, a sample is treated to lyse the cells, using known lysis buffers, electroporation, etc., with purification and/or amplification such as PCR performed as appropriate. For example, an in vitro transcription with labels covalently attached to the nucleotides is performed. Generally, the nucleic acids are labeled with biotin-FITC or PE, or with cy3 or cy5.
The target sequence can be labeled with, e.g., a fluorescent, a chemiluminescent, a chemical, or a radioactive signal, to provide a means of detecting the target sequence's specific binding to a probe. The label also can be an enzyme, such as alkaline phosphatase or horseradish peroxidase, which when provided with an appropriate substrate produces a product that is detected. Alternatively, the label is a labeled compound or small molecule, such as an enzyme inhibitor, that binds but is not catalyzed or altered by the enzyme. The label also can be a moiety or compound, such as, an epitope tag or biotin which specifically binds to streptavidin. For the example of biotin, the streptavidin is labeled as described above, thereby, providing a detectable signal for the bound target sequence. Unbound labeled streptavidin is typically removed prior to analysis.
As will be appreciated by those in the art, these assays can be direct hybridization assays or can comprise “sandwich assays”, which include the use of multiple probes, as is generally outlined in U.S. Pat. Nos. 5,681,702; 5,597,909; 5,545,730; 5,594,117; 5,591,584; 5,571,670; 5,580,731; 5,571,670; 5,591,584; 5,624,802; 5,635,352; 5,594,118; 5,359,100; 5,124,246; and 5,681,697. In this embodiment, in general, the target nucleic acid is prepared as outlined above, and then added to the biochip comprising a plurality of nucleic acid probes, under conditions that allow the formation of a hybridization complex.
A variety of hybridization conditions are used in the present invention, including high, moderate and low stringency conditions as outlined above. The assays are generally run under stringency conditions which allow formation of the label probe hybridization complex only in the presence of target. Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration pH, organic solvent concentration, etc. These parameters may also be used to control non-specific binding, as is generally outlined in U.S. Pat. No. 5,681,697. Thus, it can be desirable to perform certain steps at higher stringency conditions to reduce non-specific binding.
The reactions outlined herein can be accomplished in a variety of ways. Components of the reaction can be added simultaneously, or sequentially, in different orders, with preferred embodiments outlined below. In addition, the reaction may include a variety of other reagents. These include salts, buffers, neutral proteins, e.g. albumin, detergents, etc. which can be used to facilitate optimal hybridization and detection, and/or reduce nonspecific or background interactions. Reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may also be used as appropriate, depending on the sample preparation methods and purity of the target. The assay data are analyzed to determine the expression levels of individual genes, and changes in expression levels as between states, forming a gene expression profile.
Biological Activity-related Assays
The invention provides methods identify or screen for a compound that modulates the activity of a cancer-related gene or protein of the invention. The methods comprise adding a test compound, as defined above, to a cell comprising a cancer protein of the invention. The cells contain a recombinant nucleic acid that encodes a cancer protein of the invention. In another embodiment, a library of candidate agents is tested on a plurality of cells.
In one aspect, the assays are evaluated in the presence or absence or previous or subsequent exposure of physiological signals, e.g. hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (i.e., cell-cell contacts). In another example, the determinations are made at different stages of the cell cycle process. In this way, compounds that modulate genes or proteins of the invention are identified. Compounds with pharmacological activity are able to enhance or interfere with the activity of the cancer protein of the invention. Once identified, similar structures are evaluated to identify critical structural features of the compound.
In one embodiment, a method of modulating (e.g., inhibiting) cancer cell division is provided; the method comprises administration of a cancer modulator. In another embodiment, a method of modulating (e.g., inhibiting) cancer is provided; the method comprises administration of a cancer modulator. In a further embodiment, methods of treating cells or individuals with cancer are provided; the method comprises administration of a cancer modulator.
In one embodiment, a method for modulating the status of a cell that expresses a gene of the invention is provided. As used herein status comprises such art-accepted parameters such as growth, proliferation, survival, function, apoptosis, senescence, location, enzymatic activity, signal transduction, etc. of a cell. In one embodiment, a cancer inhibitor is an antibody as discussed above. In another embodiment, the cancer inhibitor is an antisense molecule. A variety of cell growth, proliferation, and metastasis assays are known to those of skill in the art, as described herein.
High Throughput Screening to Identify Modulators
The assays to identify suitable modulators are amenable to high throughput screening. Preferred assays thus detect enhancement or inhibition of cancer gene transcription, inhibition or enhancement of polypeptide expression, and inhibition or enhancement of polypeptide activity.
In one embodiment, modulators evaluated in high throughput screening methods are proteins, often naturally occurring proteins or fragments of naturally occurring proteins. Thus, e.g., cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, are used. In this way, libraries of proteins are made for screening in the methods of the invention. Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred. Particularly useful test compound will be directed to the class of proteins to which the target belongs, e.g., substrates for enzymes, or ligands and receptors.
Use of Soft Agar Growth and Colony Formation to Identify and Characterize Modulators
Normal cells require a solid substrate to attach and grow. When cells are transformed, they lose this phenotype and grow detached from the substrate. For example, transformed cells can grow in stirred suspension culture or suspended in semi-solid media, such as semi-solid or soft agar. The transformed cells, when transfected with tumor suppressor genes, can regenerate normal phenotype and once again require a solid substrate to attach to and grow. Soft agar growth or colony formation in assays are used to identify modulators of cancer sequences, which when expressed in host cells, inhibit abnormal cellular proliferation and transformation. A modulator reduces or eliminates the host cells' ability to grow suspended in solid or semisolid media, such as agar.
Techniques for soft agar growth or colony formation in suspension assays are described in Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd ed., 1994). See also, the methods section of Garkavtsev et al. (1996), supra.
Evaluation of Contact Inhibition and Growth Density Limitation to Identify and Characterize Modulators
Normal cells typically grow in a flat and organized pattern in cell culture until they touch other cells. When the cells touch one another, they are contact inhibited and stop growing. Transformed cells, however, are not contact inhibited and continue to grow to high densities in disorganized foci. Thus, transformed cells grow to a higher saturation density than corresponding normal cells. This is detected morphologically by the formation of a disoriented monolayer of cells or cells in foci. Alternatively, labeling index with (3H)-thymidine at saturation density is used to measure density limitation of growth, similarly an MTT or Alamar blue assay will reveal proliferation capacity of cells and the the ability of modulators to affect same. See Freshney (1994), supra. Transformed cells, when transfected with tumor suppressor genes, can regenerate a normal phenotype and become contact inhibited and would grow to a lower density.
In this assay, labeling index with 3H)-thymidine at saturation density is a preferred method of measuring density limitation of growth. Transformed host cells are transfected with a cancer-associated sequence and are grown for 24 hours at saturation density in non-limiting medium conditions. The percentage of cells labeling with (3H)-thymidine is determined by incorporated cpm.
Contact independent growth is used to identify modulators of cancer sequences, which had led to abnormal cellular proliferation and transformation. A modulator reduces or eliminates contact independent growth, and returns the cells to a normal phenotype.
Evaluation of Growth Factor or Serum Dependence to Identify and Characterize Modulators
Transformed cells have lower serum dependence than their normal counterparts (see, e.g., Temin, J. Natl. Cancer Inst. 37:167-175 (1966); Eagle et al., J. Exp. Med 131:836-879 (1970)); Freshney, supra. This is in part due to release of various growth factors by the transformed cells. The degree of growth factor or serum dependence of transformed host cells can be compared with that of control. For example, growth factor or serum dependence of a cell is monitored in methods to identify and characterize compounds that modulate cancer-associated sequences of the invention.
Use of Tumor-specific Marker Levels to Identify and Characterize Modulators
Tumor cells release an increased amount of certain factors (hereinafter “tumor specific markers”) than their normal counterparts. For example, plasminogen activator (PA) is released from human glioma at a higher level than from normal brain cells (see, e.g., Gullino, Angiogenesis, Tumor Vascularization, and Potential Interference with Tumor Growth, in Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985)). Similarly, Tumor Angiogenesis Factor (TAF) is released at a higher level in tumor cells than their normal counterparts. See, e.g., Folkman, Angiogenesis and Cancer, Sem Cancer Biol. (1992)), while bFGF is released from endothelial tumors (Ensoli, B et al).
Various techniques which measure the release of these factors are described in Freshney (1994), supra. Also, see, Unkless et al., J. Biol. Chem. 249:4295-4305 (1974); Strickland & Beers, J. Biol. Chem. 251:5694-5702 (1976); Whur et al., Br. J. Cancer 42:305 312 (1980); Gullino, Angiogenesis, Tumor Vascularization, and Potential Interference with Tumor Growth, in Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985); Freshney, Anticancer Res. 5:111-130 (1985). For example, tumor specific marker levels are monitored in methods to identify and characterize compounds that modulate cancer-associated sequences of the invention.
Invasiveness into Matrigel to Identify and Characterize Modulators
The degree of invasiveness into Matrigel or an extracellular matrix constituent can be used as an assay to identify and characterize compounds that modulate cancer associated sequences. Tumor cells exhibit a positive correlation between malignancy and invasiveness of cells into Matrigel or some other extracellular matrix constituent. In this assay, tumorigenic cells are typically used as host cells. Expression of a tumor suppressor gene in these host cells would decrease invasiveness of the host cells. Techniques described in Cancer Res. 1999; 59:6010; Freshney (1994), supra, can be used. Briefly, the level of invasion of host cells is measured by using filters coated with Matrigel or some other extracellular matrix constituent. Penetration into the gel, or through to the distal side of the filter, is rated as invasiveness, and rated histologically by number of cells and distance moved, or by prelabeling the cells with 125I and counting the radioactivity on the distal side of the filter or bottom of the dish. See, e.g., Freshney (1984), supra.
Evaluation of Tumor Growth In Vivo to Identify and Characterize Modulators
Effects of cancer-associated sequences on cell growth are tested in transgenic or immune-suppressed organisms. Transgenic organisms are prepared in a variety of art-accepted ways. For example, knock-out transgenic organisms, e.g., mammals such as mice, are made, in which a cancer gene is disrupted or in which a cancer gene is inserted. Knock-out transgenic mice are made by insertion of a marker gene or other heterologous gene into the endogenous cancer gene site in the mouse genome via homologous recombination. Such mice can also be made by substituting the endogenous cancer gene with a mutated version of the cancer gene, or by mutating the endogenous cancer gene, e.g., by exposure to carcinogens.
To prepare transgenic chimeric animals, e.g., mice, a DNA construct is introduced into the nuclei of embryonic stem cells. Cells containing the newly engineered genetic lesion are injected into a host mouse embryo, which is re-implanted into a recipient female. Some of these embryos develop into chimeric mice that possess germ cells some of which are derived from the mutant cell line. Therefore, by breeding the chimeric mice it is possible to obtain a new line of mice containing the introduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288 (1989)). Chimeric mice can be derived according to U.S. Pat. No. 6,365,797, issued 2 Apr. 2002; U.S. Pat. No. 6,107,540 issued 22 Aug. 2000; Hogan et al., Manipulating the Mouse Embryo: A laboratory Manual, Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed., IRL Press, Washington, D.C., (1987).
Alternatively, various immune-suppressed or immune-deficient host animals can be used. For example, a genetically athymic “nude” mouse (see, e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921 (1974)), a SCID mouse, a thymectomized mouse, or an irradiated mouse (see, e.g., Bradley et al., Br. J. Cancer 38:263 (1978); Selby et al., Br. J. Cancer 41:52 (1980)) can be used as a host. Transplantable tumor cells (typically about 106 cells) injected into isogenic hosts produce invasive tumors in a high proportion of cases, while normal cells of similar origin will not. In hosts which developed invasive tumors, cells expressing cancer-associated sequences are injected subcutaneously or orthotopically. Mice are then separated into groups, including control groups and treated experimental groups) e.g. treated with a modulator). After a suitable length of time, preferably 4-8 weeks, tumor growth is measured (e.g., by volume or by its two largest dimensions, or weight) and compared to the control. Tumors that have statistically significant reduction (using, e.g., Student's T test) are said to have inhibited growth.
In Vitro Assays to Identify and Characterize Modulators
Assays to identify compounds with modulating activity can be performed in vitro. For example, a cancer polypeptide is first contacted with a potential modulator and incubated for a suitable amount of time, e.g., from 0.5 to 48 hours. In one embodiment, the cancer polypeptide levels are determined in vitro by measuring the level of protein or mRNA. The level of protein is measured using immunoassays such as Western blotting, ELISA and the like with an antibody that selectively binds to the cancer polypeptide or a fragment thereof. For measurement of mRNA, amplification, e.g., using PCR, LCR, or hybridization assays, e.g., Northern hybridization, RNAse protection, dot blotting, are preferred. The level of protein or mRNA is detected using directly or indirectly labeled detection agents, e.g., fluorescently or radioactively labeled nucleic acids, radioactively or enzymatically labeled antibodies, and the like, as described herein.
Alternatively, a reporter gene system can be devised using a cancer protein promoter operably linked to a reporter gene such as luciferase, green fluorescent protein, CAT, or P-gal. The reporter construct is typically transfected into a cell. After treatment with a potential modulator, the amount of reporter gene transcription, translation, or activity is measured according to standard techniques known to those of skill in the art (Davis G F, supra; Gonzalez, J. & Negulescu, P. Curr. Opin. Biotechnol. 1998: 9:624).
As outlined above, in vitro screens are done on individual genes and gene products. That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of the expression of the gene or the gene product itself is performed.
In one embodiment, screening for modulators of expression of specific gene(s) is performed. Typically, the expression of only one or a few genes is evaluated. In another embodiment, screens are designed to first find compounds that bind to differentially expressed proteins. These compounds are then evaluated for the ability to modulate differentially expressed activity. Moreover, once initial candidate compounds are identified, variants can be further screened to better evaluate structure activity relationships.
Binding Assays to Identify and Characterize Modulators
In binding assays in accordance with the invention, a purified or isolated gene product of the invention is generally used. For example, antibodies are generated to a protein of the invention, and immunoassays are run to determine the amount and/or location of protein. Alternatively, cells comprising the cancer proteins are used in the assays.
Thus, the methods comprise combining a cancer protein of the invention and a candidate compound such as a ligand, and determining the binding of the compound to the cancer protein of the invention. Preferred embodiments utilize the human cancer protein; animal models of human disease of can also be developed and used. Also, other analogous mammalian proteins also can be used as appreciated by those of skill in the art. Moreover, in some embodiments variant or derivative cancer proteins are used.
Generally, the cancer protein of the invention, or the ligand, is non-diffusibly bound to an insoluble support. The support can, e.g., be one having isolated sample receiving areas (a microtiter plate, an array, etc.). The insoluble supports can be made of any composition'to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports can be solid or porous and of any convenient shape.
Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharide, nylon, nitrocellulose, or Teflon“ ”, etc. Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. The particular manner of binding of the composition to the support is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is nondiffusable. Preferred methods of binding include the use of antibodies which do not sterically block either the ligand binding site or activation sequence when attaching the protein to the support, direct binding to “sticky” or ionic supports, chemical crosslinking, the synthesis of the protein or agent on the surface, etc. Following binding of the protein or ligand/binding agent to the support, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.
Once a cancer protein of the invention is bound to the support, and a test compound is added to the assay. Alternatively, the candidate binding agent is bound to the support and the cancer protein of the invention is then added. Binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc.
Of particular interest are assays to identify agents that have a low toxicity for human cells. A wide variety of assays can be used for this purpose, including proliferation assays, cAMP assays, labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like.
A determination of binding of the test compound (ligand, binding agent, modulator, etc.) to a cancer protein of the invention can be done in a number of ways. The test compound can be labeled, and binding determined directly, e.g., by attaching all or a portion of the cancer protein of the invention to a solid support, adding a labeled candidate compound (e.g., a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support. Various blocking and washing steps can be utilized as appropriate.
In certain embodiments, only one of the components is labeled, e.g., a protein of the invention or ligands labeled. Alternatively, more than one component is labeled with different labels, e.g., I125, for the proteins and a fluorophor for the compound. Proximity reagents, e.g., quenching or energy transfer reagents are also useful.
Competitive Binding to Identify and Characterize Modulators
In one embodiment, the binding of the “test compound” is determined by competitive binding assay with a “competitor.” The competitor is a binding moiety that binds to the target molecule (e.g., a cancer protein of the invention). Competitors include compounds such as antibodies, peptides, binding partners, ligands, etc. Under certain circumstances, the competitive binding between the test compound and the competitor displaces the test compound. In one embodiment, the test compound is labeled. Either the test compound, the competitor, or both, is added to the protein for a time sufficient to allow binding. Incubations are performed at a temperature that facilitates optimal activity, typically between four and 40° C. Incubation periods are typically optimized, e.g., to facilitate rapid high throughput screening; typically between zero and one hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.
In one embodiment, the competitor is added first, followed by the test compound. Displacement of the competitor is an indication that the test compound is binding to the cancer protein and thus is capable of binding to, and potentially modulating, the activity of the cancer protein. In this embodiment, either component can be labeled. Thus, e.g., if the competitor is labeled, the presence of label in the post-test compound wash solution indicates displacement by the test compound. Alternatively, if the test compound is labeled, the presence of the label on the support indicates displacement.
In an alternative embodiment, the test compound is added first, with incubation and washing, followed by the competitor. The absence of binding by the competitor indicates that the test compound binds to the cancer protein with higher affinity than the competitor. Thus, if the test compound is labeled, the presence of the label on the support, coupled with a lack of competitor binding, indicates that the test compound binds to and thus potentially modulates the cancer protein of the invention.
Accordingly, the competitive binding methods comprise differential screening to identity agents that are capable of modulating the activity of the cancer proteins of the invention. In this embodiment, the methods comprise combining a cancer protein and a competitor in a first sample. A second sample comprises a test compound, the cancer protein, and a competitor. The binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the cancer protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the cancer protein.
Alternatively, differential screening is used to identify drug candidates that bind to the native cancer protein, but cannot bind to modified cancer proteins. For example the structure of the cancer protein is modeled and used in rational drug design to synthesize agents that interact with that site, agents which generally do not bind to site-modified proteins. Moreover, such drug candidates that affect the activity of a native cancer protein are also identified by screening drugs for the ability to either enhance or reduce the activity of such proteins.
Positive controls and negative controls can be used in the assays. Preferably control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples occurs for a time sufficient to allow for the binding of the agent to the protein. Following incubation, samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples can be counted in a scintillation counter to determine the amount of bound compound.
A variety of other reagents can be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc. which are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., can be used. The mixture of components is added in an order that provides for the requisite binding.
Use of Polynucleotides to Down-regulate or Inhibit a Protein of the Invention.
Polynucleotide modulators of cancer can be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand-binding molecule, as described in WO 91/04753. Suitable ligand-binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell. Alternatively, a polynucleotide modulator of cancer can be introduced into a cell containing the target nucleic acid sequence, e.g., by formation of a polynucleotide-lipid complex, as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models may also be used in screening assays as discussed above, in addition to methods of treatment.
Inhibitory and Antisense Nucleotides
In certain embodiments, the activity of a cancer-associated protein is down-regulated, or entirely inhibited, by the use of antisense polynucleotide or inhibitory small nuclear RNA (snRNA), i.e., a nucleic acid complementary to, and which can preferably hybridize specifically to, a coding mRNA nucleic acid sequence, e.g., a cancer protein of the invention, mRNA, or a subsequence thereof. Binding of the antisense polynucleotide to the mRNA reduces the translation and/or stability of the mRNA.
In the context of this invention, antisense polynucleotides can comprise naturally occurring nucleotides, or synthetic species formed from naturally occurring subunits or their close homologs. Antisense polynucleotides may also have altered sugar moieties or inter-sugar linkages. Exemplary among these are the phosphorothioate and other sulfur containing species which are known for use in the art. Analogs are comprised by this invention so long as they function effectively to hybridize with nucleotides of the invention. See, e.g., Isis Pharmaceuticals, Carlsbad, Calif.; Sequitor, Inc., Natick, Mass.
Such antisense polynucleotides can readily be synthesized using recombinant means, or can be synthesized in vitro. Equipment for such synthesis is sold by several vendors, including Applied Biosystems. The preparation of other oligonucleotides such as phosphorothioates and alkylated derivatives is also well known to those of skill in the art.
Antisense molecules as used herein include antisense or sense oligonucleotides. Sense oligonucleotides can, e.g., be employed to block transcription by binding to the anti-sense strand. The antisense and sense oligonucleotide comprise a single stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences for cancer molecules. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment generally at least about 12 nucleotides, preferably from about 12 to 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, e.g., Stein & Cohen (Cancer Res. 48:2659 (1988 and van der Krol et al. (BioTechniques 6:958 (1988)).
Ribozymes
In addition to antisense polynucleotides, ribozymes can be used to target and inhibit transcription of cancer-associated nucleotide sequences. A ribozyme is an RNA molecule that catalytically cleaves other RNA molecules. Different kinds of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, RNase P, and axhead ribozymes (see, e.g., Castanotto et al., Adv. in Pharmacology 25: 289-317 (1994) for a general review of the properties of different ribozymes).
The general features of hairpin ribozymes are described, e.g., in Hampel et al., Nucl. Acids Res. 18:299-304 (1990); European Patent Publication No. 0360257; U.S. Pat. No. 5,254,678. Methods of preparing are well known to those of skill in the art (see, e.g., WO 94/26877; Ojwang et al., Proc. Natl. Acad. Sci. USA 90:6340-6344 (1993); Yamada et al., Human Gene Therapy 1:3945 (1994); Leavitt et al., Proc. Natl. Acad. Sci. USA 92:699-703 (1995); Leavitt et al., Human Gene Therapy 5: 1151-120 (1994); and Yamada et al., Virology 205: 121-126 (1994)).
Use of Modulators in Phenotypic Screening
In one embodiment, a test compound is administered to a population of cancer cells, which have an associated cancer expression profile. By “administration” or “contacting” herein is meant that the modulator is added to the cells in such a manner as to allow the modulator to act upon the cell, whether by uptake and intracellular action, or by action at the cell surface. In some embodiments, a nucleic acid encoding a proteinaceous agent (i.e., a peptide) is put into a viral construct such as an adenoviral or retroviral construct, and added to the cell, such that expression of the peptide agent is accomplished, e.g., PCT US97/01019. Regulatable gene therapy systems can also be used. Once the modulator has been administered to the cells, the cells are washed if desired and are allowed to incubate under preferably physiological conditions for some period. The cells are then harvested and a new gene expression profile is generated. Thus, e.g., cancer tissue is screened for agents that modulate, e.g., induce or suppress, the cancer phenotype. A change in at least one gene, preferably many, of the expression profile indicates that the agent has an effect on cancer activity. Similarly, altering a biological function or a signaling pathway is indicative of modulator activity. By defining such a signature for the cancer phenotype, screens for new drugs that alter the phenotype are devised. With this approach, the drug target need not be known and need not be represented in the original gene/protein expression screening platform, nor does the level of transcript for the target protein need to change. The modulator inhibiting function will serve as a surrogate marker.
As outlined above, screens are done to assess genes or gene products. That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of either the expression of the gene or the gene product itself is performed.
Use of Modulators to Affect Peptides of the Invention
Measurements of cancer polypeptide activity, or of the cancer phenotype are performed using a variety of assays. For example, the effects of modulators upon the function of a cancer polypeptide(s) are measured by examining parameters described above. A physiological change that affects activity is used to assess the influence of a test compound on the polypeptides of this invention. When the functional outcomes are determined using intact cells or animals, a variety of effects can be assesses such as, in the case of a cancer associated with solid tumors, tumor growth, tumor metastasis, neovascularization, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., by Northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as cGNIP.
Methods of Identifying Characterizing Cancer-associated Sequences
Expression of various gene sequences is correlated with cancer. Accordingly, disorders based on mutant or variant cancer genes are determined. In one embodiment, the invention provides methods for identifying cells containing variant cancer genes, e.g., determining the presence of, all or part, the sequence of at least one endogenous cancer gene in a cell. This is accomplished using any number of sequencing techniques. The invention comprises methods of identifying the cancer genotype of an individual, e.g., determining all or part of the sequence of at least one gene of the invention in the individual. This is generally done in at least one tissue of the individual, e.g., a tissue set forth in Table I, and may include the evaluation of a number of tissues or different samples of the same tissue. The method may include comparing the sequence of the sequenced gene to a known cancer gene, i.e., a wild-type gene to determine the presence of family members, homologies, mutations or variants. The sequence of all or part of the gene can then be compared to the sequence of a known cancer gene to determine if any differences exist. This is done using any number of known homology programs, such as BLAST, Bestfit, etc. The presence of a difference in the sequence between the cancer gene of the patient and the known cancer gene correlates with a disease state or a propensity for a disease state, as outlined herein.
In a preferred embodiment, the cancer genes are used as probes to determine the number of copies of the cancer gene in the genome. The cancer genes are used as probes to determine the chromosomal localization of the cancer genes. Information such as chromosomal localization finds use in providing a diagnosis or prognosis in particular when chromosomal abnormalities such as translocations, and the like are identified in the cancer gene locus.
XIV.) RNAi and Therapeutic Use of Small Interfering RNA (siRNAs)
The present invention is also directed towards siRNA oligonucleotides, particularly double stranded RNAs encompassing at least a fragment of the 158P1D7 coding region or 5′′ UTR regions, or complement, or any antisense oligonucleotide specific to the 158P1D7 sequence. In one embodiment such oligonucleotides are used to elucidate a function of 158P1D7, or are used to screen for or evaluate modulators of 158P1D7 function or expression. In another embodiment, gene expression of 158P1D7 is reduced by using siRNA transfection and results in significantly diminished proliferative capacity of transformed cancer cells that endogenously express the antigen; cells treated with specific 158P1D7 siRNAs show reduced survival as measured, e.g., by a metabolic readout of cell viability, correlating to the reduced proliferative capacity. Thus, 158P1D7 siRNA compositions comprise siRNA (double stranded RNA) that correspond to the nucleic acid ORF sequence of the 158P1D7 protein or subsequences thereof; these subsequences are generally 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more than 35 contiguous RNA nucleotides in length and contain sequences that are complementary and non-complementary to at least a portion of the mRNA coding sequence In a preferred embodiment, the subsequences are 19-25 nucleotides in length, most preferably 21-23 nucleotides in length.
RNA interference is a novel approach to silencing genes in vitro and in vivo, thus small double stranded RNAs (siRNAs) are valuable therapeutic agents. The power of siRNAs to silence specific gene activities has now been brought to animal models of disease and is used in humans as well. For example, hydrodynamic infusion of a solution of siRNA into a mouse with a siRNA against a particular target has been proven to be therapeutically effective.
The pioneering work by Song et al. indicates that one type of entirely natural nucleic acid, small interfering RNAs (siRNAs), served as therapeutic agents even without further chemical modification (Song, E., et al. “RNA interference targeting Fas protects mice from fulminant hepatitis” Nat. Med. 9(3): 347-51(2003)). This work provided the first in vivo evidence that infusion of siRNAs into an animal could alleviate disease. In that case, the authors gave mice injections of siRNA designed to silence the FAS protein (a cell death receptor that when over-activated during inflammatory response induces hepatocytes and other cells to die). The next day, the animals were given an antibody specific to Fas. Control mice died of acute liver failure within a few days, while over 80% of the siRNA-treated mice remained free from serious disease and survived. About 80% to 90% of their liver cells incorporated the naked siRNA oligonucleotides. Furthermore, the RNA molecules functioned for 10 days before losing effect after 3 weeks.
For use in human therapy, siRNA is delivered by efficient systems that induce long-lasting RNAi activity. A major caveat for clinical use is delivering siRNAs to the appropriate cells. Hepatocytes seem to be particularly receptive to exogenous RNA. Today, targets located in the liver are attractive because liver is an organ that can be readily targeted by nucleic acid molecules and viral vectors. However, other tissue and organs targets are preferred as well.
Formulations of siRNAs with compounds that promote transit across cell membranes are used to improve administration of siRNAs in therapy. Chemically modified synthetic siRNA, that are resistant to nucleases and have serum stability have concomitant enhanced duration of RNAi effects, are an additional embodiment.
Thus, siRNA technology is a therapeutic for human malignancy by delivery of siRNA molecules directed to 158P1D7 to individuals with the cancers, such as those listed in Table 1. Such administration of siRNAs leads to reduced growth of cancer cells expressing 158P1D7, and provides an anti-tumor therapy, lessening the morbidity and/or mortality associated with malignancy.
The effectiveness of this modality of gene product knockdown is significant when measured in vitro or in vivo. Effectiveness in vitro is readily demonstrable through application of siRNAs to cells in culture (as described above) or to aliquots of cancer patient biopsies when in vitro methods are used to detect the reduced expression of 158P1D7 protein.
XV.) Kits/Articles of Manufacture
For use in the laboratory, prognostic, prophylactic, diagnostic and therapeutic applications described herein, kits are within the scope of the invention. Such kits can comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in the method, along with a label or insert comprising instructions for use, such as a use described herein. For example, the container(s) can comprise a probe that is or can be detectably labeled. Such probe can be an antibody or polynucleotide specific for a protein or a gene or message of the invention, respectively. Where the method utilizes nucleic acid hybridization to detect the target nucleic acid, the kit can also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence. Kits can comprise a container comprising a reporter, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, fluorescent, or radioisotope label; such a reporter can be used with, e.g., a nucleic acid or antibody. The kit can include all or part of the amino acid sequences in FIG. 2 or FIG. 3 or analogs thereof, or a nucleic acid molecule that encodes such amino acid sequences.
The kit of the invention will typically comprise the container described above and one or more other containers associated therewith that comprise materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes; carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package inserts with instructions for use.
A label can be present on or with the container to indicate that the composition is used for a specific therapy or non-therapeutic application, such as a prognostic, prophylactic, diagnostic or laboratory application, and can also indicate directions for either in vivo or in vitro use, such as those described herein. Directions and or other information can also be included on an insert(s) or label(s) which is included with or on the kit. The label can be on or associated with the container. A label a can be on a container when letters, numbers or other characters forming the label are molded or etched into the container itself; a label can be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. The label can indicate that the composition is used for diagnosing, treating, prophylaxing or prognosing a condition, such as a neoplasia of a tissue set forth in Table I.
The terms “kit” and “article of manufacture” can be used as synonyms.
In another embodiment of the invention, an article(s) of manufacture containing compositions, such as amino acid sequence(s), small molecule(s), nucleic acid sequence(s), and/or antibody(s), e.g., materials useful for the diagnosis, prognosis, prophylaxis and/or treatment of neoplasias of tissues such as those set forth in Table I is provided. The article of manufacture typically comprises at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass, metal or plastic. The container can hold amino acid sequence(s), small molecule(s), nucleic acid sequence(s), cell population(s) and/or antibody(s). In one embodiment, the container holds a polynucleotide for use in examining the mRNA expression profile of a cell, together with reagents used for this purpose. In another embodiment a container comprises an antibody, binding fragment thereof or specific binding protein for use in evaluating protein expression of 158P1D7 in cells and tissues, or for relevant laboratory, prognostic, diagnostic, prophylactic and therapeutic purposes; indications and/or directions for such uses can be included on or with such container, as can reagents and other compositions or tools used for these purposes. In another embodiment, a container comprises materials for eliciting a cellular or humoral immune response, together with associated indications and/or directions. In another embodiment, a container comprises materials for adoptive immunotherapy, such as cytotoxic T cells (CTL) or helper T cells (HTL), together with associated indications and/or directions; reagents and other compositions or tools used for such purpose can also be included.
The container can alternatively hold a composition that is effective for treating, diagnosis, prognosing or prophylaxing a condition and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agents in the composition can be an antibody capable of specifically binding 158P1D7 and modulating the function of 158P1D7.
The article of manufacture can further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringers solution and/or dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, stirrers, needles, syringes, and/or package inserts with indications and/or instructions for use.
EXAMPLES
Various aspects of the invention are further described and illustrated by way of the several examples that follow, none of which are intended to limit the scope of the invention.
Example 1
SSH-Generated Isolation of a cDNA Fragment of the 158P1D7 Gene
To isolate genes that are over-expressed in bladder cancer we used the Suppression Subtractive Hybridization (SSH) procedure using cDNA derived from bladder cancer tissues, including invasive transitional cell carcinoma. The 158P1D7 SSH cDNA sequence was derived from a bladder cancer pool minus normal bladder cDNA subtraction. Included in the driver were also cDNAs derived from 9 other normal tissues. The 158P1D7 cDNA was identified as highly expressed in the bladder cancer tissue pool, with lower expression seen in a restricted set of normal tissues.
The SSH DNA sequence of 231 bp (FIG. 1) has high homology (230/231 identity) to a hypothetical protein FLJ22774 (GenBank accession XM—033183) derived from a chromosome 13 genomic clone. A 158P1D7 cDNA clone (TurboScript3PX) of 2,555 bp was isolated from bladder cancer cDNA, revealing an ORF of 841 amino acids (FIG. 2 and FIG. 3).
The 158P1D7 protein has a signal sequence and a transmembrane domain and is predicted to be localized to the cell surface using the PSORT-I program (URL psort.nibb.ac.jp:8800/form.html). Amino acid sequence analysis of 158P1D7 reveals 100% identity over 798 amino acid region to a human hypothetical protein FLJ22774 (GenBank Accession XP—033182)(FIG. 4).
Materials and Methods
Human Tissues:
The bladder cancer patient tissues were purchased from several sources such as from the NDRI (Philadelphia, Pa.). mRNA for some normal tissues were purchased from Clontech, Palo Alto, Calif.
RNA Isolation:
Tissues were homogenized in Trizol reagent (Life Technologies, Gibco BRL) using 10 ml/g tissue isolate total RNA. Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA Mini and Midi kits. Total and mRNA were quantified by spectrophotometric analysis (O.D. 260/280 nm) and analyzed by gel electrophoresis.
Oligonucleotides:
The following HPLC purified oligonucleotides were used.









DPNCDN (cDNA synthesis primer):




5′TTTTGATCAAGCTT303′
(SEQ ID NO: 28)


 


Adaptor 1:


5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3′
(SEQ ID NO: 29)


 


3′GGCCCGTCCTAG5′
(SEQ ID NO: 30)


 


Adaptor 2:


5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3′
(SEQ ID NO: 31)


 


3′CGGCTCCTAG5′
(SEQ ID NO: 32)


 


PCR primer 1:


5′CTAATACGACTCACTATAGGGC3′
(SEQ ID NO: 33)


 


Nested primer (NP)1:


5′TCGAGCGGCCGCCCGGGCAGGA3′
(SEQ ID NO: 34)


 


Nested primer (NP)2:


5′AGCGTGGTCGCGGCCGAGGA3′
(SEQ ID NO: 35)






Suppression Subtractive Hybridization:
Suppression Subtractive Hybridization (SSH) was used to identify cDNAs corresponding to genes that may be differentially expressed in bladder cancer. The SSH reaction utilized cDNA from bladder cancer and normal tissues.
The gene 158P1D7 sequence was derived from a bladder cancer pool minus normal bladder cDNA subtraction. The SSH DNA sequence (FIG. 1) was identified.
The cDNA derived from of pool of normal bladder tissues was used as the source of the “driver” cDNA, while the cDNA from a pool of bladder cancer tissues was used as the source of the “tester” cDNA. Double stranded cDNAs corresponding to tester and driver cDNAs were synthesized from 2 μg of poly(A), RNA isolated from the relevant xenograft tissue, as described above, using CLONTECH's PCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotide DPNCDN as primer. First- and second-strand synthesis were carried out as described in the Kit's user manual protocol (CLONTECH Protocol No. PT1117-1, Catalog No. K1804-1). The resulting cDNA was digested with Dpn II for 3 hrs at 37° C. Digested cDNA was extracted with phenol/chloroform (1:1) and ethanol precipitated.
Driver cDNA was generated by combining in a 1:1 ratio Dpn II digested cDNA from the relevant tissue source (see above) with a mix of digested cDNAs derived from the nine normal tissues: stomach, skeletal muscle, lung, brain, liver, kidney, pancreas, small intestine, and heart.
Tester cDNA was generated by diluting 1 μl of Dpn II digested cDNA from the relevant tissue source (see above) (400 ng) in 5 μl of water. The diluted cDNA (2 μl, 160 ng) was then ligated to 2 μl of Adaptor 1 and Adaptor 2 (10 μM), in separate ligation reactions, in a total volume of 10 μl at 16° C. overnight, using 400 u of T4 DNA ligase (CLONTECH). Ligation was terminated with 1 μl of 0.2 M EDTA and heating at 72° C. for 5 min.
The first hybridization was performed by adding 1.5 μl (600 ng) of driver cDNA to each of two tubes containing 1.5 μl (20 ng) Adaptor 1- and Adaptor 2-ligated tester cDNA. In a final volume of 4 μl, the samples were overlaid with mineral oil, denatured in an MJ Research thermal cycler at 98° C. for 1.5 minutes, and then were allowed to hybridize for 8 hrs at 68° C. The two hybridizations were then mixed together with an additional 1 μl of fresh denatured driver cDNA and were allowed to hybridize overnight at 68° C. The second hybridization was then diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA, heated at 70° C. for 7 min. and stored at −20° C.
PCR Amplification, Cloning and Sequencing of Gene Fragments Generated from SSH:
To amplify gene fragments resulting from SSH reactions, two PCR amplifications were performed. In the primary PCR reaction 1 μl of the diluted final hybridization mix was added to 1 μl of PCR primer 1 (10 μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (CLONTECH) and 0.5 μl 50× Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5 min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C. for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions were performed for each experiment. The products were pooled and diluted 1:10 with water. For the secondary PCR reaction, 1 μl from the pooled and diluted primary PCR reaction was added to the same reaction mix as used for PCR 1, except that primers NP1 and NP2 (10 μM) were used instead of PCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10 sec, 68° C. for 30 sec. and 72° C. for 1.5 minutes. The PCR products were analyzed using 2% agarose gel electrophoresis.
The PCR products were inserted into pCR2.1 using the T/A vector cloning kit (Invitrogen). Transformed E. coli were subjected to blue/white and ampicillin selection. White colonies were picked and arrayed into 96 well plates and were grown in liquid culture overnight. To identify inserts, PCR amplification was performed on 1 ml of bacterial culture using the conditions of PCR1 and NP1 and NP2 as primers. PCR products were analyzed using 2% agarose gel electrophoresis.
Bacterial clones were stored in 20% glycerol in a 96 well format. Plasmid DNA was prepared, sequenced, and subjected to nucleic acid homology searches of the GenBank, dBest, and NCI-CGAP databases.
RT-PCR Expression Analysis:
First strand cDNAs can be generated from 1 μg of mRNA with oligo (dT)12-18 priming using the Gibco-BRL Superscript Preamplificaton system. The manufacturers protocol was used which included an incubation for 50 min at 42° C. with reverse transcriptase followed by RNAse H treatment at 37° C. for 20 min. After completing the reaction, the volume can be increased to 200 μl with water prior to normalization. First strand cDNAs from 16 different normal human tissues can be obtained from Clontech.
Normalization of the first strand cDNAs from multiple tissues was performed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ ID NO: 36) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO: 37) to amplify P-actin. First strand cDNA (5 II) were amplified in a total volume of 50 μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgCl2, 50 mM KCl, pH8.3) and 1× Klentaq DNA polymerase (Clontech). Five μl of the PCR reaction can be removed at 18, 20, and 22 cycles and used for agarose gel electrophoresis. PCR was performed using an MJ Research thermal cycler under the following conditions: Initial denaturation can be at 94° C. for 15 sec, followed by a 18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C. for 5 sec. A final extension at 72° C. was carried out for 2 min. After agarose gel electrophoresis, the band intensities of the 283 b.p. β-actin bands from multiple tissues were compared by visual inspection. Dilution factors for the first strand cDNAs were calculated to result in equal β-actin band intensities in all tissues after 22 cycles of PCR. Three rounds of normalization can be required to achieve equal band intensities in all tissues after 22 cycles of PCR.
To determine expression levels of the 158P1D7 gene, 5 μl of normalized first strand cDNA were analyzed by PCR using 26, and 30 cycles of amplification. Semi-quantitative expression analysis can be achieved by comparing the PCR products at cycle numbers that give light band intensities. The primers used for RT-PCR were designed using the 158P1D7 SSH sequence and are listed below.











158P1D7.1





5′ ATAAGCTTTCAATGTTGCGCTCCT 3′
(SEQ ID NO: 38)



 



158P1D7.2



5′ TGTCAACTAAGACCACGTCCATTC 3′
(SEQ ID NO: 39)






A typical RT-PCR expression analysis is shown in FIG. 6. RT-PCR expression analysis was performed on first strand cDNAs generated using pools of tissues from multiple samples. The cDNAs were shown to be normalized using beta-actin PCR. Expression of 158P1D7 was observed in bladder cancer pool.
Example 2
Full Length Cloning of 158P1D7
The 158P1D7 SSH cDNA sequence was derived from a bladder cancer pool minus normal bladder cDNA subtraction. The SSH cDNA sequence (FIG. 1) was designated 158P1D7. The full-length cDNA clone 158P1D7-clone TurboScript3PX (FIG. 2) was cloned from bladder cancer pool cDNA.
158P1D7 clone cDNA was deposited under the terms of the Budapest Treaty on 22 Aug. 2001, with the American Type Culture Collection (ATCC; 10801 University Blvd., Manassas, Va. 20110-2209 USA) as plasmid p158P1D7-Turbo/3PX, and has been assigned Accession No. PTA-3662.
Example 3
Chromosomal Mapping of 158P1D7
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.).
158P1D7 maps to chromosome 13, using 158P1D7 sequence and the NCBI BLAST tool: (world wide web URL ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.html&&ORG=Hs). This is a region of frequent amplification in bladder cancer (Prat et al., Urology 2001 May; 57(5):986-92; Muscheck et al., Carcinogenesis 2000 September; 21(9):1721-26) and is associated with rapid tumor cell proliferation in advanced bladder cancer (Tomovska et al., Int J Oncol 2001 June; 18(6):1239-44).
Example 4
Expression Analysis of 158P1D7 in Normal Tissues and Patient Specimens
Analysis of 158P1D7 by RT-PCR is shown in FIG. 6. Strong expression of 158P1D7 is observed in bladder cancer pool and breast cancer pool. Lower levels of expression are observed in VP1, VP2, xenograft pool, prostate cancer pool, colon cancer pool, lung cancer pool, ovary cancer pool, and metastasis pool.
Extensive Northern blot analysis of 158P1D7 in 16 human normal tissues confirms the expression observed by RT-PCR (FIG. 7). Two transcripts of approximately 4.6 and 4.2 kb are detected in prostate and, to lower levels, in heart, placenta, liver, small intestine and colon.
Northern blot analysis on patient tumor specimens shows expression of 158P1D7 in most bladder tumor tissues tested and in the bladder cancer cell line SCaBER (FIGS. 8A and 8B). The expression detected in normal adjacent tissues (isolated from patients) but not in normal tissues (isolated from a healthy donor) may indicate that these tissues are not fully normal and that 158P1D7 may be expressed in early stage tumors. Expression of 158P1D7 is also detected in 2 of 4 lung cancer cell lines, and in all 3 lung cancer tissues tested (FIG. 9). In breast cancer samples, 158P1D7 expression is observed in the MCF7 and CAMA-1 breast cancer cell lines, in breast tumor tissues isolated from breast cancer patients, but not in normal breast tissues (FIG. 10). 158P1D7 shows expression in melanoma cancer. RNA was extracted from normal skin cell line Detroit-551, and from the melanoma cancer cell line A375. Northern blots with 10 ug of total RNA were probed with the 158P1D7 DNA probe. Results show expression of 158P1D7 in the melanoma cancer cell line but not in the normal cell line (FIG. 20). 158P1D7 shows expression in cervical cancer patient specimens. First strand cDNA was prepared from normal cervix, cervical cancer cell line Hela, and a panel of cervical cancer patient specimens. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 158P1D7, was performed at 26 and 30 cycles of amplification. Results show expression of 158P1D7 in 5 out of 14 tumor specimens tested but not in normal cervix nor in the cell line (FIG. 21).
The restricted expression of 158P1D7 in normal tissues and the expression detected in prostate cancer, bladder cancer, colon cancer, lung cancer, ovarian cancer, breast cancer, melanoma cancer, and cervical cancer suggest that 158P1D7 is a potential therapeutic target and a diagnostic marker for human cancers.
Example 5
Production of Recombinant 158P1D7 in Prokaryotic Systems
To express recombinant 158P1D7 and 158P1D7 variants in prokaryotic cells, the full or partial length 158P1D7 and 158P1D7 variant cDNA sequences are cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 158P1D7 variants are expressed: the full length sequence presented in FIGS. 2 and 3, or any 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from 158P1D7, variants, or analogs thereof.
A. In Vitro Transcription and Translation Constructs:
pCRII: To generate 158P1D7 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 158P1D7 cDNA. The pCRII vector has Sp6 and T7 promoters flanking the insert to drive the transcription of 158P1D7 RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze the cell and tissue expression of 158P1D7 at the RNA level. Transcribed 158P1D7 RNA representing the cDNA amino acid coding region of the 158P1D7 gene is used in in vitro translation systems such as the TnTTM Coupled Reticulolysate System (Promega, Corp., Madison, Wis.) to synthesize 158P1D7 protein.
B. Bacterial Constructs:
pGEX Constructs: To generate recombinant 158P1D7 proteins in bacteria that are fused to the Glutathione S-transferase (GST) protein, all or parts of the 158P1D7 cDNA protein coding sequence are cloned into the pGEX family of GST-fusion vectors (Amersham Pharmacia Biotech, Piscataway, N.J.). These constructs allow controlled expression of recombinant 158P1D7 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 158P1D7-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 158P1D7 proteins that are fused to maltose-binding protein (MBP), all or parts of the 158P1D7 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 158P1D7 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 158P1D7. 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. Amino acids 356-608 of 158P1D7 variant 1 have been cloned into the pMALc2X vector.
pET Constructs: To express 158P1D7 in bacterial cells, all or parts of the 158P1D7 cDNA protein coding sequence are cloned into the pET family of vectors (Novagen, Madison, Wis.). These vectors allow tightly controlled expression of recombinant 158P1D7 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 158P1D7 protein are expressed as amino-terminal fusions to NusA.
C. Yeast Constructs:
pESC Constructs: To express 158P1D7 in the yeast species Saccharomyces cerevisiae for generation of recombinant protein and functional studies, all or parts of the 158P1D7 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 158P1D7. 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 158P1D7 in the yeast species Saccharomyces pombe, all or parts of the 158P1D7 cDNA protein coding sequence are cloned into the pESP family of vectors. These vectors allow controlled high level of expression of a 158P1D7 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 6
Production of Recombinant 158P1D7 in Eukaryotic Systems
A. Mammalian Constructs:
To express recombinant 158P1D7 in eukaryotic cells, the full or partial length 158P1D7 cDNA sequences were cloned into any one of a variety of expression vectors known in the art. One or more of the following regions of 158P1D7 were expressed in these constructs, amino acids 1 to 841, 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 158P1D7 v.1; amino acids 1 to 732 of v.3; amino acids 1 to 395 of v.4; amino acids 1 to 529 of v.6; 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 158P1D7 variants, or analogs thereof.
The constructs can be transfected into any one of a wide variety of mammalian cells such as 293T cells. Transfected 293T cell lysates can be probed with the anti-158P1D7 polyclonal serum, described herein.
pcDNA4/HisMax Constructs: To express 158P1D7 in mammalian cells, a 158P1D7 ORF, or portions thereof, of 158P1D7 are cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, Calif.). Protein expression is driven from the cytomegalovirus (CMV) promoter and the SP16 translational enhancer. The recombinant protein has XpressTM and six histidine (6× His) epitopes fused to the amino-terminus. The pcDNA4/HisMax vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Zeocin resistance gene allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli. 
pcDNA3.1/MycHis Constructs: To express 158P1D7 in mammalian cells, a 158P1D7 ORF, or portions thereof, of 158P1D7 with a consensus Kozak translation initiation site was cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, Calif.). Protein expression was driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the myc epitope and 6× His epitope fused to the carboxyl-terminus. The pcDNA3.1/MycHis vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability, along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The Neomycin resistance gene can be used, as it allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin permits selection and maintenance of the plasmid in E. coli. 
The complete ORF of 158P1D7 v.1 was cloned into the pcDNA3.1/MycHis construct to generate 158P1D7.pcDNA3.1/MycHis. FIG. 23 shows expression of 158P1D7.pcDNA3.1/MycHis following transfection into 293T cells. 293T cells were transfected with either 158P1D7.pcDNA3.1/MycHis or pcDNA3.1/MycHis vector control. Forty hours later, cells were collected and analyzed by flow cytometry using anti-158P1D7 monoclonal antibodies. Results show expression of 158P1D7 from the 158P1D7.pcDNA3.1/MycHis construct on the surface of transfected cells.
pcDNA3.1/CT-GFP-TOPO Construct: To express 158P1D7 in mammalian cells and to allow detection of the recombinant proteins using fluorescence, a 158P1D7 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 158P1D7 protein.
PAPtag: A 158P1D7 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 158P1D7 protein while fusing the IgGK signal sequence to the amino-terminus. Constructs are also generated in which alkaline phosphatase with an amino-terminal IgGK signal sequence is fused to the amino-terminus of a 158P1D7 protein. The resulting recombinant 158P1D7 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 158P1D7 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 158P1D7 ORF, or portions thereof, were cloned into pTag-5. This vector is similar to pAPtag but without the alkaline phosphatase fusion. This construct generated a 158P1D7 protein with an amino-terminal IgGK signal sequence and myc and 6× His epitope tags at the carboxyl-terminus that facilitate detection and affinity purification. The resulting recombinant 158P1D7 protein was optimized for secretion into the media of transfected mammalian cells, and was used as immunogen or ligand to identify proteins such as ligands or receptors that interact with the 158P1D7 proteins. Protein expression is driven from the CMV promoter. The Zeocin resistance gene present in the vector allows for selection of mammalian cells expressing the protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli. 
The extracellular domain, amino acids 16-608, 27-300, and 301-608, of 158P1D7 v.1 were cloned into the pTag5 construct to generate 158P1D7(16-608).pTag5, 158P1D7(27-300).pTag5, and 158P1D7 (301-608).pTag5 respectively. Expression and secretion of the various segments of the extracellular domain of 158P1D7 following vector transfection into 293T cells was confirmed.
PsecFc: A 158P1D7 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 generates an IgG1 Fc fusion at the carboxyl-terminus of the 158P1D7 proteins, while fusing the IgGK signal sequence to N-terminus. 158P1D7 fusions utilizing the murine IgG1 Fc region are also used. The resulting recombinant 158P1D7 proteins are optimized for secretion into the media of transfected mammalian cells, and can be used as immunogens or to identify proteins such as ligands or receptors that interact with 158P1D7 protein. Protein expression is driven from the CMV promoter. The hygromycin resistance gene present in the vector allows for selection of mammalian cells that express the recombinant protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli. 
The extracellular domain amino acids 16-608 of 158P1D7 v.1 was cloned into the psecFc construct to generate 158P1D7 (16-608).psecFc.
pSRα Constructs: To generate mammalian cell lines that express 158P1D7 constitutively, 158P1D7 ORF, or portions thereof, of 158P1D7 were cloned into pSRα constructs. Amphotropic and ecotropic retroviruses were generated by transfection of pSRα constructs into the 293T-10A1 packaging line or co-transfection of pSRα and a helper plasmid (containing deleted packaging sequences) into the 293 cells, respectively. The retrovirus is used to infect a variety of mammalian cell lines, resulting in the integration of the cloned gene, 158P1D7, into the host cell-lines. Protein expression is driven from a long terminal repeat (LTR). The Neomycin resistance gene present in the vector allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColE1 origin permit selection and maintenance of the plasmid in E. coli. The retroviral vectors can thereafter be used for infection and generation of various cell lines using, for example, PC3, NIH 3T3, TsuPr1, 293 or rat-1 cells.
The complete ORF of 158P1D7 v.1 was cloned into the pSRα construct to generate 158P1D7.pSRα. FIG. 23 shows expression of 158P1D7.pSRα following transduction into UMUC3 cells. UMUC-3 cells were transduced with either 158P1D7 pSRα or vector control. Forty hours later, cells were collected and analyzed by flow cytometry using anti-158P1D7 monoclonal antibodies. Results show expression of 158P1D7 from the 158P1D7.pSRα construct on the surface of the cells.
Additional pSRα constructs are made that fuse an epitope tag such as the FLAGTM tag to the carboxyl-terminus of 158P1D7 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: 40) is added to cloning primer at the 3′ end of the ORF. Additional pSRα constructs are made to produce both amino-terminal and carboxyl-terminal GFP and myc/6× His fusion proteins of the full-length 158P1D7 proteins.
Additional Viral Vectors: Additional constructs are made for viral-mediated delivery and expression of 158P1D7. High virus titer leading to high level expression of 158P1D7 is achieved in viral delivery systems such as adenoviral vectors and herpes amplicon vectors. A 158P1D7 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, 158P1D7 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 158P1D7 in mammalian cells, coding sequences of 158P1D7, 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 158P1D7. These vectors are thereafter used to control expression of 158P1D7 in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.
B. Baculovirus Expression Systems
To generate recombinant 158P1D7 proteins in a baculovirus expression system, 158P1D7 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-158P1D7 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 158P1D7 protein is then generated by infection of HighFive insect cells (Invitrogen) with purified baculovirus. Recombinant 158P1D7 protein can be detected using anti-158P1D7 or anti-His-tag antibody. 158P1D7 protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 158P1D7.
Example 7
Antigenicity Profiles and Secondary Structure
FIG. 11(a)-(d), FIG. 12(a)-(d), FIG. 13(a)-(d), FIG. 14(a)-(d), and FIG. 15(a)-(d) depict graphically five amino acid profiles each of 158P1D7 protein variants 1, 3, 4, and 6, each assessment available by accessing the ProtScale website located on the World Wide Web at (.expasy.ch/cgi-bin/protscale.pl) on the ExPasy molecular biology server.
These profiles: FIG. 11, Hydrophilicity, (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); FIG. 12, Hydropathicity, (Kyte J., Doolittle R. F., 1982. J. Mol. Biol. 157:105-132); FIG. 13, Percentage Accessible Residues (Janin J., 1979 Nature 277:491-492); FIG. 14, Average Flexibility, (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32:242-255); FIG. 15, Beta-turn (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294); and optionally others available in the art, such as on the ProtScale website, were used to identify antigenic regions of each of the 158P1D7 variant proteins. Each of the above amino acid profiles of 158P1D7 variants were generated using the following ProtScale parameters for analysis: 1) A window size of 9; 2) 100% weight of the window edges compared to the window center; and, 3) amino acid profile values normalized to lie between 0 and 1.
Hydrophilicity (FIG. 11), Hydropathicity (FIG. 12) and Percentage Accessible Residues (FIG. 13) 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. 14) and Beta-turn (FIG. 15) 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 158P1D7 variant proteins indicated, e.g., by the profiles set forth in FIGS. 11(a)-(d), FIG. 12(a)-(d), FIG. 13(a)-(d), FIG. 14(a)-(d), and FIG. 15(a)-(d) are used to prepare immunogens, either peptides or nucleic acids that encode them, to generate therapeutic and diagnostic anti-158P1D7 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 158P1D7 protein variants listed in FIGS. 2 and 3. In particular, peptide immunogens of the invention can comprise, a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profiles of FIG. 11; 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. 12; a peptide region of at least 5 amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profiles of FIG. 13; a peptide region of at least amino acids of FIGS. 2 and 3 in any whole number increment that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profiles on FIG. 14; 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. 15. Peptide immunogens of the invention can also comprise nucleic acids that encode any of the forgoing.
All immunogens of the invention, peptide or nucleic acid, can be embodied in human unit dose form, or comprised by a composition that includes a pharmaceutical excipient compatible with human physiology.
The secondary structure of 158P1D7 protein variants 1, 3, 4, and 6, namely the predicted presence and location of alpha helices, extended strands, and random coils, are predicted from the primary amino acid sequence using the HNN—Hierarchical Neural Network method (NPS@: Network Protein Sequence Analysis TIBS 2000 March Vol. 25, No 3 [291]:147-150 Combet C., Blanchet C., Geourjon C. and Deléage G., http://pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html), accessed from the ExPasy molecular biology server (http://www.expasy.ch/tools/). The analysis indicates that 158P1D7 variant 1 is composed of 35.32% alpha helix, 15.93% extended strand, and 48.75% random coil (FIG. 16A). Variant 3 is composed of 34.97% alpha helix, 16.94% extended strand, and 48.09% random coil (FIG. 16B). Variant 4 is composed of 24.56% alpha helix, 20.76% extended strand, and 54.68% random coil (FIG. 16C). Variant 6 is composed of 28.92% alpha helix, 17.96% extended strand, and 53.12% random coil (FIG. 16D).
Analysis for the potential presence of transmembrane domains in the 158P1D7 variant proteins was carried out using a variety of transmembrane prediction algorithms accessed from the ExPasy molecular biology server (http://www.expasy.ch/tools/). Shown graphically in FIG. 16E, 16G, 161, 16K, are the results of analysis of variants 1, 3, 4, and 6, respectively, using the TMpred program. In FIG. 16F, 16H, 16I, 16L are the results of variants 1, 3, 4, and 6, respectively, using the TMHMM program. Both the TMpred program and the TMHMM program predict the presence of 1 transmembrane domain in variant 1 and 3. Variants 4 and 6 are not predicted to contain transmembrane domains. All variants contain a stretch of hydrophobic amino acid sequence at their amino terminus that may encode a signal peptide. Analyses of 158P1D7 and 158P1D7 variants using other structural prediction programs are summarized in Table LVI.
Example 8
Generation of 158P1D7 Polyclonal Antibodies
Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. In addition to immunizing with a full length 158P1D7 protein variant, computer algorithms are employed in design of immunogens that, based on amino acid sequence analysis contain characteristics of being antigenic and available for recognition by the immune system of the immunized host (see the Example entitled “Antigenicity Profiles and Secondary Structure”). Such regions would be predicted to be hydrophilic, flexible, in beta-turn conformations, and be exposed on the surface of the protein (see, e.g., FIG. 11, FIG. 12, FIG. 13, FIG. 14, or FIG. 15 for amino acid profiles that indicate such regions of 158P1D7 protein variants 1, 3, 4, and 6).
For example, recombinant bacterial fusion proteins or peptides containing hydrophilic, flexible, beta-turn regions of 158P1D7 protein variants are used as antigens to generate polyclonal antibodies in New Zealand White rabbits or monoclonal antibodies as described in Example 9. For example, in 158P1D7 variant 1, such regions include, but are not limited to, amino acids 25-45, amino acids 250-385, and amino acids 694-730. 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 274-285 of 158P1D7 variant 1 was synthesized and conjugated to KLH. This peptide is then used as immunogen. Alternatively the immunizing agent may include all or portions of the 158P1D7 variant proteins, analogs or fusion proteins thereof. For example, the 158P1D7 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. In another embodiment, amino acids 27-300 of 158P1D7 variant 1 is fused to GST using recombinant techniques and the pGEX expression vector, expressed, purified and used to immunize a rabbit. Such fusion proteins are purified from induced bacteria using the appropriate affinity matrix.
Other recombinant bacterial fusion proteins that may be employed include maltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see the section entitled “Production of 158P1D7 in Prokaryotic Systems” and Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, P. S., Brady, W., Umes, M., Grosmaire, L., Damle, N., and Ledbetter, L.(1991) J. Exp. Med. 174, 561-566).
In addition to bacterial derived fusion proteins, mammalian expressed protein antigens are also used. These antigens are expressed from mammalian expression vectors such as the Tag5 and Fc-fusion vectors (see the section entitled “Production of Recombinant 158P1D7 in Eukaryotic Systems”), and retain post-translational modifications such as glycosylations found in native protein. In one embodiment, amino acids 16-608 of 158P1D7 variant 1 was cloned into the Tag5 mammalian secretion vector, and expressed in 293T cells. The recombinant protein was purified by metal chelate chromatography from tissue culture supernatants of 293T cells stably expressing the recombinant vector. The purified Tag5 158P1D7 variant 1 protein is then used as immunogen.
During the immunization protocol, it is useful to mix or emulsify the antigen in adjuvants that enhance the immune response of the host animal. Examples of adjuvants include, but are not limited to, complete Freund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
In a typical protocol, rabbits are initially immunized subcutaneously with up to 200 μg, typically 100-200 μg, of fusion protein or peptide conjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits are then injected subcutaneously every two weeks with up to 200 μg, typically 100-200 μg, of the immunogen in incomplete Freund's adjuvant (IFA). Test bleeds are taken approximately 7-10 days following each immunization and used to monitor the titer of the antiserum by ELISA.
To test reactivity and specificity of immune serum, such as the rabbit serum derived from immunization with the GST-fusion of 158P1D7 variant 1 protein, the full-length 158P1D7 variant 1 cDNA is cloned into pcDNA 3.1 myc-his expression vector (Invitrogen, see the Example entitled “Production of Recombinant 158P1D7 in Eukaryotic Systems”). After transfection of the constructs into 293T cells, cell lysates are probed with the anti-158P1D7 serum and with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, Calif.) to determine specific reactivity to denatured 158P1D7 protein using the Western blot technique. In addition, the immune serum is tested by fluorescence microscopy, flow cytometry and immunoprecipitation against 293T and other recombinant 158P1D7-expressing cells to determine specific recognition of native protein. Western blot, immunoprecipitation, fluorescent microscopy, and flow cytometric techniques using cells that endogenously express 158P1D7 are also carried out to test reactivity and specificity.
Anti-serum from rabbits immunized with 158P1D7 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-158P1D7 variant 1 fusion protein is first purified by passage over a column of GST protein covalently coupled to AffiGel matrix (BioRad, Hercules, Calif.). The antiserum is then affinity purified by passage over a column composed of a MBP-158P1D7 fusion protein covalently coupled to Affigel matrix. The serum is then further purified by protein G affinity chromatography to isolate the IgG fraction. Sera from other His-tagged antigens and peptide immunized rabbits as well as fusion partner depleted sera are affinity purified by passage over a column matrix composed of the original protein immunogen or free peptide.
Example 9
Generation of 158P1D7 Monoclonal Antibodies (mAbs)
In one embodiment, therapeutic mAbs to 158P1D7 variants comprise those that react with epitopes specific for each variant protein or specific to sequences in common between the variants that would bind, internalize, disrupt or modulate the biological function of the 158P1D7 variants, for example those that would disrupt the interaction with ligands and binding partners. Immunogens for generation of such mAbs include those designed to encode or contain the extracellular domain or the entire 158P1D7 protein variant sequence, regions predicted to contain functional motifs, and regions of the 158P1D7 protein variants predicted to be antigenic from computer analysis of the amino acid sequence (see, e.g., FIG. 11, FIG. 12, FIG. 13, FIG. 14, or FIG. 15, and the Example entitled “Antigenicity Profiles and Secondary Structure”). Immunogens include peptides, recombinant bacterial proteins, and mammalian expressed Tag 5 proteins and human and murine IgG FC fusion proteins. In addition, pTAG5 protein, DNA vectors encoding the pTAG5 cells engineered to express high levels of a respective 158P1D7 variant, such as 293T-158P1D7 variant 1 or 3T3, RAT, or 300.19-158P1D7 variant 1 murine Pre-B cells, are used to immunize mice.
To generate mAbs to a 158P1D7 variant, mice are first immunized intraperitoneally (IP) with, typically, 10-50 μg of protein immunogen or 107 158P1D7-expressing cells mixed in complete Freund's adjuvant. Mice are then subsequently immunized IP every 24 weeks with, typically, 10-50 μg of protein immunogen or 107 cells mixed in incomplete Freund's adjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. In addition to the above protein and cell-based immunization strategies, a DNA-based immunization protocol is employed in which a mammalian expression vector encoding a 158P1D7 variant sequence is used to immunize mice by direct injection of the plasmid DNA. For example, amino acids 16-608 of 158P1D7 of variant 1 was cloned into the Tag5 mammalian secretion vector and the recombinant vector was used as immunogen. In another example, the same amino acids were cloned into an Fc-fusion secretion vector in which the 158P1D7 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 was then used as immunogen. The plasmid immunization protocols were used in combination with purified proteins expressed from the same vector and with cells expressing the respective 158P1D7 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 158P1D7 variant 1 monoclonal antibodies, a peptide encoding amino acids 274-285 was synthesized, conjugated to KLH and used as immunogen. ELISA on free peptide was used to identify immunoreactive clones. Reactivity and specificity of the monoclonal antibodies to full length 158P1D7 variant 1 protein was monitored by Western blotting, immunoprecipitation, and flow cytometry using both recombinant and endogenous-expressing 158P1D7 variant 1 cells (See FIGS. 22, 23, 24, 25, and 28).
The binding affinity of 158P1D7 variant 1 specific monoclonal antibodies was determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which 158P1D7 variant monoclonal antibodies preferred for diagnostic or therapeutic use, as appreciated by one of skill in the art. The BIAcore system (Uppsala, Sweden) is a preferred method for determining binding affinity. The BIAcore system uses surface plasmon resonance (SPR, Welford K. 1991, Opt. Quant. Elect. 23:1; Morton and Myszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecular interactions in real time. BIAcore analysis conveniently generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants. Results of BIAcore analysis of 158P1D7 variant 1 monoclonal antibodies is shown in Table LVII.
To generate monoclonal antibodies specific for other 158P1D7 variants, immunogens are designed to encode amino acid sequences unique to the variants. In one embodiment, a peptide encoding amino acids 382-395 unique to 158P1D7 variant 4 is synthesized, coupled to KLH and used as immunogen. In another embodiment, peptides or bacterial fusion proteins are made that encompass the unique sequence generated by alternative splicing in the variants. In one example, a peptide encoding a consecutive sequence containing amino acids 682 and 683 in 158P1D7 variant 3 is used, such as amino acids 673-693. In another example, a peptide encoding a consecutive sequence containing amino acids 379-381 in 158P1D7 variant 6 is used, such as amino acids 369-391. Hybridomas are then selected that recognize the respective variant specific antigen and also recognize the full length variant protein expressed in cells. Such selection utilizes immunoassays described above such as Western blotting, immunoprecipitation, and flow cytometry.
To generate 158P1D7 monoclonal antibodies the following protocols were used. 5 Balb/c mice were immunized subcutaneously with 2 μg of peptide in Quiagen ImmuneEasyTM adjuvant. Immunizations were given 2 weeks apart. The peptide used was a 12 amino acid peptide consisting of amino acids 274-285 with the sequence EEHEDPSGSLHL (SEQ ID NO: 41) conjugated to KLH at the C′ terminal (Keyhole Limpet Hemocyanin).
B-cells from spleens of immunized mice were fused with the fusion partner Sp2/0 under the influence of polyethylene glycol. Antibody producing hybridomas were selected by screening on peptide coated ELISA plates indicating specific binding to the peptide and then by FACS on cells expressing 158P1D7. This produced and identified four 158P1D7 extra cellular domain (ECD) specific antibodies designated: M15-68(2)18.1.1; M15-68(2)22.1.1; M15-68(2)31.1.1 and M15-68(2)102.1.1.
The antibody designated M15-68(2)18.1.1 was sent (via Federal Express) to the American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, Va. 20108 on 6 Feb. 2004 and assigned Accession number ______. The characteristics of these four antibodies are set forth in Table LVII.
To clone the M15-68(2)18.1.1 antibody the following protocols were used. M15-68(2)18.1.1 hybridoma cells were lysed with Trizol reagent (Life Technologies, Gibco BRL). Total RNA was purified and quantified. First strand cDNAs was generated from total RNA with oligo (dT)12-18 priming using the Gibco-BRL Superscript Preamplification system. First strand cDNA was amplified using mouse Ig variable heavy chain primers, and mouse lg variable light chain primers. PCR products were cloned into the pCRScript vector (Stratagene, La Jolla). Several clones were sequenced and the variable heavy (VH) and variable light (VL) chain regions determined. The nucleic acid and amino acid sequences of M15-68(2)18 variable heavy and light chain regions are set forth in FIGS. 34A and 34B and FIGS. 35A and 35B.
Example 10
HLA Class I and Class II Binding Assays
HLA class I and class II binding assays using purified HLA molecules are performed in accordance with disclosed protocols (e.g., PCT publications WO94/20127 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.
Example 11
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-XLIX employ the protein sequence data from the gene product of 158P1D7 set forth in FIGS. 2 and 3.
Computer searches for epitopes bearing HLA Class I or Class II supermotifs or motifs are performed as follows. All translated 158P1D7 protein sequences are analyzed using a text string search software program to identify potential peptide sequences containing appropriate HLA binding motifs; such programs are readily produced in accordance with information in the art in view of known motif/supermotif disclosures. Furthermore, such calculations can be made mentally.
Identified A2-, A3-, and DR-supermotif sequences are scored using polynomial algorithms to predict their capacity to bind to specific HLA-Class I or Class II molecules. These polynomial algorithms account for the impact of different amino acids at different positions, and are essentially based on the premise that the overall affinity (or ΔG) of peptide-HLA molecule interactions can be approximated as a linear polynomial function of the type:

“ΔG”=a1i×a2i×a3i . . . ×ani 

where aji is a coefficient which represents the effect of the presence of a given amino acid (j) at a given position (i) along the sequence of a peptide of n amino acids. The crucial assumption of this method is that the effects at each position are essentially independent of each other (i.e., independent binding of individual side-chains). When residue j occurs at position i in the peptide, it is assumed to contribute a constant amount ji to the free energy of binding of the peptide irrespective of the sequence of the rest of the peptide.

The method of derivation of specific algorithm coefficients has been described in Gulukota et al., J. Mol. Biol. 267:1258-126, 1997; (see also Sidney et al., Human Immunol. 45:79-93, 1996; and Southwood et al., J. Immunol. 160:3363-3373, 1998). Briefly, for all i positions, anchor and non-anchor alike, the geometric mean of the average relative binding (ARB) of all peptides carrying j is calculated relative to the remainder of the group, and used as the estimate of ji. For Class II peptides, if multiple alignments are possible, only the highest scoring alignment is utilized, following an iterative procedure. To calculate an algorithm score of a given peptide in a test set, the ARB values corresponding to the sequence of the peptide are multiplied. If this product exceeds a chosen threshold, the peptide is predicted to bind. Appropriate thresholds are chosen as a function of the degree of stringency of prediction desired.
Selection of HLA-A2 Supertype Cross-reactive Peptides
Complete protein sequences from 158P1D7 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 158P1D7 protein sequence 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 158P1D7 protein 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 158P1D7 protein can also be performed to identify HLA-A1- and A24-motif-containing sequences.
High affinity and/or cross-reactive binding epitopes that bear other motif and/or supermotifs are identified using analogous methodology.
Example 12
Confirmation of Immunogenicity
Cross-reactive candidate CTL A2-supermotif-bearing peptides that are identified as described herein are selected to confirm in vitro immunogenicity. Confirmation is performed using the following methodology:
Target Cell Lines for Cellular Screening:
The 0.221A2.1 cell line, produced by transferring the HLA-A2.1 gene into the HLA-A, -B, -C null mutant human B-lymphoblastoid cell line 721.221, is used as the peptide-loaded target to measure activity of HLA-A2.1-restricted CTL. This cell line is grown in RPMI-1640 medium supplemented with antibiotics, sodium pyruvate, nonessential amino acids and 10% (v/v) heat inactivated FCS. Cells that express an antigen of interest, or transfectants comprising the gene encoding the antigen of interest, can be used as target cells to confirm the ability of peptide-specific CTLs to recognize endogenous antigen.
Primary CTL Induction Cultures:
Generation of Dendritic Cells (DC): PBMCs are thawed in RPMI with 30 μg/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640 plus 5% AB human serum, non-essential amino acids, sodium pyruvate, L-glutamine and penicillin/streptomycin). The monocytes are purified by plating 10×106 PBMC/well in a 6-well plate. After 2 hours at 37° C., the non-adherent cells are removed by gently shaking the plates and aspirating the supernatants. The wells are washed a total of three times with 3 ml RPMI to remove most of the non-adherent and loosely adherent cells. Three ml of complete medium containing 50 ng/ml of GM-CSF and 1,000 U/ml of IL-4 are then added to each well. TNFα is added to the DCs on day 6 at 75 ng/ml and the cells are used for CTL induction cultures on day 7.
Induction of CTL with DC and Peptide: CD8+ T-cells are isolated by positive selection with Dynal immunomagnetic beads (Dynabeads® M450) and the detacha-bead® reagent. Typically about 200-250×106 PBMC are processed to obtain 24×106 CD8+ T-cells (enough for a 48-well plate culture). Briefly, the PBMCs are thawed in RPMI with 30 μg/ml DNAse, washed once with PBS containing 1% human AB serum and resuspended in PBS/1% AB serum at a concentration of 20×106 cells/ml. The magnetic beads are washed 3 times with PBS/AB serum, added to the cells (140 μl beads/20×105 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 92 microglobulin in 0.25 ml RPMI/5% AB per well for 2 hours at 37° C. Peptide solution from each well is aspirated and the wells are washed once with RPMI. Most of the media is aspirated from the induction cultures (CD8+ cells) and brought to 0.5 ml with fresh media. The cells are then transferred to the wells containing the peptide-pulsed adherent cells. Twenty four hours later recombinant human IL-10 is added at a final concentration of 10 ng/ml and recombinant human IL2 is added the next day and again 2-3 days later at 50 IU/ml (Tsai et al., Critical Reviews in Immunology 18(1-2):65-75, 1998). Seven days later, the cultures are assayed for CTL activity in a 51Cr release assay. In some experiments the cultures are assayed for peptide-specific recognition in the in situ IFNγ ELISA at the time of the second restimulation followed by assay of endogenous recognition 7 days later. After expansion, activity is measured in both assays for a side-by-side comparison.
Measurement of CTL Lytic Activity by 51Cr Release.
Seven days after the second restimulation, cytotoxicity is determined in a standard (5 hr) 51Cr release assay by assaying individual wells at a single E:T. Peptide-pulsed targets are prepared by incubating the cells with 10 μg/ml peptide overnight at 37° C.
Adherent target cells are removed from culture flasks with trypsin-EDTA. Target cells are labeled with 200 μCi of 51Cr sodium chromate (Dupont, Wilmington, Del.) for 1 hour at 37° C. Labeled target cells are resuspended at 106 per ml and diluted 1:10 with K562 cells at a concentration of 3.3×106/ml (an NK-sensitive erythroblastoma cell line used to reduce non-specific lysis). Target cells (100 μl) and effectors (100 μl) are plated in 96 well round-bottom plates and incubated for 5 hours at 37° C. At that time, 100 μl of supernatant are collected from each well and percent lysis is determined according to the formula:

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

Maximum and spontaneous release are determined by incubating the labeled targets with 1% Trition 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 0.1 M 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 1 M 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 158P1D7. Briefly, PBMCs are isolated from patents, 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 13
Implementation of the Extended Supermotif to Improve the Binding Capacity of Native Epitopes by Creating Analogs
HLA motifs and supermotifs (comprising primary and/or secondary residues) are useful in the identification and preparation of highly cross-reactive naive 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 analogued 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 500 nM or less, to three of more A2 supertype alleles. The rationale for this requirement is that the WT peptides must be present endogenously in sufficient quantity to be biologically relevant. Analoged peptides have been shown to have increased immunogenicity and cross-reactivity by T cells specific for the parent epitope (see, e.g., Parkhurst et al., J. Immunol 157:2539, 1996; and Pogue et al., Proc. Natl. Acad. Sci. USA 92:8166, 1995).
In the cellular screening of these peptide analogs, it is important to confirm that analog-specific CTLs are also able to recognize the wild-type peptide and, when possible, target cells that endogenously express the epitope.
Analoging of HLA-A3 and B7-supermotif-bearing Peptides
Analogs of HLA-A3 supermotif-bearing epitopes are generated using strategies similar to those employed in analoging HLA-A2 supermotif-bearing peptides. For example, peptides binding to 3/5 of the A3-supertype molecules are engineered at primary anchor residues to possess a preferred residue (V, S, M, or A) at position 2.
The analog peptides are then tested for the ability to bind A*03 and A*11 (prototype A3 supertype alleles). Those peptides that demonstrate ≦500 nM binding capacity are then confirmed as having A3-supertype cross-reactivity.
Similarly to the A2- and A3-motif bearing peptides, peptides binding 3 or more B7-supertype alleles can be improved, where possible, to achieve increased cross-reactive binding or greater binding affinity or binding half life. B7 supermotif-bearing peptides are, for example, engineered to possess a preferred residue (V, I, L, or F) at the C-terminal primary anchor position, as demonstrated by Sidney et al. (J. Immunol. 157:3480-3490, 1996).
Analoging at primary anchor residues of other motif and/or supermotif-bearing epitopes is performed in a like manner.
The analog peptides are then be confirmed for immunogenicity, typically in a cellular screening assay. Again, it is generally important to demonstrate that analog-specific CTLs are also able to recognize the wild-type peptide and, when possible, targets that endogenously express the epitope.
Analoging at Secondary Anchor Residues
Moreover, HLA supermotifs are of value in engineering highly cross-reactive peptides and/or peptides that bind HLA molecules with increased affinity by identifying particular residues at secondary anchor positions that are associated with such properties. For example, the binding capacity of a B7 supermotif-bearing peptide with an F residue at position 1 is analyzed. The peptide is then analoged to, for example, substitute L for F at position 1. The analoged peptide is evaluated for increased binding affinity, binding half life and/or increased cross-reactivity. Such a procedure identifies analoged peptides with enhanced properties.
Engineered analogs with sufficiently improved binding capacity or cross-reactivity can also be tested for immunogenicity in HLA-B7-transgenic mice, following for example, IFA immunization or lipopeptide immunization. Analogued peptides are additionally tested for the ability to stimulate a recall response using PBMC from patients with 158P1D7-expressing tumors.
Other Analoging Strategies
Another form of peptide analoging, unrelated to anchor positions, involves the substitution of a cysteine with α-amino butyric acid. Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce binding capacity. Substitution of α-amino butyric acid for cysteine not only alleviates this problem, but has been shown to improve binding and crossbinding capabilities in some instances (see, e.g., the review by Sette et al., In: Persistent Viral Infections, Eds. R. Ahmed and 1. Chen, John Wiley & Sons, England, 1999).
Thus, by the use of single amino acid substitutions, the binding properties and/or cross-reactivity of peptide ligands for HLA supertype molecules can be modulated.
Example 14
Identification and Confirmation of 158P1D7-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 158P1D7-derived, HLA class II HTL epitopes, the 158P1D7 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 158P1D7-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. 158P1D7-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 158P1D7 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 15
Immunogenicity of 158P1D7-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 158P1D7-expressing tumors.
Example 16
Calculation of Phenotypic Frequencies of HLA-supertypes in Various Ethnic Backgrounds to Determine Breadth of Population Coverage
This example illustrates the assessment of the breadth of population coverage of a vaccine composition comprised of multiple epitopes comprising multiple supermotifs and/or motifs.
In order to analyze population coverage, gene frequencies of HLA alleles are determined. Gene frequencies for each HLA allele are calculated from antigen or allele frequencies utilizing the binomial distribution formulae gf=1−(SQRT(1−af)) (see, e.g., Sidney et al., Human Immunol. 45:79-93, 1996). To obtain overall phenotypic frequencies, cumulative gene frequencies are calculated, and the cumulative antigen frequencies derived by the use of the inverse formula [af=1−(1−Cgf)2].
Where frequency data is not available at the level of DNA typing, correspondence to the serologically defined antigen frequencies is assumed. To obtain total potential supertype population coverage no linkage disequilibrium is assumed, and only alleles confirmed to belong to each of the supertypes are included (minimal estimates). Estimates of total potential coverage achieved by inter-loci combinations are made by adding to the A coverage the proportion of the non-A covered population that could be expected to be covered by the B alleles considered (e.g., total=A+B*(1−A)). Confirmed members of the A3-like supertype are A3, A11, A31, A*3301, and A*6801. Although the A3-like supertype may also include A34, A66, and A*7401, these alleles were not included in overall frequency calculations. Likewise, confirmed members of the A2-like supertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmed alleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601, B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, and B*5602).
Population coverage achieved by combining the A2-, A3- and B7-supertypes is approximately 86% in five major ethnic groups. Coverage may be extended by including peptides bearing the A1 and A24 motifs. On average, A1 is present in 12% and A24 in 29% of the population across five different major ethnic groups (Caucasian, North American Black, Chinese, Japanese, and Hispanic). Together, these alleles are represented with an average frequency of 39% in these same ethnic populations. The total coverage across the major ethnicities when A1 and A24 are combined with the coverage of the A2-, A3- and B7-supertype alleles is >95%. 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 17
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 158P1D7 expression vectors.
The results demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized 158P1D7 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 18
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 158P1D7-derived CTL and HTL peptide vaccine compositions. The vaccine composition used herein comprise peptides to be administered to a patient with a 158P1D7-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. 159:47534761, 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 19
Selection of CTL and HTL Epitopes for Inclusion in an 158P1D7-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 158P1D7 clearance. The number of epitopes used depends on observations of patients who spontaneously clear 158P1D7. For example, if it has been observed that patients who spontaneously clear 158P1D7 generate an immune response to at least three (3) from 158P1D7 antigen, then three or four (34) 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, at URL bimas.dcrt.nih.gov/.
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 158P1D7, 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 158P1D7.
Example 20
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 158P1D7, are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from 158P1D7 to provide broad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct. The selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.
Such a construct may additionally include sequences that direct the HTL epitopes to the endoplasmic reticulum. For example, the li protein may be fused to one or more HTL epitopes as described in the art, wherein the CLIP sequence of the li protein is removed and replaced with an HLA class II epitope sequence so that HLA class II epitope is directed to the endoplasmic reticulum, where the epitope binds to an HLA class II molecules.
This example illustrates the methods to be used for construction of a minigene-bearing expression plasmid. Other expression vectors that may be used for minigene compositions are available and known to those of skill in the art.
The minigene DNA plasmid of this example contains a consensus Kozak sequence and a consensus murine kappa Ig-light chain signal sequence followed by CTL and/or HTL epitopes selected in accordance with principles disclosed herein. The sequence encodes an open reading frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.
Overlapping oligonucleotides that can, for example, average about 70 nucleotides in length with 15 nucleotide overlaps, are synthesized and HPLC-purified. The oligonucleotides encode the selected peptide epitopes as well as appropriate linker nucleotides, Kozak sequence, and signal sequence. The final multiepitope minigene is assembled by extending the overlapping oligonucleotides in three sets of reactions using PCR. A Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are performed using the following conditions: 95° C. for 15 sec, annealing temperature (5° below the lowest calculated Tm of each primer pair) for 30 sec, and 72° C. for 1 min.
For example, a minigene is prepared as follows. For a first PCR reaction, 5 μg of each of two oligonucleotides are annealed and extended: In an example using eight oligonucleotides, i.e., four pairs of primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100 μl reactions containing Pfu polymerase buffer (1x=10 mM KCL, 10 mM (NH4)2SO4, 20 mM Tris-chloride, pH 8.75, 2 mM MgSO4, 0.1% Triton X-100, 100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. The full-length dimer products are gel-purified, and two reactions containing the product of 1+2 and 3+4, and the product of 5+6 and 7+8 are mixed, annealed, and extended for 10 cycles. Half of the two reactions are then mixed, and 5 cycles of annealing and extension carried out before flanking primers are added to amplify the full length product. The full-length product is gel-purified and cloned into pCR-blunt (Invitrogen) and individual clones are screened by sequencing.
Example 21
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-AbU.S. Pat. No. -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 22
Peptide Composition for Prophylactic Uses
Vaccine compositions of the present invention can be used to prevent 158P1D7 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 158P1D7-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 158P1D7-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 23
Polyepitopic Vaccine Compositions Derived from Native 158P1D7 Sequences
A native 158P1D7 polyprotein sequence is analyzed, preferably using computer algorithms defined for each class I and/or class II supermotif or motif, to identify “relatively short” regions of the polyprotein that comprise multiple epitopes. The “relatively short” regions are preferably less in length than an entire native antigen. This relatively short sequence that contains multiple distinct or overlapping, “nested” epitopes is selected; it can be used to generate a minigene construct. The construct is engineered to express the peptide, which corresponds to the native protein sequence. The “relatively short” peptide is generally less than 250 amino acids in length, often less than 100 amino acids in length, preferably less than 75 amino acids in length, and more preferably less than 50 amino acids in length. The protein sequence of the vaccine composition is selected because it has maximal number of epitopes contained within the sequence, i.e., it has a high concentration of epitopes. As noted herein, epitope motifs may be nested or overlapping (i.e., frame shifted relative to one another). For example, with overlapping epitopes, two 9-mer epitopes and one 10-mer epitope can be present in a 10 amino acid peptide. Such a vaccine composition is administered for therapeutic or prophylactic purposes.
The vaccine composition will include, for example, multiple CTL epitopes from 158P1D7 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 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 158P1D7, 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 24
Polyepitopic Vaccine Compositions from Multiple Antigens
The 158P1D7 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 158P1D7 and such other antigens. For example, a vaccine composition can be provided as a single polypeptide that incorporates multiple epitopes from 158P1D7 as well as tumor-associated antigens that are often expressed with a target cancer associated with 158P1D7 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 25
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 158P1D7. 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, 158P1D7 HLA-A*0201-specific CTL frequencies from HLA A*0201-positive individuals at different stages of disease or following immunization comprising an 158P1D7 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 158P1D7 epitope, and thus the status of exposure to 158P1D7, or exposure to a vaccine that elicits a protective or therapeutic response.
Example 26
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 158P1D7-associated disease or who have been vaccinated with an 158P1D7 vaccine.
For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any 158P1D7 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 Umes 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 ul 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 Histocompatbility 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 158P1D7 or an 158P1D7 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 158P1D7 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 27
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 28
Phase II Trials in Patients Expressing 158P1D7
Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients having cancer that expresses 158P1D7. The main objectives of the trial are to determine an effective dose and regimen for inducing CTLs in cancer patients that express 158P1D7, 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 158P1D7.
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 158P1D7-associated disease.
Example 29
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 34 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-107 to 5×109 pfu. An alternative recombinant virus, such as an MVA, canarypox, adenovirus, or adeno-associated virus, can also be used for the booster, or the polyepitopic protein or a mixture of the peptides can be administered. For evaluation of vaccine efficacy, patient blood samples are obtained before immunization as well as at intervals following administration of the initial vaccine and booster doses of the vaccine. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity.
Analysis of the results indicates that a magnitude of response sufficient to achieve a therapeutic or protective immunity against 158P1D7 is generated.
Example 30
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 158P1D7 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/IL4. 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 ProgenipoiefinTM 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 158P1D7 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 31
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. 158P1D7. 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 158P1D7 to isolate peptides corresponding to 158P1D7 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 32
Complementary Polynucleotides
Sequences complementary to the 158P1D7-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring 158P1D7. 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 158P1D7. 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 the 158P1D7-encoding transcript.
Example 33
Purification of Naturally-occurring or Recombinant 158P1D7 Using 158P1D7 Specific Antibodies
Naturally occurring or recombinant 158P1D7 is substantially purified by immunoaffinity chromatography using antibodies specific for 158P1D7. An immunoaffinity column is constructed by covalently coupling anti-158P1D7 antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturers instructions.
Media containing 158P1D7 are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of 158P1D7 (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/158P1D7 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 34
Identification of Molecules which Interact with 158P1D7
158P1D7, 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 158P1D7, washed, and any wells with labeled 158P1D7 complex are assayed. Data obtained using different concentrations of 158P1D7 are used to calculate values for the number, affinity, and association of 158P1D7 with the candidate molecules. Throughout this application, various website data content, publications, 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 items of information are hereby incorporated by reference herein in their entireties.
Example 35
In Vivo Assay for 158P1D7 Tumor Growth Promotion
The effect of the 158P1D7 protein on tumor cell growth can be confirmed in vivo by gene overexpression in bladder cancer cells. For example, SCID mice can be injected SQ on each flank with 1×106 bladder cancer cells (such as SCaBER, UM-UC-3, HT1376, RT4, T24, TCC-SUP, J82 and SW780 cells) containing tkNeo empty vector or 158P1D7.
At least two strategies may be used: (1) Constitutive 158P1D7 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. (2) Regulated expression under control of an inducible vector system, such as ecdysone, tet, etc., can be used provided such promoters are compatible with the host cell systems. Tumor volume is then monitored at the appearance of palpable tumors and is followed over time to determine if 158P1D7-expressing cells grow at a faster rate and whether tumors produced by 158P1D7-expressing cells demonstrate characteristics of altered aggressiveness (e.g. enhanced metastasis, vascularization, reduced responsiveness to chemotherapeutic drugs). Additionally, mice can be implanted with the same cells orthotopically to determine if 158P1D7 has an effect on local growth in the bladder or on the ability of the cells to metastasize, specifically to lungs or lymph nodes (Fu, X., et al., Int. J. Cancer, 1991. 49: p. 938-939; Chang, S., et al., Anticancer Res., 1997. 17: p. 3239-3242; Peralta, E. A., et al., J. Urol., 1999. 162: p. 1806-1811). Furthermore, this assay is useful to confirm the 158P1D7 inhibitory effect of candidate therapeutic compositions, such as for example, 158P1D7 antibodies or intrabodies, and 158P1D7 antisense molecules or ribozymes.
The assay was performed using the following protocols. Male ICR-SCID mice, 5-6 weeks old (Charles River Laboratory, Wilmington, Mass.) were used and maintained in a strictly controlled environment in accordance with the NIH Guide for the Care and Use of Laboratory Animals. 158P1D7 transfected UM-UC-3 cells and parental cells were injected into the subcutaneous space of SCID mice. Each mouse received 4×106 cells suspended in 50% (v/v) of Matrigel. Tumor size was monitored through caliper measurements twice a week. The longest dimension (L) and the dimension perpendicular to it (W) were taken to calculate tumor volume according to the formula W2×L/2. The Mann-Whitney U test was used to evaluate differences of tumor growth. All tests were two sided with {acute over (α)}=0.05. The results show that 158P1D7 enhances the growth of bladder cancer in mice (FIG. 27).
Example 36
158P1D7 Monoclonal Antibody-mediated Inhibition of Bladder and Prostate Tumors In Vivo
The significant expression of 158P1D7 in cancer tissues, together with its restricted expression in normal tissues, makes 158P1D7 an excellent target for antibody therapy. In cases where the monoclonal antibody target is a cell surface protein, antibodies have been shown to be efficacious at inhibiting tumor growth (See, e.g., (Saffran, D., et al., PNAS 10:1073-1078 or URL: pnas.org/cgi/doi/10.1073/pnas.051624698). In cases where the target is not on the cell surface, such as PSA and PAP in prostate cancer, antibodies have still been shown to recognize and inhibit growth of cells expressing those proteins (Saffran, D. C., et al., Cancer and Metastasis Reviews, 1999. 18: p. 437-449). As with any cellular protein with a restricted expression profile, 158P1D7 is a target for T cell-based immunotherapy.
Accordingly, the therapeutic efficacy of anti-158P1D7 mAbs in human bladder cancer mouse models is modeled in 158P1D7-expressing bladder cancer xenografts or bladder cancer cell lines, such as those described in Example (the Example entitled “In Vivo Assay for 158P1D7 Tumor Growth Promoton”, that have been engineered to express 158P1D7.
Antibody efficacy on tumor growth and metastasis formation is confirmed, e.g., in a mouse orthotopic bladder cancer xenograft model. The antibodies can be unconjugated, as discussed in this Example, or can be conjugated to a therapeutic modality, as appreciated in the art. It is confirmed that anti-158P1D7 mAbs inhibit formation of 158P1D7-expressing bladder and prostate tumors (FIGS. 30 and 31). Anti-158P1D7 mAbs can be tested for the retardation of the growth of established orthotopic tumors and the prolonged survival of tumor-bearing mice. These results indicate the utility of anti-158P1D7 mAbs in the treatment of local and advanced stages of bladder and prostate cancers. (See, e.g., Saffran, D., et al., PNAS 10:1073-1078 or URL: pnas.org/cgi/doi/10.1073/pnas.051624698)
Administration of anti-158P1D7 mAbs retard established orthotopic tumor growth and inhibit metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies indicate that 158P1D7 is an attractive target for immunotherapy and demonstrate the therapeutic potential of anti-158P1D7 mAbs for the treatment of local and metastatic bladder cancer.
This example demonstrates that unconjugated 158P1D7 monoclonal antibodies effectively to inhibit the growth of human bladder tumors grown in SCID mice; accordingly a combination of such efficacious monoclonal antibodies is also effective.
Tumor Inhibition Using Multiple Unconjugated 158P1D7 mAbs
Materials and Methods
158P1D7 Monoclonal Antibodies:
Monoclonal antibodies are raised against 158P1D7 as described in the Example entitled “Generation of 158P1D7 Monoclonal Antibodies (mAbs).” The antibodies are characterized by ELISA, Western blot, FACS, and immunoprecipitation, in accordance with techniques known in the art, for their capacity to bind 158P1D7. Epitope mapping data for the anti-158P1D7 mAbs, as determined by ELISA and Western analysis, recognize epitopes on the 158P1D7 protein. Immunohistochemical analysis of bladder 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 bladder tumor xenografts.
Bladder Cancer Cell Lines
Bladder cancer cell lines (Scaber, J82, UM-UC-3, HT1376, RT4, T24, TCC-SUP, J82 and SW780) expressing 158P1D7 are generated by retroviral gene transfer as described in Hubert, R. S., et al., STEAP: a prostate-specific cell-surface antigen highly expressed in human prostate tumors. Proc Natl Acad Sci USA, 1999. 96(25):14523-8. Anti-158P1D7 staining is detected by using an FITC-conjugated goat anti-mouse antibody (Southern Biotechnology Associates) followed by analysis on a Coulter Epics-XL flow cytometer.
In Vivo Mouse Models.
Subcutaneous (s.c.) tumors are generated by injection of 1×106 158P1D7-expressing bladder cancer cells mixed at a 1:1 dilution with Matrigel (Collaborative Research) in the right flank of male SCID mice. To test antibody efficacy on tumor formation, i.p. antibody injections are started on the same day as tumor-cell injections. As a control, mice are injected with either purified mouse IgG (ICN) or PBS; or a purified monoclonal antibody that recognizes an irrelevant antigen not expressed in human cells. In preliminary studies, no difference is found between mouse IgG or PBS on tumor growth. Tumor sizes are determined by vernier caliper measurements, and the tumor volume is calculated as length×width×height. Mice with s.c. tumors greater than 1.5 cm in diameter are sacrificed. Circulating levels of anti-158P1D7 mAbs are determined by a capture ELISA kit (Bethyl Laboratories, Montgomery, Tex.). (See, e.g., (Saffran, D., et al., PNAS 10:1073-1078)
Orthotopic injections are performed, for example, in two alternative embodiments, under anesthesia by, for example, use of ketamine/xylazine. In a first embodiment, an intravesicular injection of bladder cancer cells is administered directly through the urethra and into the bladder (Peralta, E. A., et al., J. Urol., 1999.162:1806-1811). In a second embodiment, an incision is made through the abdominal wall, the bladder is exposed, and bladder tumor tissue pieces (1-2 mm in size) derived from a s.c. tumor are surgically glued onto the exterior wall of the bladder, termed “onplantafion” (Fu, X., et al., Int. J. Cancer, 1991. 49: 938-939; Chang, S., et al., Anticancer Res., 1997. 17: p. 3239-3242). Antibodies can be administered to groups of mice at the time of tumor injection or onplantation, or after 1-2 weeks to allow tumor establishment.
Anti-158P1D7 mAbs Inhibit Growth of 158P1D7-Expressing Bladder Cancer Tumors
In one embodiment, the effect of anti-158P1D7 mAbs on tumor formation is tested by using the bladder onplantation orthotopic model. As compared with the s.c. tumor model, the orthotopic model, which requires surgical attachment of tumor tissue directly on the bladder, results in a local tumor growth, development of metastasis in distal sites, and subsequent death (Fu, X., et al., Int. J. Cancer, 1991. 49: p. 938-939; Chang, S., et al., Anticancer Res., 1997. 17: p. 3239-3242). This feature make the orthotopic model more representative of human disease progression and allows one to follow the therapeutic effect of mAbs, as well as other therapeutic modalities, on clinically relevant end points.
Accordingly, 158P1D7-expressing tumor cells are onplanted orthotopically, and 2 days later, the mice are segregated into two groups and treated with either: a) 50-2000 μg, usually 200-500 μg, of anti-158P1D7 Ab, or b) PBS, three times per week for two to five weeks. Mice are monitored weekly for indications of tumor growth.
As noted, a major advantage of the orthotopic bladder cancer model is the ability to study the development of metastases. Formation of metastasis in mice bearing established orthotopic tumors is studied by histological analysis of tissue sections, including lung and lymph nodes (Fu, X., et al., Int. J. Cancer, 1991. 49:938-939; Chang, S., et al., Anticancer Res., 1997. 17:3239-3242). Additionally, IHC analysis using anti-158P1D7 antibodies can be performed on the tissue sections.
Mice bearing established orthotopic 158P1D7-expressing bladder tumors are administered 1000 μg injections of either anti-158P1D7 mAb or PBS over a 4-week period. Mice in both groups are allowed to establish a high tumor burden (1-2 weeks growth), to ensure a high frequency of metastasis formation in mouse lungs and lymph nodes. Mice are then sacrificed and their local bladder tumor and lung and lymph node tissue are analyzed for the presence of tumor cells by histology and IHC analysis.
In another embodiment, the effect of anti-158P1D7 mAbs on tumor growth was tested using the following protocols. Male ICR-SCID mice, 5-6 weeks old (Charles River Laboratory, Wilmington, Mass.) were used and were maintained in a strictly-controlled environment in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
UG-B1, a patient bladder cancer, was used to establish xenograft models. Stock tumors regularly maintained in SCID mice were sterilely dissected, minced, and digested using Pronase (Calbiochem, San Diego, Calif.). Cell suspensions generated were incubated overnight at 37° C. to obtain a homogeneous single-cell suspension. Each mouse received 2.5×106 cells at the subcutaneous site of right flank. Murine monoclonal antibodies to 158P1D7 were tested at a dose of 500 μg/mouse in the study. PBS was used as control. MAbs were dosed intra-peritoneally twice a week for a total of 12 doses, starting on the same day of tumor cell injection. Tumor size was monitored through caliper measurements twice a week. The longest dimension (L) and the dimension perpendicular to it (W) were taken to calculate tumor volume according to the formula: W2×L/2. The results show that Anti-158P1D7 mAbs are capable of inhibiting the growth of human bladder carcinoma in mice (FIG. 30).
Anti-158P1D7 mAbs Retard the Growth of Established 158P1D7-Expressing Prostate Cancer Tumors
In another embodiment, the effect of anti-158P1D7 mAbs on tumor growth was tested using the following protocols. Male ICR-SCID mice, 5-6 weeks old (Charles River Laboratory, Wilmington, Mass.) were used and were maintained in a strictly-controlled environment in accordance with the NIH Guide for the Care and Use of Laboratory Animals. LAPC-9AD, an androgen-dependent human prostate cancer, was used to establish xenograft models. Stock tumors were regularly maintained in SCID mice. At the day of implantation, stock tumors were harvested and trimmed of necrotic tissues and minced to 1 mm3 pieces. Each mouse received 4 pieces of tissues at the subcutaneous site of right flank. Murine monoclonal antibodies to 158P1D7 were tested at a dose of 500 μg/mouse and 500 μg/mouse respectively. PBS and anti-KLH monoclonal antibody were used as controls. The study cohort consisted of 4 groups with 6 mice in each group. MAbs were dosed intra-peritoneally twice a week for a total of 8 doses. Treatment was started when tumor volume reached 45 mm3. Tumor size was monitored through caliper measurements twice a week. The longest dimension (L) and the dimension perpendicular to it (W) were taken to calculate tumor volume according to the formula: W2×L/2. The Student's t test and the Mann-Whitney U test, where applicable, were used to evaluate differences of tumor growth. All tests were two-sided with a=0.05. The results show that Anti-158P1D7 mAbs are capable of retarding the growth of established human prostate carcinoma in mice (FIG. 31).
These studies demonstrate a broad anti-tumor efficacy of anti-158P1D7 antibodies on initiation and progression of bladder cancer and prostate cancer and indicate that 158P1D7 antibodies to be efficacious in inhibiting and retarding the growth of 158P1D7-expressing tissues (Table I) in mouse models. Anti-158P1D7 antibodies inhibit tumor formation and retard the growth of already established tumors and prolong the survival of treated mice. Moreover, anti-158P1D7 mAbs demonstrate a dramatic inhibitory effect on the spread of local bladder tumor to distal sites, even in the presence of a large tumor burden. Thus, anti-158P1D7 mAbs are efficacious on major clinically relevant end points including lessened tumor growth, lessened metastasis, and prolongation of survival.
Example 37
Homology Comparison of 158P1D7 to Known Sequences
The 158P1D7 protein has 841 amino acids with calculated molecular weight of 95.1 kDa, and pl of 6.07. 158P1D7 is predicted to be a plasma membrane protein (0.46 PSORT http://psort.nibb.ac.jp/form.html) with a possibility of it being a nuclear protein (65% by PSORT http://psort.nibb.ac.jp/form2.html). 158P1D7 has a potential cleavage site between aa 626 and 627 and a potential signal site at aa 3-25.
158P1D7 contains a single transmembrane region from amino acids 611-633 with high probability that the amino-terminus resides outside, consistent with the topology of a Type 1 transmembrane protein (located on the World Wide Web at .cbs.dtu.dk/services/TMHMM). Also visualized is a short hydrophobic stretch from amino acids 3-25, consistent with the existence of an amino-terminal signal peptide. 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), 158P1D7 contains a primary transmembrane region from amino acids 609-633 and a secondary transmembrane region from amino acids 3-25 (contiguous amino acids with values greater than 0 on the plot have high probability of being transmembrane regions) with an orientation in which the amino terminus resides inside and the carboxyl terminus outside. An alternative model is also predicted that 158P1D7 is a Type 1 transmembrane protein in which the amino-terminus resides outside and the protein contains a secondary transmembrane domain signal peptide from amino acids 3-25 and a primary transmembrane domain from aa615-633. The transmembrane prediction algorithms are accessed through the ExPasy molecular biology server located on the World Wide Web at (.expasy.ch/tools/).
By use of the PubMed website of the N.C.B.I. located on the World Wide Web at (.ncbi.nlm.nih.gov/entrez), it was found at the protein level that 158P1D7 shows best homology to the hypothetical protein FLJ22774 (PubMed record: gi 14149932) of unknown function, with 97% identity and 97% homology (FIG. 4 and FIG. 5A). The 158P1D7 protein demonstrates homology to a human protein similar to IGFALS (insulin-like growth factor binding protein, acid labile subunit) (PubMed record: gi 6691962) with 36% identity and 52% homology (FIG. 5B), to Slit proteins with 25% identity and 39% homology and to the leucine-rich repeat transmembrane family of proteins FLRT (Fibronectin-like domain-containing leucine-rich transmembrane protein), including FLRT2 with 26% identity and 43% homology, and FLRT3 with 34% identity and 53% homology.
Insulin-like growth factors (IGF) have been shown to play an important role in tumor growth including prostate, breast, brain and ovarian cancer (O'Brian et al., Urology. 2001, 58:1; Wang J et al Oncogene. 2001, 20:3857; Helle S et al., Br J Cancer. 2001, 85:74). IGFs produce their oncogenic effect by binding to specific cell surface receptors and activating survival as well as mitogenic pathways (Babajko S et al., Med Pediatr Oncol. 2001, 36:154; Scalia P et al., J Cell Biochem. 2001, 82:610). The activity of insulin-like growth factors is regulated by IGF binding proteins (IGF-BP) and the acid labile subunit (ALS) of IGF-BP (Zeslawski W et al., EMBO J. 2001, 20:3638; Jones J I. and Clemmons D R. Endocr. Rev. 1995,16: 3). In the plasma, most IGFs exist as a ternary complex containing IGF-BP and ALS (Jones J I. and Clemmons D R. Endocr. Rev. 1995, 16: 3). Association with ALS allows the retention of the ternary complex in the vasculature and extends its lifespan (Ueki I et al., Proc Natl Acad Sci USA 2000, 97:6868). Studies in mice demonstrate the contribution of ALS to cell growth by showing that mice carrying mutant ALS exhibit a growth deficit (Ueki I et al., Proc Natl Acad Sci USA 2000, 97:6868), indicating that ALS plays a critical role in the growth of tumor cells. The 158P1D7 protein serves as an IGF-ALS-like protein in that it facilitates the formation of the IGF ternary complex. The 158P1D7-induced IGF complex formation leads to increased growth of tumor cells expressing 158P1D7 which facilitates the growth of this malignancy in vivo. The induction of the IGF complex allows one to assay for monoclonal antibodies with neutralizing ability to disrupt, or enhancing capacity to help form, the ternary interaction.
Slit proteins were first identified in Drosophila as secreted proteins that regulate axon guidance and orientation (Rajagopalan S et al., Cell. 2000, 103:1033; Chen J et al., J. Neurosci. 2001, 21:1548). Mammalian homologs were cloned in mice and humans, where they are shown to regulate migration and chemotaxis (Wu J et al., Nature. 2001, 410:948; Brose K and Tessier M, Curr Opin Neurobiol. 2001, 10:95). Slit proteins localize at two distinct subcellular sites within epithelial cells depending on cell stage, with Slit 3 predominantly localizing in the mitochondria and targeting to the cell surface in more confluent cells (Little M H et al., Am J Physiol Cell Physiol. 2001, 281:C486). The differential Slit localization suggests that Slit may function differently whether it is secreted, associated with the cell surface or retained in the mitochondria. The 158P1D7 protein functions as a Slit-like protein in that it binds to Roundabout receptors (Robos) on the surface of cells. 158P1D7 has homology (83% identity along entire length) with the murine Slitrk6 gene, a member of a new family of Leucine Rich Receptors (LRRs). The Slit family of LRRs is involved in neurite outgrowth and axonal guidance during development. These proteins also play a role in organ development by providing cues for branching morphogenesis in lung, kidney and other organs. The crystal structure for several LRRs has been determined. These proteins are shaped like a horseshoe with LRRs on both sides of a central flexible region. This horseshoe shape likely forms a central pocket where other proteins (binding partners) can interact. The term binding partner includes ligands, receptors, substrates, antibodies, and other molecules that interact with the 158P1D7 polypeptide through contact or proximity between particular portions of the binding partner and the 158P1D7 polypeptide. Binding partners for 158P1D7 polypeptides are expressed on both epithelial and mesenchymal cells within an organ. Known binding partners for the Slit family of LRRs include both the Robo family of genes and glypicans. Both of these potential protein interacting partners are aberrantly expressed in human cancers. Robos are Ig-like proteins that act as adhesion molecules. Interaction of specific Robo and Slit proteins results in cell migration with the ultimate outcome being either repulsion or attraction depending on intracellular signaling cascades. Mutations that disrupt interaction of Slit with Robo result in failure to repel migrating neurons during development. Moreover, mutations that disrupt functional interactions lead to organ failure and hyperproliferation in the developing lung. Mutational analysis has further shown that the LRR region is required for biologic activity of these receptors. 158P1D7 is overexpressed in a variety of human cancers including those derived from bladder and lung. Aberrant expression of this protein leads to enhanced cell growth, survival, increased metastasis and angiogenesis by disrupting or promoting protein interactions between 158P1D7 and specific binding partners on the surface of adjacent cells. Binding of 158P1D7 to Robo receptors (Robo-1, -2, -3 and 4) is observed in vitro, both as recombinant proteins and as cell surface molecules. Biological effects are induced when the Robo-1, -2, -3 or -4 receptors or glypican-binding partners binds to 158P1D7 on the cell surface. These activities are detected by adhesion, enhanced migration or repulsion in cell based assays. The interaction between 158P1D7 and Robo receptors leads to increased adhesion between 158P1D7-expressing tumor cells and endothelium or other cell types expressing Robo receptors, leading to spreading and metastasis of tumor cells as well as enhanced angiogenesis. Further, the association between 158P1D7 and Robo receptors allows one to screen for monoclonal antibodies with the ability to block (or enhance) the interaction in an in vitro assay. Such antibodies have a modulating effect on growth of 158P1D7 expressing tumors.
The FLRT (Fibronectin-like domain-containing leucine-rich transmembrane protein) family of transmembrane proteins has three members, FLRT1, FLRT2 and FLRT3, which contain 10 leucine-rich repeats flanked by cysteine-rich domains, a fibronectin/collagen-like motif and an intracellular tail (Lacy S E et al., Genomics 1999, 62:417). Based on overall structure of the three proteins, a role in cell adhesion and receptor signaling is predicted. A Xenopus laevis ortholog of FLRT3 (XFLRT3) was identified that shows co-expression with FGFs (fibroblast growth factors) and is induced after activation and reduced following inhibition of signal transduction through the FGFs (Bottcher R T et al., Nature Cell Biol 2004, 6:38). The interaction between FGFRs (FGF receptors) and XFLRT3 indicates that XFLRT3 modulates FGF-induced signal transduction through the MAP kinase pathway. The 158P1D7 protein forms a complex with FGFRs that induces modulation of FGF-induced signal transduction through the MAP kinase (ERK-1 and ERK-2) pathway. FGF-induced signals are potentiated by expression of 158P1D7, which leads to an increase in the proliferative capacity of the cells. This significantly promotes unregulated growth of cancer cells expressing 158P1D7, contributing to their growth advantage in vivo. The interaction between 158P1D7 protein and FGFR allows one to screen for monoclonal antibodies with the ability to disrupt (or enhance) the association of these two molecules. Such antibodies have a modulating effect on growth of 158P1D7 expressing tumors.
Example 38
Identification and Confirmation of Signal Transduction Pathways
Many mammalian proteins have been reported to interact with signaling molecules and to participate in regulating signaling pathways. (J. Neurochem. 2001; 76:217-223). In particular, IGF and IGF-BP have been shown to regulate mitogenic and survival pathways (Babajko S et al., Med Pediatr Oncol. 2001, 36:154; Scalia P et al., J Cell Biochem. 2001, 82:610). Using immunoprecipitation and Western blotting techniques, proteins are identified that associate with 158P1D7 and mediate signaling events. Several pathways known to play a role in cancer biology are regulated by 158P1D7, including phospholipid pathways such as PI3K, AKT, etc, adhesion and migration pathways, including FAK, Rho, Rac-1, etc, as well as mitogenic/survival cascades such as ERK, p38, etc. (Cell Growth Differ. 2000, 11:279; J Biol. Chem. 1999, 274:801; Oncogene. 2000, 19:3003, J. Cell Biol. 1997, 138:913.). Bioinformatic analysis revealed that 158P1D7 can become phosphorylated by serine/threonine as well as tyrosine kinases. Thus, the phosphorylation of 158P1D7 is provided by the present invention to lead to activation of the above listed pathways.
Using, e.g., Western blotting techniques, the ability of 158P1D7 to regulate these pathways is confirmed. Cells expressing or lacking 158P1D7 are either left untreated or stimulated with cytokines, hormones and anti-integrin antibodies. Cell lysates are analyzed using anti-phospho-specific antibodies (Cell Signaling, Santa Cruz Biotechnology) in order to detect phosphorylation and regulation of ERK, p38, AKT, PI3K, PLC and other signaling molecules. When 158P1D7 plays a role in the regulation of signaling pathways, whether individually or communally, it is used as a target for diagnostic, prognostic, preventative and therapeutic purposes.
To confirm that 158P1D7 directly or indirectly activates known signal transduction pathways in cells, luciferase (luc) based transcriptional reporter assays are carded out in cells expressing individual genes. These transcriptional reporters contain consensus-binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways. The reporters and examples of these associated transcription factors, signal transduction pathways, and activation stimuli are listed below:

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


Gene-mediated effects are assayed in cells showing mRNA expression. Luciferase reporter plasmids are 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 158P1D7 are mapped and used for the identification and validation of therapeutic targets. When 158P1D7 is involved in cell signaling, it is used as target for diagnostic, prognostic, preventative and therapeutic purposes.
Example 39
Involvement in Tumor Progression
The 158P1D7 gene can contribute to the growth of cancer cells. The role of 158P1D7 in tumor growth is confirmed in a variety of primary and transfected cell lines including prostate, colon, bladder and kidney cell lines as well as NIH 3T3 cells engineered to stably express 158P1D7. Parental cells lacking 158P1D7 and cells expressing 158P1D7 are evaluated for cell growth using a well-documented proliferation assay (see, e.g., 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 158P1D7 in the transformation process, its effect in colony forming assays is investigated. Parental NIH3T3 cells lacking 158P1D7 are compared to NHI-3T3 cells expressing 158P1D7, 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 158P1D7 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, colon, bladder and kidney cell lines lacking 158P1D7 are compared to cells expressing 158P1D7, respectively. 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.
158P1D7 can also play a role in cell cycle and apoptosis. Parental cells and cells expressing 158P1D7 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 158P1D7, including normal and tumor bladder cells. Engineered and parental cells are treated with various chemotherapeutic agents, such as paclitaxel, gemcitabine, 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 158P1D7 can play a critical role in regulating tumor progression and tumor load.
When 158P1D7 plays a role in cell growth, transformation, invasion or apoptosis, it is used as a target for diagnostic, prognostic, preventative and therapeutic purposes.
Example 40
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). 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 effect of 158P1D7 on angiogenesis is confirmed. For example, endothelial cells engineered to express 158P1D7 are evaluated using tube formation and proliferation assays. The effect of 158P1D7 is also confirmed in animal models in vivo. For example, cells either expressing or lacking 158P1D7 are implanted subcutaneously in immunocompromised mice. Endothelial cell migration and angiogenesis are evaluated 5-15 days later using immunohistochemistry techniques. When 158P1D7 affects angiogenesis, it is used as a target for diagnostic, prognostic, preventative and therapeutic purposes.
Example 41
Regulation of Transcription
The above-indicated localization of 158P1D7 to the nucleus and its similarity to IGF-BP which has been found to activate signaling pathways and to regulate essential cellular functions, support the present invention use of 158P1D7 based on its role in the transcriptional regulation of eukaryotic genes. Regulation of gene expression is confirmed, e.g., by studying gene expression in cells expressing or lacking 158P1D7. For this purpose, two types of experiments are performed.
In the first set of experiments, RNA from parental and 158P1D7-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 or androgen 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 (e.g., 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.
When 158P1D7 plays a role in gene regulation, it is used as a target for diagnostic, prognostic, preventative and therapeutic purposes.
Example 42
Subcellular Localization of 158P1D7
The cellular location of 158P1D7 is assessed using subcellular fractionation techniques widely used in cellular biology (Storrie B, et al. Methods Enzymol. 1990;182:203-25). A variety of cell lines, including prostate, kidney and bladder cell lines as well as cell lines engineered to express 158P1D7 are separated into nuclear, cytosolic and membrane fractions. Gene expression and location in nuclei, heavy membranes (lysosomes, peroxisomes, and mitochondria), light membranes (plasma membrane and endoplasmic reticulum), and soluble protein fractions are tested using Western blotting techniques.
Alternatively, 293T cells are transfected with an expression vector encoding individual genes, HIS-tagged (PCDNA 3.1 MYC/HIS, Invitrogen) and the subcellular localization of these genes is determined as described above. In short, the transfected cells are harvested and subjected to a differential subcellular fractionation protocol (Pemberton, P. A. et al., 1997, J of Histochemistry and Cytochemistry, 45:1697-1706). Location of the HIS-tagged genes is followed by Western blotting.
Using 158P1D7 antibodies, it is possible to demonstrate cellular localization by immunofluorescence and immunohistochemistry. For example, cells expressing or lacking 158P1D7 are adhered to a microscope slide and stained with anti-158P1D7 specific Ab. Cells are incubated with an FITC-coupled secondary anti-species Ab, and analyzed by fluorescent microscopy. Alternatively, cells and tissues lacking or expressing 158P1D7 are analyzed by IHC as described herein.
When 158P1D7 is localized to specific cell compartments, it is used as a target for diagnostic, preventative and therapeutic purposes.
Example 43
Involvement of 158P1D7 in Protein Trafficking
Due to its similarity to Slit proteins, 158P1D7 can regulate intracellular trafficking and retention into mitochondrial and/or nuclear compartments. Its role in the trafficking of proteins can be confirmed using well-established methods (Valetti C. et al. Mol Biol Cell. 1999, 10:4107). For example, FITC-conjugated α2-macroglobulin is incubated with 158P1D7-expressing and 158P1D7-negative cells. The location and uptake of FITC-α2-macroglobulin is visualized using a fluorescent microscope. In another approach, the co-localization of 158P1D7 with vesicular proteins is confirmed by co-precipitation and Western blotting techniques and fluorescent microscopy.
Alternatively, 158P1D7-expressing and 158P1D7-lacking cells are compared using bodipy-ceramide labeled bovine serum albumine (Huber L et al. Mol. Cell. Biol. 1995, 15:918). Briefly, cells are allowed to take up the labeled BSA and are placed intermittently at 4° C. and 18° C. to allow for trafficking to take place. Cells are examined under fluorescent microscopy, at different time points, for the presence of labeled BSA in specific vesicular compartments, including Golgi, endoplasmic reticulum, etc.
In another embodiment, the effect of 158P1D7 on membrane transport is examined using biotin-avidin complexes. Cells either expressing or lacking 158P1D7 are transiently incubated with biotin. The cells are placed at 4° C. or transiently warmed to 37° C. for various periods of time. The cells are fractionated and examined by avidin affinity precipitation for the presence of biotin in specific cellular compartments. Using such assay systems, proteins, antibodies and small molecules are identified that modify the effect of 158P1D7 on vesicular transport. When 158P1D7 plays a role in intracellular trafficking, 158P1D7 is a target for diagnostic, prognostic, preventative and therapeutic purposes.
Example 44
Protein-Protein Association
IGF and IGF-BP proteins have been shown to interact with other proteins, thereby forming protein complexes that can regulate protein localization, biological activity, gene transcription, and cell transformation (Zeslawski W et al., EMBO J. 2001, 20:3638; Yu H, Rohan T, J Natl Cancer Inst. 2000, 92:1472). Using immunoprecipitation techniques as well as two yeast hybrid systems, proteins are identified that associate with 158P1D7. Immunoprecipitates from cells expressing 158P1D7 and cells lacking 158P1D7 are compared for specific protein-protein associations.
Studies are performed to determine the extent of the association of 158P1D7 with receptors, such as the EGF and IGF receptors, and with intracellular proteins, such as IGF-BP, cytoskeletal proteins etc. Studies comparing 158P1D7 positive and 158P1D7 negative cells, as well as studies comparing unstimulated/resting cells and cells treated with epithelial cell activators, such as cytokines, growth factors and anti-integrin Ab reveal unique protein-protein 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 158P1D7-DNA-binding domain fusion protein and a reporter construct. Protein-protein interaction is detected by colorimetric reporter activity. Specific association with surface receptors and effector molecules directs one of skill to the mode of action of 158P1D7, 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 158P1D7.
When 158P1D7 associates with proteins or small molecules it is used as a target for diagnostic, prognostic, preventative and therapeutic purposes.
Example 45
Transcript Variants of 158P1D7
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., URL www.doubletwist.com/products/c11_agentsOverview.jhtml). 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 (URL compbio.oml.gov/Grail-bin/EmptyGrailForm) and GenScan (URL genes.mit.edu/GENSCAN.html). For a general discussion of splice variant identification protocols see., e.g., Southan, C., A genomic perspective on human proteases, FEBS Left. 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, EurJ Biochem. 1997 Oct. 1; 249(1):1-7. For PCR-based Validation: Wellmann S, et al., Specific reverse transcription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCycler technology, Clin Chem. 2001 April; 47(4):654-60; Jia, H. P., et al., Discovery of new human beta-defensins using a genomics-based approach, Gene. 2001 Jan. 24; 263(1-2):211-8. For PCR-based and 5′ RACE Validation: Brigle, K. E., et al., Organization of the murine reduced folate carrier gene and identification of variant splice forms, Biochem Biophys Acta. 1997 Aug. 7; 1353(2): 191-8).
It is known in the art that genomic regions are modulated in cancers. When the genomic region to which a gene maps is modulated in a particular cancer, the alternative transcripts or splice variants of the gene are modulated as well. Disclosed herein is that 158P1D7 has a particular expression profile related to cancer. Alternative transcripts and splice variants of 158P1D7 may also be involved in cancers in the same or different tissues, thus serving as tumor-associated markers/antigens.
Using the full-length gene and EST sequences, four transcript variants were identified, designated as 158P1D7 v.3, v.4, v.5 and v.6. The boundaries of the exon in the original transcript, 158P1D7 v.1 were shown in Table BILL-I. Compared with 158P1D7v.1, transcript variant 158P1D7v.3 has spliced out 2069-2395 from variant 158P1D7 v.1, as shown in FIG. 12. Variant 158P1D7 v.4 spliced out 1162-2096 of variant 158P1D7 v.1. Variant 158P1D7 v.5 added one exon to the 5′ and extended 2 bp to the 5′ end and 288 bp to the 3′ end of variant 158P1D7 v.1. Theoretically, each different combination of exons in spatial order, e.g. exon 1 of v.5 and exons 1 and 2 of v.3 or v.4, is a potential splice variant.
The variants of 158P1D7 include those that lack a transmembrane motif, but include a signal peptide indicating that they are secreted proteins (v.4 and v.6). Secreted proteins such as v.4 and v.6 serve as biomarkers of cancer existence and progression. The levels of such variant proteins in the serum of cancer patients serves as a prognostic marker of cancer disease or its progression, particularly of cancers such as those listed in Table I. Moreover, such secreted proteins are targets of monoclonal antibodies and related binding molecules. Accordingly, secreted proteins such as these serve as targets for diagnostics, prognostics, prophylactics and therapeutics for human malignancies. Targeting of secreted variants of 158P1D7 is particularly preferred when they have pathogy-related or cancer-related effects on cells/tissues.
Tables LI (a)-(d) through LIV(a)-(d) are set forth on a variant-by-variant bases. Tables LI(a)-(d) shows nucleotide sequence of the transcript variant. Tables L11(a)-(d) shows the alignment of the transcript variant with nucleic acid sequence of 158P1D7 v.1. Tables LIII (a)-(d) lays out amino acid translation of the transcript variant for the identified reading frame orientation. Tables LIV(a)-(d) displays alignments of the amino acid sequence encoded by the splice variant with that of 158P1D7 v.1.
Example 46
Single Nucleotide Polymorphisms of 158P1D7
A Single Nucleotide Polymorphism (SNP) is a single base pair variation in a nucleotide sequence at a specific location. At any given point of the genome, there are four possible nucleotide base pairs: A/T, C/G, G/C and T/A. Genotype refers to the specific base pair sequence of one or more locations in the genome of an individual. Haplotype refers to the base pair sequence of more than one location on the same DNA molecule (or the same chromosome in higher organisms), often in the context of one gene or in the context of several tightly linked genes. SNP that occurs on a cDNA is called cSNP. This cSNP may change amino acids of the protein encoded by the gene and thus change the functions of the protein. Some SNP cause inherited diseases; others contribute to quantitative variations in phenotype and reactions to environmental factors including diet and drugs among individuals. Therefore, SNP and/or combinations of alleles (called haplotypes) have many applications, including diagnosis of inherited diseases, determination of drug reactions and dosage, identification of genes responsible for diseases, and analysis of the genetic relationship between individuals (P. Nowotny, J. M. Kwon and A. M. Goate, “SNP analysis to dissect human traits,” Curr. Opin. Neurobiol. 2001 October; 11(5):637-641; M. Pirmohamed and B. K. Park, “Genetic susceptibility to adverse drug reactions,” Trends Pharmacol. Sci. 2001 June; 22 (6):298-305Allan, E. Lai and A. Roses, “The use of single nucleotide polymorphisms in the isolation of common disease genes,” Pharmacogenomics. 2000 February; 1(1):3947; R. Judson, J. C. Stephens and A. Windemuth, “The predictive power of haplotypes in clinical response,” Pharmacogenomics. 2000 February; 1(1):15-26).
SNP are identified by a variety of art-accepted methods (P. Bean, “The promising voyage of SNP target discovery,” Am. Clin. Lab. 2001 October-November; 20(9):18-20; K. M. Weiss, “In search of human variation,” Genome Res. 1998 July; 8(7):691-697; M. M. She, “Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies,” Clin. Chem. 2001 February; 47(2):164-172). For example, SNP can be identified by sequencing DNA fragments that show polymorphism by gel-based methods such as restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). They can also be discovered by direct sequencing of DNA samples pooled from different individuals or by comparing sequences from different DNA samples. With the rapid accumulation of sequence data in public and private databases, one can discover SNP by comparing sequences using computer programs (Z. Gu, L. Hillier and P. Y. Kwok, “Single nucleotide polymorphism hunting in cyberspace,” Hum. Mutat. 1998; 12(4):221-225). SNP can be verified and genotype or haplotype of an individual can be determined by a variety of methods including direct sequencing and high throughput microarrays (P. Y. Kwok, “Methods for genotyping single nucleotide polymorphisms,” Annu. Rev. Genomics Hum. Genet. 2001; 2:235-258; M. Kokoris, K. Dix, K. Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft, “High-throughput SNP genotyping with the Masscode system,” Mol. Diagn. 2000 December; 5(4):329-340).
Using the methods described above, one SNP was identified in the original transcript, 158P1D7 v.1, at positions 1546 (A/G). The transcripts or proteins with alternative allele was designated as variant 158P1D7 v.2. FIG. 17 shows the schematic alignment of the SNP variants. FIG. 18 shows the schematic alignment of protein variants, corresponding to nucleotide variants. Nucleotide variants that code for the same amino acid sequence as v.1 are not shown in FIG. 18. These alleles of the SNP, though shown separately here, can occur in different combinations (haplotypes) and in any one of the transcript variants (such as 158P1D7 v.5) that contains the site of the SNP.
Example 47
Therapeutic and Diagnostic Use of Anti-158P1D7 Antibodies in Humans
Anti-158P1D7 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-158P1D7 mAb show strong extensive staining in carcinoma but significantly lower or undetectable levels in normal tissues. Detection of 158P1D7 in carcinoma and in metastatic disease demonstrates the usefulness of the mAb as a diagnostic and/or prognostic indicator. Anti-158P1D7 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-158P1D7 mAb specifically binds to carcinoma cells. Thus, anti-158P1D7 antibodies are used in diagnostic whole body imaging applications, such as radioimmunoscintigraphy and radioimmunotherapy, (see, e.g., Potamianos S., et. al. Anticancer Res 20(2A):925-948 (2000)) for the detection of localized and metastatic cancers that exhibit expression of 158P1D7. Shedding or release of an extracellular domain of 158P1D7 into the extracellular milieu, such as that seen for alkaline phosphodiesterase B10 (Meerson, N. R., Hepatology 27:563-568 (1998)), allows diagnostic detection of 158P1D7 by anti-158P1D7 antibodies in serum and/or urine samples from suspect patients.
Anti-158P1D7 antibodies that specifically bind 158P1D7 are used in therapeutic applications for the treatment of cancers that express 158P1D7. Anti-158P1D7 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-158P1D7 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 “158P1D7 Monoclonal Antibody-mediated Inhibition of Bladder and Lung Tumors In Vivo”). Either conjugated and unconjugated anti-158P1D7 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 48
Human Clinical Trials for the Treatment and Diagnosis of Human Carcinomas Through Use of Human Anti-158P1D7 Antibodies In Vivo
Antibodies are used in accordance with the present invention which recognize an epitope on 158P1D7, and are used in the treatment of certain tumors such as those listed in Table I. Based upon a number of factors, including 158P1D7 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-158P1D7 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-158P1D7 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-158P1D7 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-158P1D7 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-158P1D7 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 158P1D7. In connection with the use of the anti-158P1D7 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)-158P1D7 antibody is used as an imaging agent in a Phase I human clinical trial in patients having a carcinoma that expresses 158P1D7 (by analogy see, e.g., Divgi etal. 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-158P1D7 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-158P1D7 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-158P1D7 antibodies that are fully human antibodies, as compared to the chimeric antibody, have slower clearance; accordingly, dosing in patients with such fully human anti-158P1D7 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-158P1D7 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-158P1D7 antibodies in connection with adjunctive therapy, monotherapy, and as an imaging agent. Trials initially demonstrate safety and thereafter confirm efficacy in repeat doses. Trails are open label comparing standard chemotherapy with standard therapy plus anti-158P1D7 antibodies. As will be appreciated, one criteria that can be utilized in connection with enrollment of patients is 158P1D7 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 158P1D7. Standard tests and follow-up are utilized to monitor each of these safety concerns. Anti-158P1D7 antibodies are found to be safe upon human administration.
Example 49
Human Clinical Trial Adjunctive Therapy with Human Anti-158P1D7 Antibody and Chemotherapeutic Agent
A phase I human clinical trial is initiated to assess the safety of six intravenous doses of a human anti-158P1D7 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-158P1D7 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-158P1D7 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 158P1D7. 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-158P1D7 antibodies are demonstrated to be safe and efficacious, Phase II trials confirm the efficacy and refine optimum dosing.
Example 50
Human Clinical Trial: Monotherapy with Human Anti-158P1D7 Antibody
Anti-158P1D7 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-158P1D7 antibodies.
Example 51
Human Clinical Trial: Diagnostic Imaging with Anti-158P1D7 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-158P1D7 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 52
RNA Interfernece (RNAi)
RNA interference (RNAi) technology is implemented to a variety of cell assays relevant to oncology. RNAi is a post-transcriptional gene silencing mechanism activated by double-stranded RNA (dsRNA). RNAi induces specific mRNA degradation leading to changes in protein expression and subsequently in gene function. In mammalian cells, these dsRNAs called short interfering RNA (siRNA) have the correct composition to activate the RNAi pathway targeting for degradation, specifically some mRNAs. See, Elbashir S. M., et. al., Duplexes of 21-nucleotide RNAs Mediate RNA interference in Cultured Mammalian Cells, Nature 411(6836):494-8 (2001). Thus, RNAi technology is used successfully in mammalian cells to silence targeted genes.
Loss of cell proliferation control is a hallmark of cancerous cells; thus, assessing the role of 158P1D7 in cell survival/proliferation assays is relevant. Accordingly, RNAi was used to investigate the function of the 158P1D7 antigen. To generate siRNA for 158P1D7, algorithms were used that predict oligonucleotides that exhibit the critical molecular parameters (G:C content, melting temperature, etc.) and have the ability to significantly reduce the expression levels of the 158P1D7 protein when introduced into cells. Accordingly, one targeted sequence for the 158P1D7 siRNA is: 5′ AAGCTCATTCTAGCGGGAAAT 3′ (SEQ ID NO: 42)(oligo 158P1D7.b). In accordance with this Example, 158P1D7 siRNA compositions are used that comprise siRNA (double stranded, short interfering RNA) that correspond to the nucleic acid ORF sequence of the 158P1D7 protein or subsequences thereof. Thus, siRNA subsequences are used in this manner are generally 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more than 35 contiguous RNA nucleotides in length. These siRNA sequences are complementary and non-complementary to at least a portion of the mRNA coding sequence. In a preferred embodiment, the subsequences are 19-25 nucleotides in length, most preferably 21-23 nucleotides in length. In preferred embodiments, these siRNA achieve knockdown of 158P1D7 antigen in cells expressing the protein and have functional effects as described below.
The selected siRNA (158P1D7.b oligo) was tested in numerous cell lines in the survival/proliferation MTS assay (measures cellular metabolic activity). Tetrazolium-based colorimetric assays (i.e., MTS) detect viable cells exclusively, since living cells are metabolically active and therefore can reduce tetrazolium salts to colored formazan compounds; dead cells, however do not. Moreover, this 158P1D7.b oligo achieved knockdown of 158P1D7 antigen in cells expressing the protein and had functional effects as described below using the following protocols.
Mammalian siRNA transfections: The day before siRNA transfection, the different cell lines were plated in media (RPMI 1640 with 10% FBS w/o antibiotics) at 2×103 cells/well in 80 μl (96 well plate format) for the survival/MTS assay. In parallel with the 158P1D7 specific siRNA oligo, the following sequences were included in every experiment as controls: a) Mock transfected cells with Lipofectamine 2000 (Invitrogen, Carisbad, Calif.) and annealing buffer (no siRNA); b) Luciferase-4 specific siRNA (targeted sequence: 5′-AAGGGACGAAGACGAACACUUCTT-3′) (SEQ ID NO: 43); and, c) Eg5 specific siRNA (targeted sequence: 5′-AACTGAAGACCTGAAGACAATAA-3′) (SEQ ID NO: 44). SiRNAs were used at 10 nM and 1 μg/ml Lipofectamine 2000 final concentration.
The procedure was as follows: The siRNAs were first diluted in OPTIMEM (serum-free transfection media, Invitrogen) at 0.1 uM μM (10-fold concentrated) and incubated 5-10 min RT. Lipofectamine 2000 was diluted at 10 μg/ml (10-fold concentrated) for the total number transfections and incubated 5-10 minutes at room temperature (RT). Appropriate amounts of diluted 10-fold concentrated Lipofectamine 2000 were mixed 1:1 with diluted 10-fold concentrated siRNA and incubated at RT for 20-30′ (5-fold concentrated transfection solution). 20 μls of the 5-fold concentrated transfection solutions were added to the respective samples and incubated at 37° C. for 96 hours before analysis.
MTS assay: The MTS assay is a colon metric method for determining the number of viable cells in proliferation, cytotoxicity or chemosensitivity assays based on a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS(b)] and an electron coupling reagent (phenazine ethosulfate; PES). Assays were performed by adding a small amount of the Solution Reagent directly to culture wells, incubating for 14 hours and then recording absorbance at 490 nm with a 96-well plate reader. The quantity of colored formazan product as measured by the amount of 490 nm absorbance is directly proportional to the mitochondrial activity and/or the number of living cells in culture.
In order to address the function of 158P1D7 in cells, 158P1D7 was silenced by transfecting the endogenously expressing 158P1D7 cell lines (LNCaP and PC3) with the 158P1D7 specific siRNA (158P1D7.b) along with negative siRNA controls (Luc4, targeted sequence not represented in the human genome) and a positive siRNA control (targeting Eg5) (FIG. 29). The results indicated that when these cells are treated with siRNA specifically targeting the 158P1D7 mRNA, the resulting “158P1D7 deficient cells” showed diminished cell viability or proliferation as measured by this assay (see oligo 158P1D7.b treated cells). This effect is likely caused by an active induction of apoptosis. The reduced viability is measured by the increased release (and activity) of a mitochondrial enzyme that occurs predominantly in apoptotic cells.
As control, 3T3 cells, a cell line with no detectable expression of 158P1D7 mRNA, was also treated with the panel of siRNAs (including oligo 158P1D7.b) and no phenotype was observed. This result reflects the fact that the specific protein knockdown in the LNCaP and PC3 cells is not a function of general toxicity, since the 3T3 cells did not respond to the 158P1D7.b oligo. The differential response of the three cell lines to the Eg5 control is a reflection of differences in levels of cell transfection and responsiveness of the cell lines to oligo treatment (FIG. 29).
Together, these data indicate that 158P1D7 plays an important role in the proliferation of cancer cells and that the lack of 158P1D7 clearly decreases the survival potential of these cells. It is to be noted that 158P1D7 is constitutively expressed in many tumor cell lines. 158P1D7 serves a role in malignancy; it expression is a primary indicator of disease, where such disease is often characterized by high rates of uncontrolled cell proliferation and diminished apoptosis. Correlating cellular phenotype with gene knockdown following RNAi treatments is important, and allows one to draw valid conclusions and rule out toxicity or other non-specific effects of these reagents. To this end, assays to measure the levels of expression of both protein and mRNA for the target after RNAi treatments are important, including Western blotting, FACS staining with antibody, immunoprecipitation, Northern blotting or RT-PCR (Taqman or standard methods). Any phenotypic effect of the siRNAs in these assays should be correlated with the protein and/or mRNA knockdown levels in the same cell lines. Knockdown of 158P1D7 is achieved using the 158P1D7.b oligo as measured by Western blotting and RT-PCR analysis.
A method to analyze 158P1D7 related cell proliferation is the measurement of DNA synthesis as a marker for proliferation. Labeled DNA precursors (i.e. 3H-Thymidine) are used and their incorporation to DNA is quantified. Incorporation of the labeled precursor into DNA is directly proportional to the amount of cell division occurring in the culture. Another method used to measure cell proliferation is performing clonogenic assays. In these assays, a defined number of cells are plated onto the appropriate matrix and the number of colonies formed after a period of growth following siRNA treatment is counted.
In 158P1D7 cancer target validation, complementing the cell survival/proliferation analysis with apoptosis and cell cycle profiling studies are considered. The biochemical hallmark of the apoptotic process is genomic DNA fragmentation, an irreversible event that commits the cell to die. A method to observe fragmented DNA in cells is the immunological detection of histone-complexed DNA fragments by an immunoassay (i.e. cell death detection ELISA) which measures the enrichment of histone-complexed DNA fragments (mono- and oligo-nucleosomes) in the cytoplasm of apoptotic cells. This assay does not require pre-labeling of the cells and can detect DNA degradation in cells that do not proliferate in vitro (i.e. freshly isolated tumor cells).
The most important effector molecules for triggering apoptotic cell death are caspases. Caspases are proteases that when activated cleave numerous substrates at the carboxy-terminal site of an aspartate residue mediating very early stages of apoptosis upon activation. All caspases are synthesized as pro-enzymes and activation involves cleavage at aspartate residues. In particular, caspase 3 seems to play a central role in the initiation of cellular events of apoptosis. Assays for determination of caspase 3 activation detect early events of apoptosis. Following RNAi treatments, Western blot detection of active caspase 3 presence or proteolytic cleavage of products (i.e. PARP) found in apoptotic cells further support an active induction of apoptosis. Because the cellular mechanisms that result in apoptosis are complex, each has its advantages and limitations. Consideration of other criteria/endpoints such as cellular morphology, chromatin condensation, membrane blebbing, apoptotic bodies help to further support cell death as apoptotic. Since not all the gene targets that regulate cell growth are anti-apoptotic, the DNA content of permeabilized cells is measured to obtain the profile of DNA content or cell cycle profile. Nuclei of apoptotic cells contain less DNA due to the leaking out to the cytoplasm (sub-G1 population). In addition, the use of DNA stains (i.e., propidium iodide) also differentiate between the different phases of the cell cycle in the cell population due to the presence of different quantities of DNA in G0/G1, S and G2/M. In these studies the subpopulations can be quantified.
For the 158P1D7 gene, RNAi studies facilitate the understanding of the contribution of the gene product in cancer pathways. Such active RNAi molecules have use in identifying assays to screen for mAbs that are active anti-tumor therapeutics. Further, siRNA are administered as therapeutics to cancer patients for reducing the malignant growth of several cancer types, including those listed in Table 1. When 158P1D7 plays a role in cell survival, cell proliferation, tumorigenesis, or apoptosis, it is used as a target for diagnostic, prognostic, preventative and/or therapeutic purposes.
Example 53
158P1D7 Functional Assays
I. Enhanced Proliferation and Cell Cycle Modulation in 158P1D7 Expressing Cells.
Enhanced proliferation and entry into S-phase of tumor cells relative to normal cells is a hallmark of the cancer cell phenotype. To address the effect of expression of 158P1D7 on the proliferation rate of normal cells, two rodent cell lines (3T3 and Rat-1) were infected with virus containing the 158P1D7 gene and stable cells expressing 158P1D7 antigen were derived, as well as empty vector control cells expressing the selection marker neomycin (Neo). The cells were grown overnight in 0.5% FBS and then compared to cells treated with 10% FBS. The cells were evaluated for proliferation at 18-96 hr post-treatment by a 3H-thymidine incorporation assay and for cell cycle analysis by a BrdU incorporation/propidium iodide staining assay. The results in FIG. 32 show that the Rat-1 cells expressing the 158P1D7 antigen grew effectively in low serum concentrations (0.1%) compared to the Rat-1-Neo cells. Similar results were obtained for the 3T3 cells expressing 158P1D7 versus Neo only. To assess cell proliferation by another methodology, the cells were stained with BrdU and propidium iodide. Briefly, cells were labeled with 100M BrdU, washed, trypsinized and fixed in 0.4% paraformaldehyde and 70% ethanol. Anti-BrdU-FITC (Pharmigen) was added to the cells, the cells were washed and then incubated with 10 □g/ml propidium iodide for 20 min prior to washing and analysis for fluorescence at 488 nm. The results in FIG. 33 show that there was increased labeling of cells in S-phase (DNA synthesis phase of the cell cycle) in 3T3 cells that expressed the 158P1D7 antigen relative to control cells. These results confirm those measured by 3H-thymidine incorporation, and indicate that cells that express 158P1D7 antigen have an enhanced proliferative capacity and survive in low serum conditions. Accordingly, 158P1D7 expressing cells have increased potential for growth as tumor cells in vivo.
II. Recombinant Extracellular Domain (ECD) Binding to Cell Surface.
Cell-cell interactions are essential in maintaining tissue/organ integrity and homeostasis, both of which become deregulated during tumor formation and progression. Additionally, cell-cell interactions facilitate tumor cell attachment during metastasis and activation of endothelium for increased angiogenesis. To address interaction between the gene product of 158P1D7 and a putative ligand, an assay was established to identify the interaction between the extracellular domain (ECD) (amino acids 16-608) of 158P1D7 antigen and primary endothelium. Human umbilical vein endothelial cells (HUVEC) were grown in 0.1% FBS in media for 3 hr. Cells were washed, detached in 10 mM EDTA and resuspended in 10% FBS. Recombinant 158P1D7 ECD (described in Example entitled “Production of Recombinant 158P1D7 in Eukaryotic Systems”) was added to cells, and the cells were washed prior to the addition of MAb M15/X68.2.22 at 1 ug/ml. After washing, secondary Ab (anti-mouse-PE, 1:400) was added to cells for 1 hr on ice. Cells were washed and fixed in 1% formalin for 3 hr on ice, then resuspended in PBS and analyzed by flow cytometry. FIG. 26A shows that the 158P1D7 ECD bound directly to the surface of HUVEC cells as detected by the 158P1D7 specific MAb. In a similar embodiment, recombinant ECD of 158P1D7 was iodinated to high specific activity using the iodogen (1,3,4,5-tetrachloro-3a,6a-diphenylglycoluril) method. HUVEC cells at 90% confluency in 6 well plates were incubated with 1 nM of 125I-158P1D7 ECD in the presence (non-specific binding) or absence (Total binding) of 50 fold excess unlabeled ECD for 2 hours at either 4° C. or 37° C. Cells were washed, solubilized in 0.5M NaOH, and subjected to gamma counting. The data in FIG. 26B shows specific binding of 158P1D7 ECD to HUVEC cells suggesting the presence of a 158P1D7 receptor on HUVEC cells. These results indicate that 158P1D7 antigen is involved in cell-cell interactions that facilitate tumor growth, activation of endothelium for tumor vascularization or tumor cell metastasis. The data also indicate that 158P1D7 antigen shed from the cell surface of expressing cells may bind to cells in an autocrine or paracrine fashion to induce cell effector functions.
Example 54
Detection of 158P1D7 Protein in Cancer Patient Specimens Using Immunohistochemistry
To determine the expression of 158P1D7 protein, specimens were obtained from various cancer patients and stained using an affinity purified monoclonal antibody raised against the peptide encoding amino acids 274-285 of 158P1D7 (See the Example Entitled “Generation of 158P1D7 Monoclonal Antibodies (mAbs)”), formalin fixed, paraffin embedded tissues were cut into 4 micron sections and mounted on glass slides. The sections were dewaxed, rehydrated and treated with antigen retrieval solution (Antigen Retrieval Citra Solution; BioGenex, 4600 Norris Canyon Road, San Ramon, Calif., 94583) at high temperature. Sections were then incubated in mouse monoclonal anti-158P1D7 antibody, M15-68(2)22, for 3 hours. The slides were washed three times in buffer and further incubated with DAKO EnVision+TM peroxidase-conjugated goat anti-mouse immunoglobulin secondary antibody (DAKO Corporation, Carpenteria, Calif.) for 1 hour. The sections were then washed in buffer, developed using the DAB kit (SIGMA Chemicals), counterstained using hematoxylin, and analyzed by bright field microscopy. The results showed expression of 158P1D7 in cancer patients' tissue (FIG. 36). Generally, in bladder transitional cell carcinoma expression of 158P1D7 was mainly around the cell membrane indicating that 158P1D7 is membrane associated in these tissues. 49.3% of bladder transitional cell carcinoma samples tested were positive for 158P1D7 (Table LVIII).
These results indicate that 158P1D7 is a target for diagnostic, prophylactic, prognostic and therapeutic applications in cancer.
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.
All documents and publications recited herein are hereby incorporated in their entirety as if fully set forth.







TABLE I





Tissues that Express 158P1D7 When Malignant

















Bladder, Prostate, Colon, Lung, Breast, Ovary, Skin, Cervix

















TABLE II







AMINO ACID ABBREVIATIONS











SINGLE LETTER
THREE LETTER
FULL NAME







F
Phe
phenylalanine



L
Leu
leucine



S
Ser
serine



Y
Tyr
tyrosine



C
Cys
cysteine



W
Trp
tryptophan



P
Pro
proline



H
His
histidine



Q
Gln
glutamine



R
Arg
arginine



I
Ile
isoleucine



M
Met
methionine



T
Thr
threonine



N
Asn
asparagine



K
Lys
lysine



V
Val
valine



A
Ala
alanine



D
Asp
aspartic acid



E
Glu
glutamic acid



G
Gly
glycine

















TABLE III







AMINO ACID SUBSTITUTION MATRIX


Adapted from the GCG Software 9.0 BLOSUM62 amino acid substitution matrix


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


found in related, natural proteins.


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



























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






























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



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




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





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






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







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








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









4
−3
2
1
−3
−3
−3
−3
−2
−1
3
−3
−1
I










5
−2
−1
0
−1
1
2
0
−1
−2
−3
−2
K











4
2
−3
−3
−2
−2
−2
−1
1
−2
−1
L












5
−2
−2
0
−1
−1
−1
1
−1
−1
M













6
−2
0
0
1
0
−3
−4
−2
N














7
−1
−2
−1
−1
−2
−4
−3
P















5
1
0
−1
−2
−2
−1
Q
















5
−1
−1
−3
−3
−2
R

















4
1
−2
−3
−2
S


















5
0
−2
−2
T



















4
−3
−1
V




















11
2
W





















7
Y
















TABLE IV







HLA Class I Supermotifs/Motifs











POSITION
POSITION
POSITION



2 (Primary
3 (Primary
C Terminus



Anchor)
Anchor)
(Primary Anchor)














SUPERMOTIFS





A1
TILVMS

FWY


A2
LIVMATQ

IVMATL


A3
VSMATLI

RK


A24
YFWIVLMT

FIYWLM


B7
P

VILFMWYA


B27
RHK

FYLWMIVA


B44
ED

FWYLIMVA


B58
ATS

FWYLIVMA


B62
QLIVMP

FWYMIVLA


MOTIFS


A1
TSM

Y


A1

DEAS
Y


A2.1
LMVQIAT

VLIMAT


A3
LMVISATFCGD

KYRHFA


A11
VTMLISAGNCDF

KRYH


A24
YFWM

FLIW


A*3101
MVTALIS

RK


A*3301
MVALFIST

RK


A*6801
AVTMSLI

RK


B*0702
P

LMFWYAIV


B*3501
P

LMFWYIVA


B51
P

LIVFWYAM


B*5301
P

IMFWYALV


B*5401
P

ATIVLMFWY





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













TABLE IV (B)







HLA CLASS II SUPERMOTIF









1
6
9





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
















TABLE IV (C)







HLA Class II Motifs

















MOTIFS

1° anchor 1
2
3
4
5
1° anchor 6
7
8
9





DR4
preferred
FMYLIVW
M
T

I
VSTCPALIM
MH

MH



deleterious



W


R

WDE


DR1
preferred
MFLIVWY


PAMQ

VMATSPLIC
M

AVM



deleterious

C
CH
FD
CWD

GDE
D


DR7
preferred
MFLIVWY
M
W
A

IVMSACTPL
M

IV



deleterious

C

G


GRD
N
G


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


Motif a preferred

LIVMFY


D


Motif b preferred

LIVMFAY


DNQEST

KRH


DR Supermotif

MFLIVWY




VMSTACPLI





Italicized residues indicate less preferred or “tolerated” residues













TABLE IV (D)







HLA Class I Supermotifs










SUPER-

POSITION:


















MOTIFS

1
2
3
4
5
6
7
8
C-terminus





A1


1° Anchor






1° Anchor





TILVMS






FWY


A2


1° Anchor






1° Anchor





 LIVMATQ






LIVMAT


A3
Preferred

1° Anchor
YFW


YFW
YFW
P
1° Anchor





VSMATLI
(4/5)


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



deleterious
DE (3/5);

DE




P (5/5)

(4/5)


A24


1° Anchor






1° Anchor





 YFWIVLMT






FIYWLM


B7
Preferred
FWY (5/5)
1° Anchor
FWY




FWY
1° Anchor




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




(3/5)
 VILFMWYA



deleterious
DE (3/5);



DE
G
QN
DE




P(5/5);



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




G(4/5);




A(3/5);




QN(3/5)


B27


1° Anchor






1° Anchor





RHK






 FYLWMIVA


B44


1° Anchor






1° Anchor





ED






 FWYLIMVA


B58


1° Anchor






1° Anchor





ATS






 FWYLIVMA


B62


1° Anchor






1° Anchor





QLIVMP






 FWYMIVLA





Italicized residues indicate less preferred or “tolerated” residues













TABLE IV (E)





HLA Class I Motifs


















POSITION





























9 or C-
C-




1
2
3
4
5
6
7
8
terminus
terminus





A1
preferred
GFYW
1° Anchor
DEA
YFW

P
DEQN
YFW
1° Anchor


9-mer


STM






Y



deleterious
DE

RHKLIVMP
A
G
A


A1
preferred
GRHK
ASTCLIVM
1° Anchor
GSTC

ASTC
LIVM
DE
1° Anchor


9-mer



DEAS





Y



deleterious
A
RHKDEPYFW

DE
PQN
RHK
PG
GP


A1
preferred
YFW
1° Anchor
DEAQN
A
YFWQN

PASTC
GDE
P
1° Anchor


10-mer


STM







Y



deleterious
GP

RHKGLIVM
DE
RHK
QNA
RHKYFW
RHK
A


A1
preferred
YFW
STCLIVM
1° Anchor
A
YFW

PG
G
YFW
1° Anchor


10-mer



DEAS






Y



deleterious
RHK
RHKDEPYFW


P
G

PRHK
QN


A2.1
preferred
YFW
1° Anchor
YFW
STC
YFW

A
P
1° Anchor


9-mer


LMIVQAT






VLIMAT



deleterious
DEP

DERKH


RKH
DERKH













POSITION:
C-




















1
2
3
4
5
6
7
8
9
terminus





A2.1
pre-
AYFW
1° Anchor
LVIM
G

G

FYWLVIM

1° Anchor


10-mer
ferred

LMIVQAT







VLIMAT



dele-
DEP

DE
RKHA
P

RKH
DERKH
RKH



te-



rious


A3
pre-
RHK
1° Anchor
YFW
PRHKYFW
A
YFW

P
1° Anchor



ferred

 LMVISATFCGD






KYRHFA



dele-
DEP

DE



te-



rious


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



ferred

 VTLMISAGNCD






KRYH



dele-
DEP
F




A
G



te-



rious


A24
pre-
YFWRHK
1° Anchor

STC


YFW
YFW
1° Anchor


9-mer
ferred

YFWM






FLIW



dele-
DEG

DE
G
QNP
DERHK
G
AQN



te-



rious


A24
Pre-

1° Anchor

P
YFWP

P


1° Anchor


10-mer
ferred

YFWM







FLIW



Dele-


GDE
QN
RHK
DE
A
QN
DEA



te-



rious


A3101
Pre-
RHK
1° Anchor
YFW
P

YFW
YFW
AP
1° Anchor



ferred

MVTALIS






RK



Dele-
DEP

DE

ADE
DE
DE
DE



te-



rious


A3301
Pre-

1° Anchor
YFW



AYFW

1° Anchor



ferred

 MVALFIST






RK



Dele-
GP

DE



te-



rious


A6801
Pre-
YFWSTC
1° Anchor


YFWLIV

YFW
P
1° Anchor




ferred

AVTMSLI


M



RK



dele-
GP

DEG

RHK


A



te-



rious


B0702
Pre-
RHKFWY
1° Anchor
RHK

RHK
RHK
RHK
PA
1° Anchor



ferred

P






 LMFWYAIV



dele-
DEQNP

DEP
DE
DE
GDE
QN
DE



te-



rious


B3501
Pre-
FWYLIVM
1° Anchor
FWY



FWY

1° Anchor



ferred

P






 LMFWYIVA













POSITION:
C-




























9 or C-
termi-




1
2
3
4
5
6
7
8
terminus
nus





A1
pre-
GFYW
1° Anchor
DEA
YFW

P
DEQN
YFW
1° Anchor


9-mer
ferred

STM






Y



dele-
DE

RHKLIVMP
A
G
A



te-



rious


A1
pre-
GRHK
ASTCLIVM
1° Anchor
GSTC

ASTC
LIVM
DE
1° Anchor


9-mer
ferred


DEAS





Y



dele-
A
RHKDEPYFW

DE
PQN
RHK
PG
GP



te-





rious



dele-
AGP



G
G


A



te-



rious


B51
Pre-
LIVMFWY
1° Anchor
FWY
STC
FWY

G
FWY
1° Anchor



ferred

P






 LIVFWYAM



dele-
AGPDER



DE
G
DEQN
GDE



te-
HKSTC



rious


B5301
pre-
LIVMFWY
1° Anchor
FWY
STC
FWY

LIVMFWY
FWY
1° Anchor



ferred

P






 IMFWYALV



dele-
AGPQN




G
RHKQN
DE



te-



rious


B5401
pre-
FWY
1° Anchor
FWYLIVM

LIVM

ALIVM
FWYAP
1° Anchor



ferred

P






 ATIVLMFWY



dele-
GPQNDE

GDESTC

RHKDE
DE
QNDGE
DE



te-



rious





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













TABLE IV (F)







Summary of HLA-supertypes


Overall phenotypic frequencies of HLA-supertypes in different ethnic populations








Specificity
Phenotypic frequency















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


















B7
P
AILMVFWY
43.2
55.1
57.1
43.0
49.3
49.5


A3
AILMVST
RK
37.5
42.1
45.8
52.7
43.1
44.2


A2
AILMVT
AILMVT
45.8
39.0
42.4
45.9
43.0
42.2


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


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


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


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


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


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
















TABLE IV (G)







Calculated population coverage afforded by different HLA-supertype combinations









Phenotypic frequency













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
















A2, A3 and B7
83.0
86.1
87.5
88.4
86.3
86.2


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


and A1
99.9
99.6
100.0
99.8
99.9
99.8


A2, A3, B7, A24,


B44, A1, 827, B62,


and B 58





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














TABLE V







V1-HLA-A1-9mers-



158P1D7


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
VIEPSAFSK
900.000



 


436
NLEYLYLEY
225.000


 


812
LVEQTKNEY
45.000


 


828
HAEPDYLEV
45.000


 


711
GSDAKHLQR
37.500


 


546
CTSPGHLDK
25.000


 


265
SICPTPPVY
10.000


 


351
NIESLSDLR
9.000


 


799
LMETLMYSR
9.000


 


173
ESLPPNIFR
7.500


 


650
DNSPVHLQY
6.250


 


601
LTDAVPLSV
6.250


 


174
SLPPNIFRF
5.000


 


100
IADIEIGAF
5.000


 


682
MVSPMVHVY
5.000


 


102
DIEIGAFNG
4.500


 


134
GLENLEFLQ
4.500


 


47
NCEAKGIKM
4.500


 


383
LVEYFTLEM
4.500


 


401
VLEEGSFMN
4.500


 


388
TLEMLHLGN
4.500


 


749
FQDASSLYR
3.750


 


56
VSEISVPPS
2.700


 


561
NSEILCPGL
2.700


 


431
FLGLHNLEY
2.500


 


291
INDSRMSTK
2.500


 


142
QADNNFITV
2.500


 


502
ILDDLDLLT
2.500


 


522
SCDLVGLQQ
2.500


 


223
NCDLLQLKT
2.500


 


771
ITEYLRKNI
2.250


 


232
WLENMPPQS
1.800


 


171
AIESLPPNI
1.800


 


137
NLEFLQADN
1.800


 


355
LSDLRPPPQ
1.500


 


380
KSDLVEYFT
1.500


 


59
ISVPPSRPF
1.500


 


255
GSILSRLKK
1.500


 


540
VTDDILCTS
1.250


 


308
TKAPGLIPY
1.250


 


817
KNEYFELKA
1.125


 


743
STEFLSFQD
1.125


 


359
RPPPQNPRK
1.000


 


246
VCNSPPFFK
1.000


 


417
YLNGNHLTK
1.000


 


433
GLHNLEYLY
1.000


 


785
DMEAHYPGA
0.900


 


398
RIEVLEEGS
0.900


 


701
EEEEERNEK
0.900


 


833
YLEVLEQQT
0.900


 


513
DLEDNPWDC
0.900


 


123
SLEILKEDT
0.900


 


203
FLEHIGRIL
0.900


 


36
NCEEKDGTM
0.900


 


699
HLEEEEERN
0.900


 


214
QLEDNKWAC
0.900


 


573
PSMPTQTSY
0.750


 


81
TNDFSGLTN
0.625


 


192
GNQLQTLPY
0.625


 


301
TSILKLPTK
0.600


 


631
LVLHRRRRY
0.500


 


643
QVDEQMRDN
0.500


 


610
LILGLLIMF
0.500


 


407
FMNLTRLQK
0.500


 


89
NAISIHLGF
0.500


 


187
HLDLRGNQL
0.500


 


511
QIDLEDNPW
0.500


 


627
GIVVLVLHR
0.500


 


472
QVLPPHIFS
0.500


 


593
TADTILRSL
0.500


 


337
VLSPSGLLI
0.500


 


210
ILDLQLEDN
0.500


 


615
LIMFITIVF
0.500


 


473
VLPPHIFSG
0.500


 


730
LTGSNMKYK
0.500


 


447
IKEILPGTF
0.450


 


669
TTERPSASL
0.450


 


441
YLEYNAIKE
0.450


 


802
TLMYSRPRK
0.400


 


683
VSPMVHVYR
0.300


 


547
TSPGHLDKK
0.300


 


32
DSLCNCEEK
0.300


 


723
EQENHSPLT
0.270


 


276
HEDPSGSLH
0.250


 


769
LGITEYLRK
0.250


 


76
LTMLHTNDF
0.250


 


235
NMPPQSIIG
0.250


 


196
QTLPYVGFL
0.250


 


738
KTTNQSTEF
0.250


 


372
GNIIHSLMK
0.250


 


287
ATSSINDSR
0.250


 


551
HLDKKELKA
0.250


 


825
ANLHAEPDY
0.250


 


148
ITVIEPSAF
0.250


 


729
PLTGSNMKY
0.250


 


584
VTTPATTTN
0.250


 


664
KTTHHTTER
0.250


 


526
VGLQQWIQK
0.250


 


801
ETLMYSRPR
0.250


 


297
STKTTSILK
0.250

















TABLE V







V3-HLA-A1-9mers-



158P1D7


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













7
HMGAHEELK
0.100



 


2
SLYEQHMGA
0.050


 


3
LYEQHMGAH
0.045


 


1
ASLYEQHMG
0.015


 


8
MGAHEELKL
0.013


 


6
QHMGAHEEL
0.001


 


5
EQHMGAHEE
0.000


 


4
YEQHMGAHE
0.000

















TABLE V







V4-HLA-A1-9mers-



158P1D7


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
HSLMKSILW
0.075



 


8
SILWSKASG
0.020


 


11
WSKASGRGR
0.015


 


7
KSILWSKAS
0.015


 


9
ILWSKASGR
0.010


 


5
LMKSILWSK
0.010


 


1
IIHSLMKSI
0.010


 


4
SLMKSILWS
0.005


 


12
SKASGRGRR
0.005


 


13
KASGRGRRE
0.001


 


6
MKSILWSKA
0.001


 


2
IHSLMKSIL
0.001


 


14
ASGRGRREE
0.000


 


10
LWSKASGRG
0.000

















TABLE VI







V1-HLA-A1-10mers-



158P1D7


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













56
VSEISVPPSR
27.000



 


669
TTERPSASLY
11.250


 


210
ILDLQLEDNK
10.000


 


781
QLQPDMEAHY
10.000


 


150
VIEPSAFSKL
9.000


 


171
AIESLPPNIF
9.000


 


828
HAEPDYLEVL
9.000


 


123
SLEILKEDTF
9.000


 


398
RIEVLEEGSF
9.000


 


812
LVEQTKNEYF
9.000


 


173
ESLPPNIFRF
7.500


 


546
CTSPGHLDKK
5.000


 


134
GLENLEFLQA
4.500


 


401
VLEEGSFMNL
4.500


 


380
KSDLVEYFTL
3.750


 


456
NPMPKLKVLY
2.500


 


505
DLDLLTQIDL
2.500


 


502
ILDDLDLLTQ
2.500


 


743
STEFLSFQDA
2.250


 


771
ITEYLRKNIA
2.250


 


682
MVSPMVHVYR
2.000


 


214
QLEDNKWACN
1.800


 


355
LSDLRPPPQN
1.500


 


264
ESICPTPPVY
1.500


 


753
SSLYRNILEK
1.500


 


561
NSEILCPGLV
1.350


 


601
LTDAVPLSVL
1.250


 


276
HEDPSGSLHL
1.250


 


590
TTNTADTILR
1.250


 


149
TVIEPSAFSK
1.000


 


106
GAFNGLGLLK
1.000


 


801
ETLMYSRPRK
1.000


 


545
LCTSPGHLDK
1.000


 


824
KANLHAEPDY
1.000


 


525
LVGLQQWIQK
1.000


 


300
TTSILKLPTK
1.000


 


477
HIFSGVPLTK
1.000


 


100
IADIEIGAFN
1.000


 


768
QLGITEYLRK
1.000


 


245
VVCNSPPFFK
1.000


 


721
LLEQENHSPL
0.900


 


700
LEEEEERNEK
0.900


 


102
DIEIGAFNGL
0.900


 


441
YLEYNAIKEI
0.900


 


436
NLEYLYLEYN
0.900


 


36
NCEEKDGTML
0.900


 


513
DLEDNPWDCS
0.900


 


383
LVEYFTLEML
0.900


 


388
TLEMLHLGNN
0.900


 


137
NLEFLQADNN
0.900


 


232
WLENMPPQSI
0.900


 


47
NCEAKGIKMV
0.900


 


747
LSFQDASSLY
0.750


 


711
GSDAKHLQRS
0.750


 


723
EQENHSPLTG
0.675


 


728
SPLTGSNMKY
0.625


 


830
EPDYLEVLEQ
0.625


 


435
HNLEYLYLEY
0.625


 


191
RGNQLQTLPY
0.625


 


643
QVDEQMRDNS
0.500


 


223
NCDLLQLKTW
0.500


 


142
QADNNFITVI
0.500


 


60
SVPPSRPFQL
0.500


 


765
ELQQLGITEY
0.500


 


609
VLILGLLIMF
0.500


 


453
GTFNPMPKLK
0.500


 


630
VLVLHRRRRY
0.500


 


42
GTMLINCEAK
0.500


 


472
QVLPPHIFSG
0.500


 


593
TADTILRSLT
0.500


 


337
VLSPSGLLIH
0.500


 


811
VLVEQTKNEY
0.500


 


187
HLDLRGNQLQ
0.500


 


614
LLIMFITIVF
0.500


 


603
DAVPLSVLIL
0.500


 


200
YVGFLEHIGR
0.500


 


522
SCDLVGLQQW
0.500


 


203
FLEHIGRILD
0.450


 


759
ILEKERELQQ
0.450


 


706
RNEKEGSDAK
0.450


 


785
DMEAHYPGAH
0.450


 


351
NIESLSDLRP
0.450


 


439
YLYLEYNAIK
0.400


 


59
ISVPPSRPFQ
0.300


 


727
HSPLTGSNMK
0.300


 


419
NGNHLTKLSK
0.250


 


310
APGLIPYITK
0.250


 


681
HMVSPMVHVY
0.250


 


783
QPDMEAHYPG
0.250


 


432
LGLHNLEYLY
0.250


 


119
INHNSLEILK
0.250


 


451
LPGTFNPMPK
0.250


 


371
AGNIIHSLMK
0.250


 


254
KGSILSRLKK
0.250


 


796
ELKLMETLMY
0.250


 


584
VTTPATTTNT
0.250


 


820
YFELKANLHA
0.225


 


817
KNEYFELKAN
0.225


 


793
AHEELKLMET
0.225


 


358
LRPPPQNPRK
0.200

















TABLE VI







V3-HLA-A1-10mers-



158P1D7


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













2
ASLYEQHMGA
0.075



 


8
HMGAHEELKL
0.025


 


1
SASLYEQHMG
0.010


 


7
QHMGAHEELK
0.010


 


3
SLYEQHMGAH
0.010


 


4
LYEQHMGAHE
0.009


 


9
MGAHEELKLM
0.003


 


6
EQHMGAHEEL
0.002


 


5
YEQHMGAHEE
0.000

















TABLE VI







V4-HLA-A1-10mers-



158P1D7


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


each start position is specified, the


length of peptide is 10 amino acids, and


the end position for each peptide is the


start position plus nine.









Start
Subsequence
Score













9
SILWSKASGR
0.100



 


4
HSLMKSILWS
0.075


 


8
KSILWSKASG
0.030


 


5
SLMKSILWSK
0.020


 


12
WSKASGRGRR
0.015


 


1
NIIHSLMKSI
0.010


 


2
IIHSLMKSIL
0.010


 


3
IHSLMKSILW
0.003


 


10
ILWSKASGRG
0.001


 


14
KASGRGRREE
0.001


 


11
LWSKASGRGR
0.001


 


6
LMKSILWSKA
0.001


 


7
MKSILWSKAS
0.001


 


13
SKASGRGRRE
0.000

















TABLE VII







V1-HLA-A2-9mers-



158P1D7


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













465
YLNNNLLQV
735.860



 


614
LLIMFITIV
423.695


 


193
NQLQTLPYV
330.059


 


616
IMFITIVFC
285.492


 


140
FLQADNNFI
263.950


 


415
KLYLNGNHL
239.259


 


439
YLYLEYNAI
230.356


 


611
ILGLLIMFI
224.357


 


2
KLWIHLFYS
158.832


 


429
GMFLGLHNL
131.296


 


581
YLMVTTPAT
126.833


 


463
VLYLNNNLL
116.211


 


574
SMPTQTSYL
84.856


 


71
LLNNGLTML
83.527


 


4
WIHLFYSSL
77.017


 


305
KLPTKAPGL
74.768


 


613
GLLIMFITI
73.343


 


213
LQLEDNKWA
71.455


 


826
NLHAEPDYL
57.572


 


803
LMYSRPRKV
54.652


 


501
NILDDLDLL
50.218


 


798
KLMETLMYS
50.051


 


527
GLQQWIQKL
49.134


 


158
KLNRLKVLI
36.515


 


178
NIFRFVPLT
33.135


 


225
DLLQLKTWL
32.604


 


462
KVLYLNNNL
24.206


 


767
QQLGITEYL
21.597


 


116
QLHINHNSL
21.362


 


68
QLSLLNNGL
21.362


 


502
ILDDLDLLT
20.776


 


70
SLLNNGLTM
18.382


 


470
LLQVLPPHI
17.736


 


391
MLHLGNNRI
17.736


 


164
VLILNDNAI
17.736


 


337
VLSPSGLLI
17.736


 


774
YLRKNIAQL
17.177


 


450
ILPGTFNPM
16.047


 


323
QLPGPYCPI
15.649


 


367
KLILAGNII
14.971


 


316
YITKPSTQL
13.512


 


141
LQADNNFIT
12.523


 


214
QLEDNKWAC
9.777


 


582
LMVTTPATT
9.149


 


758
NILEKEREL
8.912


 


17
SLHSQTPVL
8.759


 


182
FVPLTHLDL
8.598


 


609
VLILGLLIM
7.964


 


295
RMSTKTTSI
7.535


 


309
KAPGLIPYI
6.415


 


539
TVTDDILCT
6.149


 


618
FITIVFCAA
5.970


 


596
TILRSLTDA
5.813


 


432
LGLHNLEYL
5.437


 


479
FSGVPLTKV
4.804


 


517
NPWDCSCDL
4.745


 


544
ILCTSPGHL
4.721


 


531
WIQKLSKNT
4.713


 


597
ILRSLTDAV
4.403


 


524
DLVGLQQWI
4.304


 


290
SINDSRMST
4.201


 


681
HMVSPMVHV
3.928


 


425
KLSKGMFLG
3.479


 


608
SVLILGLLI
3.378


 


336
KVLSPSGLL
3.147


 


147
FITVIEPSA
3.142


 


48
CEAKGIKMV
3.111


 


722
LEQENHSPL
2.895


 


16
ISLHSQTPV
2.856


 


99
NIADIEIGA
2.801


 


163
KVLILNDNA
2.758


 


92
SIHLGFNNI
2.726


 


400
EVLEEGSFM
2.720


 


384
VEYFTLEML
2.547


 


442
LEYNAIKEI
2.538


 


302
SILKLPTKA
2.527


 


453
GTFNPMPKL
2.525


 


154
SAFSKLNRL
2.525


 


45
LINCEAKGI
2.439


 


393
HLGNNRIEV
2.365


 


624
CAAGIVVLV
2.222


 


833
YLEVLEQQT
2.194


 


455
FNPMPKLKV
2.088


 


621
IVFCAAGIV
2.040


 


408
MNLTRLQKL
2.017


 


646
EQMRDNSPV
1.957


 


481
GVPLTKVNL
1.869


 


780
AQLQPDMEA
1.864


 


196
QTLPYVGFL
1.805


 


604
AVPLSVLIL
1.763


 


473
VLPPHIFSG
1.690


 


487
VNLKTNQFT
1.683


 


675
ASLYEQHMV
1.680


 


612
LGLLIMFIT
1.674


 


821
FELKANLHA
1.644


 


175
LPPNIFRFV
1.613


 


494
FTHLPVSNI
1.533


 


474
LPPHIFSGV
1.466


 


709
KEGSDAKHL
1.454


 


620
TIVFCAAGI
1.435

















TABLE VII







V3-HLA-A2-9mers-



158P1D7


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













2
SLYEQHMGA
65.180



 


8
MGAHEELKL
0.237


 


6
QHMGAHEEL
0.027


 


1
ASLYEQHMG
0.002


 


4
YEQHMGAHE
0.001


 


7
HMGAHEELK
0.000


 


5
EQHMGAHEE
0.000


 


3
LYEQHMGAH
0.000

















TABLE VII







V4-HLA-A2-9mers-



158P1D7


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













1
IIHSLMKSI
5.609



 


4
SLMKSILWS
3.488


 


9
ILWSKASGR
0.210


 


8
SILWSKASG
0.038


 


6
MKSILWSKA
0.020


 


5
LMKSILWSK
0.011


 


2
IHSLMKSIL
0.010


 


7
KSILWSKAS
0.002


 


13
KASGRGRRE
0.000


 


3
HSLMKSILW
0.000


 


14
ASGRGRREE
0.000


 


11
WSKASGRGR
0.000


 


12
SKASGRGRR
0.000


 


10
LWSKASGRG
0.000

















TABLE VII







V1-HLA-A2-10mers-



158P1D7


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













613
GLLIMFITIV
922.161



 


431
FLGLHNLEYL
609.108


 


616
IMFITIVFCA
301.064


 


600
SLTDAVPLSV
285.163


 


417
YLNGNHLTKL
226.014


 


473
VLPPHIFSGV
224.653


 


70
SLLNNGLTML
181.794


 


433
GLHNLEYLYL
176.240


 


166
ILNDNAIESL
167.806


 


407
FMNLTRLQKL
163.232


 


174
SLPPNIFRFV
145.364


 


425
KLSKGMFLGL
142.060


 


581
YLMVTTPATT
126.833


 


409
NLTRLQKLYL
117.493


 


610
LILGLLIMFI
114.142


 


746
FLSFQDASSL
98.267


 


213
LQLEDNKWAC
97.424


 


141
LQADNNFITV
93.387


 


465
YLNNNLLQVL
92.666


 


369
ILAGNIIHSL
83.527


 


415
KLYLNGNHLT
83.462


 


140
FLQADNNFIT
81.516


 


158
KLNRLKVLIL
70.507


 


611
ILGLLIMFIT
69.289


 


78
MLHTNDFSGL
69.001


 


615
LIMFITIVFC
54.353


 


802
TLMYSRPRKV
51.468


 


531
WIQKLSKNTV
43.992


 


469
NLLQVLPPHI
38.601


 


67
FQLSLLNNGL
36.864


 


803
LMYSRPRKVL
34.412


 


115
KQLHINHNSL
28.049


 


462
KVLYLNNNLL
24.206


 


86
GLTNAISIHL
21.362


 


401
VLEEGSFMNL
18.106


 


44
MLINCEAKGI
17.736


 


596
TILRSLTDAV
17.338


 


621
IVFCAAGIVV
15.695


 


501
NILDDLDLLT
15.544


 


4
WIHLFYSSLL
13.512


 


486
KVNLKTNQFT
12.552


 


163
KVLILNDNAI
11.822


 


336
KVLSPSGLLI
11.822


 


60
SVPPSRPFQL
10.841


 


282
SLHLAATSSI
10.433


 


110
GLGLLKQLHI
10.433


 


766
LQQLGITEYL
9.923


 


126
ILKEDTFHGL
9.902


 


15
CISLHSQTPV
9.563


 


582
LMVTTPATTT
9.149.


 


257
ILSRLKKESI
8.691


 


517
NPWDCSCDLV
7.571


 


568
GLVNNPSMPT
7.452


 


441
YLEYNAIKEI
7.064


 


295
RMSTKTTSIL
6.326


 


678
YEQHMVSPMV
6.221


 


195
LQTLPYVGFL
6.055


 


770
GITEYLRKNI
5.881


 


322
TQLPGPYCPI
5.871


 


382
DLVEYFTLEM
5.805


 


192
GNQLQTLPYV
5.743


 


374
IIHSLMKSDL
4.993


 


647
QMRDNSPVHL
4.807


 


623
FCAAGIVVLV
4.804


 


305
KLPTKAPGLI
4.747


 


263
KESICPTPPV
4.733


 


457
PMPKLKVLYL
4.294


 


428
KGMFLGLHNL
4.153


 


2
KLWIHLFYSS
4.113


 


656
LQYSMYGHKT
4.110


 


574
SMPTQTSYLM
3.588


 


227
LQLKTWLENM
3.571


 


343
LLIHCQERNI
3.547


 


490
KTNQFTHLPV
3.381


 


220
WACNCDLLQL
3.139


 


232
WLENMPPQSI
3.071


 


738
KTTNQSTEFL
2.799


 


555
KELKALNSEI
2.627


 


721
LLEQENHSPL
2.324


 


390
EMLHLGNNRI
2.091


 


328
YCPIPCNCKV
2.088


 


212
DLQLEDNKWA
2.049


 


526
VGLQQWIQKL
2.017


 


605
VPLSVLILGL
2.017


 


798
KLMETLMYSR
1.820


 


313
LIPYITKPST
1.742


 


577
TQTSYLMVTT
1.738


 


380
KSDLVEYFTL
1.698


 


204
LEHIGRILDL
1.624


 


198
LPYVGFLEHI
1.587


 


608
SVLILGLLIM
I.517


 


108
FNGLGLLKQL
1.475


 


6
HLFYSSLLAC
1.437


 


488
NLKTNQFTHL
1.421


 


814
EQTKNEYFEL
1.413


 


825
ANLHAEPDYL
1.391


 


512
IDLEDNPWDC
1.335


 


818
NEYFELKANL
1.329


 


575
MPTQTSYLMV
1.158


 


77
TMLHTNDFSG
1.155

















TABLE VIII







V3-HLA-A2-10mers-



158P1D7


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













8
HMGAHEELKL
0.525



 


3
SLYEQHMGAH
0.292


 


9
MGAHEELKLM
0.127


 


2
ASLYEQHMGA
0.120


 


6
EQHMGAHEEL
0.080


 


5
YEQHMGAHEE
0.001


 


1
SASLYEQHMG
0.001


 


7
QHMGAHEELK
0.000


 


4
LYEQHMGAHE
0.000

















TABLE VIII







V4-HLA-A2-10mers-



158P1D7


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


each start position is specified, the


length of peptide is 10 amino acids, and


the end position for each peptide is the


start position plus nine.









Start
Subsequence
Score













1
NIIHSLMKSI
3.299



 


2
IIHSLMKSIL
2.047


 


5
SLMKSILWSK
0.951


 


6
LMKSILWSKA
0.363


 


10
ILWSKASGRG
0.137


 


9
SILWSKASGR
0.008


 


8
KSILWSKASG
0.002


 


4
HSLMKSILWS
0.001


 


7
MKSILWSKAS
0.000


 


14
KASGRGRREE
0.000


 


3
IHSLMKSILW
0.000


 


13
SKASGRGRRE
0.000


 


12
WSKASGRGRR
0.000


 


11
LWSKASGRGR
0.000

















TABLE IX







V1-A3-9mers-158P1D7



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













754
SLYRNILEK
300.000



 


417
YLNGNHLTK
60.000


 


407
FMNLTRLQK
40.000


 


433
GLHNLEYLY
36.000


 


802
TLMYSRPRK
30.000


 


43
TMLINCEAK
30.000


 


342
GLLIHCQER
18.000


 


799
LMETLMYSR
18.000


 


613
GLLIMFITI
16.200


 


429
GMFLGLHNL
13.500


 


174
SLPPNIFRF
13.500


 


768
QLGITEYLR
12.000


 


627
GIVVLVLHR
10.800


 


150
VIEPSAFSK
9.000


 


415
KLYLNGNHL
9.000


 


527
GLQQWIQKL
8.100


 


436
NLEYLYLEY
8.000


 


431
FLGLHNLEY
8.000


 


378
LMKSDLVEY
6.000


 


529
QQWIQKLSK
6.000


 


546
CTSPGHLDK
3.000


 


463
VLYLNNNLL
3.000


 


439
YLYLEYNAI
3.000


 


2
KLWIHLFYS
2.700


 


367
KLILAGNII
2.700


 


297
STKTTSILK
2.000


 


6
HLFYSSLLA
2.000


 


632
VLHRRRRYK
2.000


 


409
NLTRLQKLY
2.000


 


611
ILGLLIMFI
1.800


 


337
VLSPSGLLI
1.800


 


305
KLPTKAPGL
1.800


 


390
EMLHLGNNR
1.800


 


158
KLNRLKVLI
1.800


 


682
MVSPMVHVY
1.800


 


616
IMFITIVFC
1.500


 


659
SMYGHKTTH
1.500


 


628
IVVLVLHRR
1.350


 


614
LLIMFITIV
1.350


 


323
QLPGPYCPI
1.350


 


610
LILGLLIMF
1.350


 


729
PLTGSNMKY
1.200


 


453
GTFNPMPKL
1.012


 


228
QLKTWLENM
0.900


 


450
ILPGTFNPM
0.900


 


615
LIMFITIVF
0.900


 


609
VLILGLLIM
0.900


 


255
GSILSRLKK
0.900


 


482
VPLTKVNLK
0.900


 


774
YLRKNIAQL
0.900


 


164
VLILNDNAI
0.900


 


655
HLQYSMYGH
0.900


 


86
GLTNAISIH
0.900


 


71
LLNNGLTML
0.900


 


656
LQYSMYGHK
0.900


 


246
VCNSPPFFK
0.900


 


798
KLMETLMYS
0.810


 


730
LTGSNMKYK
0.750


 


681
HMVSPMVHV
0.675


 


469
NLLQVLPPH
0.675


 


312
GLIPYITKP 
0.608


 


295
RMSTKTTSI
0.600


 


630
VLVLHRRRR
0.600


 


140
FLQADNNFI
0.600


 


826
NLHAEPDYL
0.600


 


391
MLHLGNNRI
0.600


 


68
QLSLLNNGL
0.600


 


465
YLNNNLLQV
0.600


 


574
SMPTQTSYL
0.600


 


70
SLLNNGLTM
0.600


 


488
NLKTNQFTH
0.600


 


664
KTTHHTTER
0.600


 


116
QLHINHNSL
0.600


 


17
SLHSQTPVL
0.600


 


187
HLDLRGNQL
0.600


 


265
SICPTPPVY
0.600


 


486
KVNLKTNQF
0.600


 


470
LLQVLPPHI
0.600


 


110
GLGLLKQLH
0.600


 


676
SLYEQHMVS
0.600


 


214
QLEDNKWAC
0.600


 


781
QLQPDMEAH
0.450


 


11
SLLACISLH
0.450


 


178
NIFRFVPLT
0.450


 


524
DLVGLQQWI
0.405


 


20
SQTPVLSSR
0.405


 


393
HLGNNRIEV
0.400


 


551
HLDKKELKA
0.400


 


351
NIESLSDLR
0.400


 


457
PMPKLKVLY
0.400


 


812
LVEQTKNEY
0.400


 


113
LLKQLHINH
0.400


 


372
GNIIHSLMK
0.360


 


604
AVPLSVLIL
0.360


 


741
NQSTEFLSF
0.360


 


328
YCPIPCNCK
0.300


 


287
ATSSINDSR
0.300


 


738
KTTNQSTEF
0.300


 


728
SPLTGSNMK
0.300


 


359
RPPPQNPRK
0.300

















TABLE IX







V3-A3-9mers-158P1D7



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













7
HMGAHEELK
20.000



 


2
SLYEQHMGA
3.000


 


6
QHMGAHEEL
0.001


 


8
MGAHEELKL
0.001


 


5
EQHMGAHEE
0.000


 


1
ASLYEQHMG
0.000


 


3
LYEQHMGAH
0.000


 


4
YEQHMGAHE
0.000

















TABLE IX







V4-A3-9mers-158P1D7



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
LMKSILWSK
135.000



 


9
ILWSKASGR
20.000


 


4
SLMKSILWS
0.180


 


1
IIHSLMKSI
0.045


 


3
HSLMKSILW
0.003


 


8
SILWSKASG
0.003


 


11
WSKASGRGR
0.002


 


7
KSILWSKAS
0.001


 


12
SKASGRGRR
0.001


 


2
IHSLMKSIL
0.001


 


6
MKSILWSKA
0.000


 


13
KASGRGRRE
0.000


 


14
ASGRGRREE
0.000


 


10
LWSKASGRG
0.000

















TABLE X







V1-HLA-A3-10mers-



158P1D7


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













439
YLYLEYNAIK
300.000



 


798
KLMETLMYSR
121.500


 


632
VLHRRRRYKK
60.000


 


768
QLGITEYLRK
40.000


 


477
HIFSGVPLTK
30.000


 


210
ILDLQLEDNK
20.000


 


481
GVPLTKVNLK
18.000


 


681
HMVSPMVHVY
18.000


 


616
IMFITIVFCA
13.500


 


149
TVIEPSAFSK
13.500


 


158
KLNRLKVLIL
10.800


 


425
KLSKGMFLGL
10.800


 


815
QTKNEYFELK
9.000


 


609
VLILGLLIMF
9.000


 


245
VVCNSPPFFK
9.000


 


614
LLIMFITIVF
9.000


 


811
VLVEQTKNEY
9.000


 


377
SLMKSDLVEY
9.000


 


453
GTFNPMPKLK
7.500


 


781
QLQPDMEAHY
6.000


 


655
HLQYSMYGHK
6.000


 


378
LMKSDLVEYF
6.000


 


75
GLTMLHTNDF
6.000


 


106
GAFNGLGLLK
6.000


 


2
KLWIHLFYSS
5.400


 


86
GLTNAISIHL
5.400


 


401
VLEEGSFMNL
5.400


 


42
GTMLINCEAK
4.500


 


613
GLLIMFITIV
4.050


 


627
GIVVLVLHRR
4.050


 


525
LVGLQQWIQK
4.000


 


134
GLENLEFLQA
3.600


 


433
GLHNLEYLYL
3.600


 


110
GLGLLKQLHI
3.600


 


6
HLFYSSLLAC
3.000


 


470
LLQVLPPHIF
3.000


 


194
QLQTLPYVGF
3.000


 


290
SINDSRMSTK
3.000


 


126
ILKEDTFHGL
2.700


 


357
DLRPPPQNPR
2.700


 


796
ELKLMETLMY
2.400


 


546
CTSPGHLDKK
2.250


 


803
LMYSRPRKVL
2.250


 


729
PLTGSNMKYK
2.250


 


369
ILAGNIIHSL
2.025


 


123
SLEILKEDTF
2.000


 


765
ELQQLGITEY
1.800


 


112
GLLKQLHINH
1.800


 


367
KLILAGNIIH
1.800


 


78
MLHTNDFSGL
1.800


 


488
NLKTNQFTHL
1.800


 


300
TTSILKLPTK
1.500


 


659
SMYGHKTTHH
1.500


 


415
KLYLNGNHLT
1.500


 


568
GLVNNPSMPT
1.350


 


473
VLPPHIFSGV
1.350


 


70
SLLNNGLTML
1.350


 


417
YLNGNHLTKL
1.350


 


528
LQQWIQKLSK
1.200


 


409
NLTRLQKLYL
1.200


 


197
TLPYVGFLEH
1.200


 


94
HLGFNNIADI
0.900


 


407
FMNLTRLQKL
0.900


 


166
ILNDNAIESL
0.900


 


393
HLGNNRIEVL
0.900


 


465
YLNNNLLQVL
0.900


 


469
NLLQVLPPHI
0.900


 


682
MVSPMVHVYR
0.900


 


431
FLGLHNLEYL
0.900


 


337
VLSPSGLLIH
0.900


 


232
WLENMPPQSI
0.900


 


767
QQLGITEYLR
0.810


 


382
DLVEYFTLEM
0.810


 


200
YVGFLEHIGR
0.800


 


611
ILGLLIMFIT
0.675


 


45
LINCEAKGIK
0.600


 


600
SLTDAVPLSV
0.600


 


182
FVPLTHLDLR
0.600


 


574
SMPTQTSYLM
0.600


 


647
QMRDNSPVHL
0.600


 


295
RMSTKTTSIL
0.600


 


310
APGLIPYITK
0.600


 


282
SLHLAATSSI
0.600


 


422
HLTKLSKGMF
0.600


 


721
LLEQENHSPL
0.600


 


746
FLSFQDASSL
0.600


 


630
VLVLHRRRRY
0.600


 


257
ILSRLKKESI
0.600


 


336
KVLSPSGLLI
0.540


 


305
KLPTKAPGLI
0.540


 


801
ETLMYSRPRK
0.450


 


753
SSLYRNILEK
0.450


 


551
HLDKKELKAL
0.450


 


44
MLINCEAKGI
0.450


 


441
YLEYNAIKEI
0.450


 


189
DLRGNQLQTL
0.405


 


610
LILGLLIMFI
0.405


 


545
LCTSPGHLDK
0.400


 


451
LPGTFNPMPK
0.400


 


71
LLNNGLTMLH
0.400

















TABLE X







V3-HLA-A3-10mers-



158P1D7


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













8
HMGAHEELKL
1.200



 


3
SLYEQHMGAH
0.675


 


7
QHMGAHEELK
0.045


 


6
EQHMGAHEEL
0.005


 


2
ASLYEQHMGA
0.003


 


1
SASLYEQHMG
0.000


 


9
MGAHEELKLM
0.000


 


5
YEQHMGAHEE
0.000


 


4
LYEQHMGAHE
0.000

















TABLE X







V4-HLA-A3-10mers-



158P1D7


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













5
SLMKSILWSK
202.500



 


9
SILWSKASGR
0.600


 


6
LMKSILWSKA 
0.200


 


1
NIIHSLMKSI
0.068


 


2
IIHSLMKSIL
0.060


 


10
ILWSKASGRG
0.030


 


12
WSKASGRGRR
0.006


 


4
HSLMKSILWS
0.001


 


8
KSILWSKASG
0.000


 


11
LWSKASGRGR
0.000


 


3
IHSLMKSILW
0.000


 


14
KASGRGRREE
0.000


 


7
MKSILWSKAS
0.000


 


13
SKASGRGRRE
0.000

















TABLE XI







V1-A11-9mers-158P1D7



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













529
QQWIQKLSK
2.400



 


297
STKTTSILK
2.000


 


546
CTSPGHLDK
2.000


 


754
SLYRNILEK
1.600


 


656
LQYSMYGHK
1.200


 


150
VIEPSAFSK
1.200


 


407
FMNLTRLQK
0.800


 


802
TLMYSRPRK
0.800


 


417
YLNGNHLTK
0.800


 


627
GIVVLVLHR
0.720


 


628
IVVLVLHRR
0.600


 


440
LYLEYNAIK
0.600


 


246
VCNSPPFFK
0.600


 


359
RPPPQNPRK
0.600


 


664
KTTHHTTER
0.600


 


43
TMLINCEAK
0.600


 


730
LTGSNMKYK
0.500


 


478
IFSGVPLTK
0.400


 


107
AFNGLGLLK
0.400


 


372
GNIIHSLMK
0.360


 


342
GLLIHCQER
0.360


 


482
VPLTKVNLK
0.300


 


728
SPLTGSNMK
0.300


 


420
GNHLTKLSK
0.240


 


749
FQDASSLYR
0.240


 


287
ATSSINDSR
0.200


 


790
YPGAHEELK
0.200


 


328
YCPIPCNCK
0.200


 


255
GSILSRLKK
0.180


 


799
LMETLMYSR
0.160


 


768
QLGITEYLR
0.160


 


20
SQTPVLSSR
0.120


 


454
TFNPMPKLK
0.100


 


550
GHLDKKELK
0.090


 


809
RKVLVEQTK
0.090


 


336
KVLSPSGLL
0.090


 


462
KVLYLNNNL
0.090


 


163
KVLILNDNA
0.090


 


252
FFKGSILSR
0.080


 


351
NIESLSDLR
0.080


 


769
LGITEYLRK
0.060


 


526
VGLQQWIQK
0.060


 


453
GTFNPMPKL
0.060


 


42
GTMLINCEA
0.060


 


629
VVLVLHRRR
0.060


 


608
SVLILGLLI
0.060


 


183
VPLTHLDLR
0.060


 


486
KVNLKTNQF
0.060


 


481
GVPLTKVNL
0.060


 


707
NEKEGSDAK
0.060


 


291
INDSRMSTK
0.040


 


182
FVPLTHLDL
0.040


 


383
LVEYFTLEM
0.040


 


120
NHNSLEILK
0.040


 


604
AVPLSVLIL
0.040


 


633
LHRRRRYKK
0.040


 


222
CNCDLLQLK
0.040


 


621
IVFCAAGIV
0.040


 


46
INCEAKGIK
0.040


 


632
VLHRRRRYK
0.040


 


390
EMLHLGNNR
0.036


 


613
GLLIMFITI
0.036


 


301
TSILKLPTK
0.030


 


211
LDLQLEDNK
0.030


 


738
KTTNQSTEF
0.030


 


815
QTKNEYFEL
0.030


 


711
GSDAKHLQR
0.024


 


433
GLHNLEYLY
0.024


 


429
GMFLGLHNL
0.024


 


415
KLYLNGNHL
0.024


 


816
TKNEYFELK
0.020


 


155
AFSKLNRLK
0.020


 


690
YRSPSFGPK
0.020


 


682
MVSPMVHVY
0.020


 


87
LTNAISIHL
0.020


 


601
LTDAVPLSV
0.020


 


245
VVCNSPPFF
0.020


 


812
LVEQTKNEY
0.020


 


547
TSPGHLDKK
0.020


 


76
LTMLHTNDF
0.020


 


410
LTRLQKLYL
0.020


 


698
KHLEEEEER
0.018


 


367
KLILAGNII
0.018


 


57
SEISVPPSR
0.018


 


780
AQLQPDMEA
0.018


 


701
EEEEERNEK
0.018


 


615
LIMFITIVF
0.016


 


201
VGFLEHIGR
0.016


 


6
HLFYSSLLA
0.016


 


591
TNTADTILR
0.016


 


196
QTLPYVGFL
0.015


 


148
ITVIEPSAF
0.015


 


630
VLVLHRRRR
0.012


 


641
KKQVDEQMR
0.012


 


86
GLTNAISIH
0.012


 


527
GLQQWIQKL
0.012


 


170
SLLNNGLTM
0.012


 


174
SLPPNIFRF
0.012


 


488
NLKTNQFTH
0.012


 


368
LILAGNIIH
0.012

















TABLE XI







V3-A11-9mers-158P1D7



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













7
HMGAHEELK
0.400



 


2
SLYEQHMGA
0.016


 


3
LYEQHMGAH
0.004


 


8
MGAHEELKL
0.000


 


6
QHMGAHEEL
0:000


 


5
EQHMGAHEE
0.000


 


4
YEQHMGAHE
0.000


 


1
ASLYEQHMG
0.000

















TABLE XI







V4-A11-9mers-158P1D7



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
LMKSILWSK
0.800



 


9
ILWSKASGR
0.160


 


12
SKASGRGRR
0.004


 


1
IIHSLMKSI
0.002


 


4
SLMKSILWS
0.002


 


3
HSLMKSILW
0.001


 


8
SILWSKASG
0.001


 


11
WSKASGRGR
0.000


 


6
MKSILWSKA
0.000


 


2
IHSLMKSIL
0.000


 


13
KASGRGRRE
0.000


 


7
KSILWSKAS
0.000


 


10
LWSKASGRG
0.000


 


14
ASGRGRREE
0.000

















TABLE XII







V1-HLA-A11-10mers-



158P1D7


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












149
TVIEPSAFSK
9.000


 


245
VVCNSPPRRK
6.000


 


42
GTMLINCEAK
6.000


 


481
GVPLTKVNLK
6.000


 


525
LVGLQQWIQK
4.000


 


453
GTFNPMPKLK
3.000


 


106
GAFNGLGLLK
2.400


 


477
HIFSGVPLTK
1.600


 


416
LYLNGNHLTK
1.200


 


528
LQQWIQKLSK
1.200


 


815
QTKNEYFELK
1.000


 


300
TTSILKLPTK
1.000


 


546
CTSPGHLDKK
1.000


 


798
KLMETLMYSR
0.960


 


200
YVGFLEHIGR
0.800


 


406
SFMNLTRLQK
0.800


 


439
YLYLEYNAIK
0.800


 


768
QLGITEYLRK
0.800


 


632
VLHRRRRYKK
0.800


 


801
ETLMYSRPRK
0.450


 


310
APGLIPYITK
0.400


 


789
HYPGAHEELK
0.400


 


655
HLQYSMYGHK
0.400


 


451
LPGTFNPMPK
0.400


 


689
VYRSPSFGPK
0.400


 


545
LCTSPGHLDK
0.400


 


210
ILDLQLEDNK
0.400


 


590
TTNTADTILR
0.400


 


290
SINDSRMSTK
0.400


 


45
LINCEAKGIK
0.400


 


682
MVSPMVHVYR
0.400


 


182
FVPLTHLDLR
0.400


 


767
QQLGITEYLR
0.360


 


627
GIVVLVLHRR
0.360


 


631
LVLHRRRRYK
0.300


 


221
ACNCDLLQLK
0.200


 


336
KVLSPSGLLI
0.180


 


706
RNEKEGSDAK
0.120


 


254
KGSILSRLKK
0.120


 


462
KVLYLNNNLL
0.090


 


163
KVLILNDNAI
0.090


 


621
IVFCAAGIVV
0.080


 


748
SFQDASSLYR
0.080


 


119
INGNSLEILK
0.080


 


753
SSLYRNILEK
0.060


 


60
SVPPSRPFQL
0.060


 


490
KTNQFTHLPV
0.060


 


700
LEEEEERNEK
0.060


 


628
IVVLVLHRRR
0.060


 


608
SVLILGLLIM
0.060


 


629
VVLVLHRRRR
0.060


 


296
MSTKTTSILK
0.040


 


755
LYRNILEKER
0.040


 


327
PYCPIPCNCK
0.040


 


154
SAFSKLNRLK
0.040


 


286
AATSSINDSR
0.040


 


371
AGNIIHSLMK
0.040


 


419
NGNHLTKLSK
0.040


 


350
RNIESLSDLR
0.036


 


112
GLLKQLHINH
0.036


 


367
KLILAGNIIH
0.036


 


738
KTTNQSTEFL
0.030


 


115
KQLHINHNSL
0.027


 


433
GLHNLEYLYL
0.024


 


52
GIKMVSEISV
0.024


 


110
GLGLLKQLHI
0.024


 


172
IESLPPNIFR
0.024


 


158
KLNRLKVLIL
0.024


 


134
GLENLEFLQA
0.024


 


616
IMFITIVFCA
0.024


 


425
KLSKGMFLGL
0.024


 


357
DLRPPPQNPR
0.024


 


86
GLTNAISIHL
0.024


 


152
EPSAFSKLNR
0.024


 


389
LEMLHLGNNR
0.024


 


297
STKTTSILKL
0.020


 


812
LVEQTKNEYF
0.020


 


727
HSPLTGSNMK
0.020


 


686
MVHVYRSPSF
0.020


 


383
LVEYFTLEML
0.020


 


358
LRPPPQNPRK
0.020


 


31
CDSLCNCEEK
0.020


 


729
PLTGSNMKYK
0.020


 


423
LTKLSKGMFL
0.020


 


613
GLLIMFITIV
0.018


 


181
RFVPLTHLDL
0.018


 


251
PFFKGSILSR
0.016


 


178
NIFRFVPLTH
0.016


 


619
ITIVFCAAGI
0.015


 


626
AGIVVLVLHR
0.012


 


640
KKKQVDEQMR
0.012


 


141
LQADNNFITV
0.012


 


688
HVYRSPSFGP
0.012


 


75
GLTMLHTNDR
0.012


 


609
VLILGLLIMF
0.012


 


464
LYLNNNLLQV
0.012


 


614
LLIMFITIVF
0.012


 


96
GFNNIADIEI
0.012


 


295
RMSTKTTSIL
0.012


 


610
LILGLLIMFI
0.012

















TABLE XII







V3-HLA-A11-10mers-



158P1D7


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













7
QHMGAHEELK
0.040



 


3
SLYEQHMGAH
0.008


 


8
HMGAHEELKL
0.008


 


6
EQHMGAHEEL
0.002


 


2
ASLYEQHMGA
0.001


 


4
LYEQHMGAHE
0.000


 


1
SASLYEQHMG
0.000


 


9
MGAHEELKLM
0.000


 


5
YEQHMGAHEE
0.000

















TABLE XII







V4-HLA-A11-10mers-



158P1D7


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













5
SLMKSILWSK
1.600



 


9
SILWSKASGR
0.120


 


2
IIHSLMKSIL
0.004


 


12
WSKASGRGRR
0.004


 


6
LMKSILWSKA
0.004


 


1
NIIHSLMKSI
0.003


 


10
ILWSKASGRG
0.001


 


11
LWSKASGRGR
0.000


 


3
IHSLMKSILW
0.000


 


8
KSILWSKASG
0.000


 


4
HSLMKSILWS
0.000


 


14
KASGRGRREE
0.000


 


7
MKSILWSKAS
0.000


 


13
SKASGRGRRE
0.000

















TABLE XIII







V1-HLA-A24-9mers-



158P1D7


Each peptide is a portion of SEQ


ID NO: 3: each start position is


specified, the length of peoptide is 9


amino acids, and the end position


for each peptide is the start


position plus eight.









Start
Subsequence
Score













443
EYNAIKEIL
420.000



 


789
HYPGAHEEL
330.000


 


819
EYFELKANL
288.000


 


804
MYSRPRKVL
200.000


 


8
FYSSLLACI
60.000


 


386
YFTLEMLHL
20.000


 


139
EFLQADNNF
18.000


 


462
KVLYLNNNL
17.280


 


350
RNIESLSDL
14.400


 


599
RSLTDAVPL
12.000


 


336
KVLSPSGLL
12.000


 


305
KLPTKAPGL
12.000


 


736
KYKTTNQST
12.000


 


580
SYLMVTTPA
10.500


 


415
KLYLNGNHL
9.600


 


272
VYEEHEDPS
9.000


 


202
GFLEHIGRI
9.000


 


438
EYLYLEYNA
9.000


 


466
LNNNLLQVL
8.640


 


767
QQLGITEYL
8.400


 


203
FLEHIGRIL
8.400


 


607
LSVLILGLL
8.400


 


87
LTNAISIHL
8.400


 


537
KNTVTDDIL
8.000


 


219
KWACNCDLL
8.000


 


758
NILEKEREL
7.920


 


408
MNLTRLQKL
7.920


 


527
GLQQWIQKL
7.920


 


416
LYLNGNHLT
7.500


 


199
PYVGFLEHI
7.500


 


486
KVNLKTNQF
7.200


 


109
NGLGLLKQL
7.200


 


196
QTLPYVGFL
7.200


 


133
HGLENLEFL
7.200


 


225
DLLQLKTWL
7.200


 


83
DFSGLTNAI
7.200


 


456
NPMPKLKVL
7.200


 


561
NSEILCPGL
7.200


 


501
NILDDLDLL
7.200


 


500
SNILDDLDL
6.000


 


221
ACNCDLLQL
6.000


 


71
LLNNGLTML
6.000


 


604
AVPLSVLIL
6.000


 


182
FVPLTHLDL
6.000


 


347
CQERNIESL
6.000


 


669
TTERPSASL
6.000


 


10
SSLLACISL
6.000


 


590
TTNTADTIL
6.000


 


481
GVPLTKVNL
6.000


 


432
LGLHNLEYL
6.000


 


61
VPPSRPFQL
6.000


 


394
LGNNRIEVL
6.000


 


574
SMPTQTSYL
6.000


 


739
TTNQSTEFL
6.000


 


68
QLSLLNNGL
5.760


 


625
AAGIVVLVL
5.600


 


370
LAGNIIHSL
5.600


 


593
TADTILRSL
5.600


 


657
QYSMYGHKT
5.500


 


154
SAFSKLNRL
4.800


 


517
NPWDCSCDL
4.800


 


463
VLYLNNNLL
4.800


 


752
ASSLYRNIL
4.800


 


207
IGRILDLQL
4.800


 


713
DAKHLQRSL
4.800


 


116
QLHINHNSL
4.800


 


187
HLDLRGNQL
4.800


 


426
LSKGMFLGL
4.800


 


453
GTFNPMPKL
4.400


 


815
QTKNEYFEL
4.400


 


418
LNGNHLTKL
4.400


 


738
KTTNQSTEF
4.400


 


615
LIMFITIVF
4.200


 


89
NAISIHLGF
4.000


 


4
WIHLFYSSL
4.000


 


26
SSRGSCDSL
4.000


 


106
GAFNGLGLL
4.000


 


826
NLHAEPDYL
4.000


 


429
GMFLGLHNL
4.000


 


544
ILCTSPGHL
4.00


 


458
MPKLKVLYL
4.000


 


159
LNRLKVLIL
4.000


 


692
SPSFGPKHL
4.000


 


623
FCAAGIVVL
4.000


 


296
MSTKTTSIL
4.000


 


17
SLHSQTPVL
4.000


 


747
LSFQDASSL
4.000


 


316
YITKPSTQL
4.000


 


119
INGNSLEIL
4.000


 


520
DCSCDLVGL
4.000


 


405
GSFMNLTRL
4.000


 


105
IGAFNGLGL
4.000


 


774
YLRKNIAQL
4.000


 


410
LTRLQKLYL
4.000


 


167
LNDNAIESL
4.000


 


130
DTFHGLENL
4.000


 


309
KAPGLIPYI
3.600


 


158
KLNRLKVLI
3.600


 


76
LTMLHTNDF
3.600


 


59
ISVPPSRPF
3.600

















TABLE XIII







V3-HLA-A24-9mers



158P1D7


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
MGAHEELKL
4.400



 


3
LYEQHMGAH
0.750


 


6
QHMGAHEEL
0.660


 


2
SLYEQHMGA
0.120


 


1
ASLYEQHMG
0.015


 


5
EQHMGAHEE
0.011


 


7
HMGAHEELK
0.010


 


4
YEQHMGAHE
0.002

















TABLE XIII







V3-HLA-A24-9mers-



158P1D7


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













1
IIHSLMKSI
1.200



 


2
IHSLMKSIL
0.400


 


7
KSILWSKAS
0.300


 


4
SLMKSILWS
0.150


 


3
HSLMKSILW
0.150


 


13
KASGRGRRE
0.020


 


8
SILWSKASG
0.015


 


5
LMKSILWSK
0.014


 


6
MKSILWSKA
0.013


 


14
ASGRGRREE
0.011


 


10
LWSKASGRG
0.010


 


11
WSKASGRGR
0.010


 


9
ILWSKASGR
0.010


 


12
SKASGRGRR
0.001

















TABLE XIV







V1-HLA-A24-10mers-



158P1D7


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













773
EYLRKNIAQL
300.000



 


385
EYFTLEMLHL
200.000


 


438
EYLYLEYNAI
90.000


 


181
RFVPLTHLDL
72.000


 


202
GFLEHIGRIL
50.400


 


677
LYEQHMVSPM
37.500


 


315
PYITKPSTQL
30.000


 


252
FFKGSILSRL
28.000


 


622
VFCAAGIVVL
20.000


 


179
IFRFVPLTHL
20.000


 


359
RPPPQNPRKL
15.840


 


462
KVLYLNNNLL
14.400


 


115
KQLHINHNSL
14.400


 


757
RNILEKEREL
13.200


 


832
DYLEVLEQQT
12.960


 


691
RSPSFGPKHL
12.000


 


428
KGMFLGLHNL
12.000


 


158
KLNRLKVLIL
12.000


 


131
TFHGLENLEF
11.000


 


425
KLSKGMFLGL
9.600


 


150
VIEPSAFSKL
9.504


 


139
EFLQADNNFI
9.000


 


102
DIEIGAFNGL
8.640


 


465
YLNNNLLQVL
8.640


 


67
FQLSLLNNGL
8.640


 


401
VLEEGSFMNL
8.640


 


497
LPVSNILDDL
8.400


 


766
LQQLGITEYL
8.400


 


96
GFNNIADIEI
8.250


 


738
KTTNQSTEFL
8.000


 


380
KSDLVEYFTL
8.000


 


295
RMSTKTTSIL
8.000


 


526
VGLQQWIQKL
7.920


 


407
FMNLTRLQKL
7.920


 


580
SYLMVTTPAT
7.500


 


464
LYLNNNLLQV
7.500


 


828
HAEPDYLEVL
7.200


 


329
CPIPCNCKVL
7.200


 


36
NCEEKDGTML
7.200


 


346
HCQERNIESL
7.200


 


166
ILNDNAIESL
7.200


 


60
SVPPSRPFQL
7.200


 


605
VPLSVLILGL
7.200


 


480
SGVPLTKVNL
7.200


 


603
DAVPLSVLIL
7.200


 


494
FTHLPVSNIL
6.720


 


592
NTADTILRSL
6.720


 


417
YLNGNHLTKL
6.600


 


118
HINHNSLEIL
6.000


 


500
SNILDDLDLL
6.000


 


455
FNPMPKLKVL
6.000


 


70
SLLNNGLTML
6.000


 


16
ISLHSQTPVL
6.000


 


8
FYSSLLACIS
6.000


 


543
DILCTSPGHL
6.000


 


249
SPPFFKGSIL
6.000


 


3
LWIHLFYSSL
6.000


 


825
ANLHAEPDYL
6.000


 


398
RIEVLEEGSF
6.000


 


499
VSNILDDLDL
6.000


 


721
LLEQENHSPL
6.000


 


383
LVEYFTLEML
6.000


 


7
LFYSSLLACI
6.000


 


516
DNPWDCSCDL
6.000


 


560
LNSEILCPGL
5.760


 


126
ILKEDTFHGL
5.760


 


624
CAAGIVVLVL
5.600


 


86
GLTNAISIHL
5.600


 


369
ILAGNIIHSL
5.600


 


657
QYSMYGHKTT
5.000


 


804
MYSRPRKVLV
5.000


 


660
MYGHKTTHHT
5.000


 


493
QFTHLPVSNI
5.000


 


647
QMRDNSPVHL
4.800


 


206
HIGRILDLQL
4.800


 


488
NLKTNQFTHL
4.800


 


108
FNGLGLLKQL
4.800


 


668
HTTERPSASL
4.800


 


189
DLRGNQLQTL
4.800


 


78
MLHTNDFSGL
4.800


 


751
DASSLYRNIL
4.800


 


548
SPGHLDKKEL
4.400


 


790
YPGAHEELKL
4.400


 


297
STKTTSILKL
4.400


 


814
EQTKNEYFEL
4.400


 


614
LLIMFITIVF
4.200


 


217
DNKWACNCDL
4.000


 


9
YSSLLACISL
4.000


 


409
NLTRLQKLYL
4.000


 


713
DAKHLQRSLL
4.000


 


105
IGAFNGLGLL
4.000


 


431
FLGLHNLEYL
4.000


 


433
GLHNLEYLYL
4.000


 


551
HLDKKELKAL
4.000


 


556
ELKALNSEIL
4.000


 


374
IIHSLMKSDL
4.000


 


601
LTDAVPLSVL
4.000


 


104
EIFAFNGLGL
4.000


 


393
HLGNNRIEVL
4.000


 


404
EGSFMNLTRL
4.000

















TABLE XIV







V3-HLA-A24-10mers-



158P1D7


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













8
HMGAHEELKL
4.400



 


6
EQHMGAHEEL
4.400


 


4
LYEQHMGAHE
0.750


 


9
MGAHEELKLM
0.500


 


2
ASLYEQHMGA
0.150


 


3
SLYEQHMGAH
0.012


 


1
SASLYEQHMG
0.010


 


5
YEQHMGAHEE
0.002


 


7
QHMGAHEELK
0.002

















TABLE XIV







V4-HLA-A24-10mers-



158P1D7


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













2
IIHSLMKSIL
4.000



 


1
NIIHSLMKSI
1.800


 


4
HSLMKSILWS
0.150


 


6
LMKSILWSKA
0.132


 


8
KSILWSKASG
0.030


 


14
KASGRGRREE
0.022


 


5
SLMKSILWSK
0.021


 


9
SILWSKASGR
0.015


 


10
ILWSKASGRG
0.010


 


3
IHSLMKSILW
0.010


 


7
MKSILWSKAS
0.010


 


12
WSKASGRGRR
0.010


 


11
LWSKASGRGR
0.010


 


13
SKASGRGRRE
0.001

















TABLE XV







V1-HLA-B7-9mers-



158P1D7


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













456
NPMPKLKVL
240.000



 


458
MPKLKVLYL
80.000


 


692
SPSFGPKHL
80.000


 


61
VPPSRPFQL
80.000


 


517
NPWDCSCDL
80.000


 


604
AVPLSVLIL
60.000


 


26
SSRGSCDSL
40.000


 


207
IGRILDLQL
40.000


 


410
LTRLQKLYL
40.000


 


159
LNRLKVLIL
40.000


 


774
YLRKNIAQL
40.000


 


625
AAGIVVLVL
36.000


 


336
KVLSPSGLL
30.000


 


481
GVPLTKVNL
20.000


 


182
FVPLTHLDL
20.000


 


462
KVLYLNNNL
20.000


 


652
SPVHLQYSM
20.000


 


575
MPTQTSYLM
20.000


 


752
ASSLYRNIL
18.000


 


370
LAGNIIHSL
12.000


 


154
SAFSKLNRL
12.000


 


713
DAKHLQRSL
12.000


 


221
ACNCDLLQL
12.000


 


106
GAFNGLGLL
12.000


 


249
SPPFFKGSI
8.000


 


306
LPTKAPGLI
8.000


 


250
PPFFKGSIL
8.000


 


360
PPPQNPRKL
8.000


 


453
GTFNPMPKL
6.000


 


310
APGLIPYIT
6.000


 


316
YITKPSTQL
6.000


 


400
EVLEEGSFM
5.000


 


429
GMFLGLHNL
4.000


 


418
LNGNHLTKL
4.000


 


544
ILCTSPGHL
4.000


 


826
NLHAEPDYL
4.000


 


350
RNIESLSDL
4.000


 


4
WIHLFYSSL
4.000


 


501
NILDDLDLL
4.000


 


109
NGLGLLKQL
4.000


 


607
LSVLILGLL
4.000


 


71
LLNNGLTML
4.000


 


599
RSLTDAVPL
4.000


 


739
TTNQSTEFL
4.000


 


87
LTNAISIHL
4.000


 


130
DTFHGLENL
4.000


 


415
KLYLNGNHL
4.000


 


175
LPPNIFRFV
4.000


 


105
IGAFNGLGL
4.000


 


296
MSTKTTSIL
4.000


 


63
PSRPFQLSL
4.000


 


590
TTNTADTIL
4.000


 


767
QQLGITEYL
4.000


 


133
HGLENLEFL
4.000


 


500
SNILDDLDL
4.000


 


305
KLPTKAPGL
4.000


 


394
LGNNRIEVL
4.000


 


815
QTKNEYFEL
4.000


 


466
LNNNLLQVL
4.000


 


520
DCSCDLVGL
4.000


 


747
LSFQDASSL
4.000


 


623
FCAAGIVVL
4.000


 


574
SMPTQTSYL
4.000


 


527
GLQQWIQKL
4.000


 


426
LSKGMFLGL
4.000


 


329
CPIPCNCKV
4.000


 


474
LPPHIFSGV
4.000


 


10
SSLLACISL
4.000


 


68
QLSLLNNGL
4.000


 


405
GSFMNLTRL
4.000


 


758
NILEKEREL
4.000


 


17
SLHSQTPVL
4.000


 


225
DLLQLKTWL
4.000


 


119
INHNSLEIL
4.000


 


408
MNLTRLQKL
4.000


 


463
VLYLNNNLL
4.000


 


537
KNTVTDDIL
4.000


 


116
QLHINHNSL
4.000


 


196
QLTPYVGFL
4.000


 


432
LGLHNLEYL
4.000


 


258
LSRLKKESI
4.000


 


593
TADTILRSL
3.600


 


792
GAHEELKLM
3.000


 


674
SASLYEQHM
3.000


 


371
AGNIIHSLM
3.000


 


597
ILRSLTDAV
2.000


 


608
SVLILGLLI
2.000


 


807
RPRKVLVEQ
2.000


 


805
YSRPRKVLV
2.000


 


498
PVSNILDDL
2.000


 


364
NPRKLILAG
2.000


 


339
SPSGLLIHC
2.000


 


586
TPATTTNTA
2.000


 


278
DPSGSLHLA
2.000


 


314
IPYITKPST
2.000


 


714
AKHLQRSLL
1.800


 


361
PPQNPRKLI
1.800


 


669
TTERPSASL
1.800


 


234
ENMPPQSIL
1.800


 


383
LVEYFTLEM
1.500

















TABLE XV







V3-HLA-B7-9mers-



158P1D7


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
MGAHEELKL
4.000



 


6
QHMGAHEEL
1.200


 


2
SLYEQHMGA
0.100


 


1
ASLYEQHMG
0.030


 


5
EQHMGAHEE
0.010


 


7
HMGAHEELK
0.010


 


4
YEQHMGAHE
0.001


 


3
LYEQHMGAH
0.000

















TABLE XV







V4-HLA-B7-9mers-



158P1D7


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












1
IIHSLMKSI
0.400


 


2
IHSLMKSIL
0.400


 


4
SLMKSILWS
0.060


 


14
ASGRGRREE
0.045


 


13
KASGRGRRE
0.030


 


3
HSLMKSILW
0.020


 


7
KSILWSKAS
0.020


 


8
SILWSKASG
0.010


 


11
WSKASGRGR
0.010


 


9
ILWSKASGR
0.010


 


6
MKSILWSKA
0.010


 


5
LMKSILWSK
0.010


 


12
SKASGRGRR
0.002


 


10
LWSKASGRG
0.001

















TABLE XVI







V1-HLA-B7-10mers-158P1D7



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













249
SPPFFKGSIL
80.000



 


548
SPGHLDKKEL
80.000


 


497
LPVSNILDDL
80.000


 


329
CPIPCNCKVL
80.000


 


790
YPGAHEELKL
80.000


 


605
VPLSVLILGL
80.000


 


359
RPPPQNPRKL
80.000


 


189
DLRGNQLQTL
40.000


 


647
QMRDNSPVHL
40.000


 


566
CPGLVNNPSM
20.000


 


807
RPRKVLVEQT
20.000


 


462
KVLYLNNNLL
20.000


 


60
SVPPSRPFQL
20.000


 


713
DAKHLQRSLL
18.000


 


751
DASSLYRNIL
18.000


 


603
DAVPLSVLIL
12.000


 


624
CAAGIVVLVL
12.000


 


428
KGMFLGLHNL
12.000


 


825
ANLHAEPDYL
12.000


 


220
WACNCDLLQL
12.000


 


803
LMYSRPRKVL
9.000


 


198
LPYVGFLEHI
8.000


 


361
PPQNPRKLIL
8.000


 


176
PPNIFRFVPL
8.000


 


475
PPHIFSGVPL
8.000


 


62
PPSRPFQLSL
8.000


 


179
IFRFVPLTHL
6.000


 


668
HTTERPSASL
6.000


 


383
LVEYFTLEML
6.000


 


608
SVLILGLLIM
5.000


 


393
HLGNNRIEVL
4.000


 


589
TTTNTADTIL
4.000


 


783
KTTNQSTEFL
4.000


 


78
MLHTNDFSGL
4.000


 


16
ISLHSQTPVL
4.000


 


9
YSSLLACISL
4.000


 


814
EQTKNEYFEL
4.000


 


407
FMNLTRLQKL
4.000


 


575
MPTQTSYLMV
4.000


 


4
WIHLFYSSLL
4.000


 


417
YLNGNHLTKL
4.000


 


63
PSRPFQLSLL
4.000


 


757
RNILEKEREL
4.000


 


108
FNGLGLLKQL
4.000


 


409
NLTRLQKLYL
4.000


 


556
ELKALNSEIL
4.000


 


166
ILNDNAIESL
4.000


 


217
DNKWACNCDL
4.000


 


364
NPRKLILAGN
4.000


 


295
RMSTKTTSIL
4.000


 


517
NPWDCSCDLV
4.000


 


499
VSNILDDLDL
4.000


 


465
YLNNNLLQVL
4.000


 


104
EIGAFNGLGL
4.000


 


346
HCQERNIESL
4.000


 


691
RSPSFGPKHL
4.000


 


433
GLHNLEYLYL
4.000


 


126
ILKEDTFHGL
4.000


 


526
VGLQQWIQKL
4.000


 


488
NLKTNQFTHL
4.000


 


297
STKTTSILKL
4.000


 


115
KQLHINHNSL
4.000


 


560
LNSEILCPGL
4.000


 


334
NCKVLSPSGL
4.000


 


156
FSKLNRLKVL
4.000


 


195
LQTLPYVGFL
4.000


 


86
GLTNAISIHL
4.000


 


592
NTADTILRSL
4.000


 


431
FLGLHNLEYL
4.000


 


746
FLSFQDASSL
4.000


 


423
LTKLSKGMFL
4.000


 


158
KLNRLKVLIL
4.000


 


369
ILAGNIIHSL
4.000


 


206
HIGRILDLQL
4.000


 


516
DNPWDCSCDL
4.000


 


494
FTHLPVSNIL
4.000


 


500
SNILDDLDLL
4.000


 


404
EGSFMNLTRL
4.000


 


766
LQQLGITEYL
4.000


 


455
FNPMPKLKVL
4.000


 


480
SGVPLTKVNL
4.000


 


105
IGAFNGLGLL
4.000


 


25
LSSRGSCDSL
4.000


 


236
MPPQSIIGDV
4.000


 


67
FQLSLLNNGL
4.000


 


374
IIHSLMKSDL
4.000


 


543
DILCTSPGHL
4.000


 


70
SLLNNGLTML
4.000


 


118
HINHNSLEIL
4.000


 


425
KLSKGMFLGL
4.000


 


828
HAEPDYLEVL
3.600


 


287
ATSSINDSRM
3.000


 


370
LAGNIIHSLM
3.000


 


22
TPVLSSRGSC
3.000


 


278
DPSGSLHLAA
2.000


 


324
LPGPYCPIPC
2.000


 


482
VPLTKVNLKT
2.000


 


163
KVLILNDNAI
2.000


 


326
GPYCPIPCNC
2.000


 


336
KVLSPSGLLI
2.000

















TABLE XVI







V3-HLA-B7-10mers-158P1D7



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













8
HMGAHEELKL
4.000



 


6
EQHMGAHEEL
4.000


 


9
MGAHEELKLM
1.000


 


2
ASLYEQHMGA
0.300


 


1
SASLYEQHMG
0.030


 


3
SLYEQHMGAH
0.010


 


7
QHMGAHEELK
0.003


 


5
YEQHMGAHEE
0.001


 


4
LYEQHMGAHE
0.000

















TABLE XVI







V4-HLA-B7-10mers-



158P1D7


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













2
IIHSLMKSIL
4.000



 


1
NIIHSLMKSI
0.400


 


6
LMKSILWSKA
0.100


 


14
KASGRGRREE
0.045


 


5
SLMKSILWSK
0.030


 


4
HSLMKSILWS
0.020


 


12
WSKASGRGRR
0.015


 


8
KSILWSKASG
0.010


 


10
ILWSKASGRG
0.010


 


9
SILWSKASGR
0.010


 


7
MKSILWSKAS
0.002


 


3
IHSLMKSILW
0.002


 


13
SKASGRGRRE
0.001


 


11
LWSKASGRGR
0.001

















TABLE XVII







VI-HLA-B35-9mers-



158P1D7


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













458
MPKLKVLYL
60.000



 


652
SPVHLQYSM
40.000


 


575
MPTQTSYLM
40.000


 


517
NPWDCSCDL
40.000


 


456
NPMPKLKVL
20.000


 


692
SPSFGPKHL
20.000


 


61
VPPSRPFQL
20.000


 


792
GAHEELKLM
18.000


 


26
SSRGSCDSL
15.000


 


426
LSKGMFLGL
15.000


 


599
RSLTDAVPL
15.000


 


727
HSPLTGSNM
10.000


 


288
TSSINDSRM
10.000


 


713
DAKHLQRSL
9.000


 


378
LMKSDLVEY
9.000


 


306
LPTKAPGLI
8.000


 


249
SPPFFKGSI
8.000


 


747
LSFQDASSL
7.500


 


228
QLKTWLENM
6.000


 


674
SASLYEQHM
6.000


 


400
EVLEEGSFM
6.000


 


258
LSRLKKESI
6.000


 


796
ELKLMETLM
6.000


 


752
ASSLYRNIL
5.000


 


607
LSVLILGLL
5.000


 


10
SSLLACISL
5.000


 


59
ISVPPSRPF
5.000


 


296
MSTKTTSIL
5.000


 


405
GSFMNLTRL
5.000


 


815
QTKNEYFEL
4.500


 


350
RNIESLSDL
4.000


 


329
CPIPCNCKV
4.000


 


474
LPPHIFSGV
4.000


 


782
LQPDMEAHY
4.000


 


65
RPFQLSLLN
4.000


 


175
LPPNIFRFV
4.000


 


805
YSRPRKVLV
3.000


 


774
YLRKNIAQL
3.000


 


154
SAFSKLNRL
3.000


 


410
LTRLQKLYL
3.000


 


207
IGRILDLQL
3.000


 


370
LAGNIIHSL
3.000


 


106
GAFNGLGLL
3.000


 


156
FSKLNRLKV
3.000


 


501
NILDDLDLL
3.000


 


423
LTKLSKGMF
3.000


 


625
AAGIVVLVL
3.000


 


159
LNRLKVLIL
3.000


 


89
NAISIHLGF
3.000


 


309
KAPGLIPYI
2.400


 


609
VLILGLLIM
2.000


 


339
SPSGLLIHC
2.000


 


450
ILPGTFNPM
2.000


 


415
KLYLNGNHL
2.000


 


133
HGLENLEFL
2.000


 


360
PPPQNPRKL
2.000


 


278
DPSGSLHLA
2.000


 


738
KTTNQSTEF
2.000


 


422
HLTKLSKGM
2.000


 


586
TPATTTNTA
2.000


 


314
IPYITKPST
2.000


 


310
APGLIPYIT
2.000


 


336
KVLSPSGLL
2.000


 


778
NAIQLQPDM
2.000


 


766
LQQLGITEY
2.000


 


326
GPYCPIPCN
2.000


 


409
NLTRLQKLY
2.000


 


631
LVLHRRRRY
2.000


 


70
SLLNNGLTM
2.000


 


265
SICPTPPVY
2.000


 


572
NPSMPTQTS
2.000


 


462
KVLYLNNNL
2.000


 


305
KLPTKAPGL
2.000


 


192
GNQLQTLPY
2.000


 


825
ANLHAEPDY
2.000


 


566
CPGLVNNPS
2.000


 


684
SPMVHVYRS
2.000


 


250
PPFFKGSIL
2.000


 


433
GLHNLEYLY
2.000


 


486
KVNLKTNQF
2.000


 


331
IPCNCKVLS
2.000


 


537
KNTVTDDIL
2.000


 


431
FLGLHNLEY
2.000


 


758
NILEKEREL
2.000


 


22
TPVLSSRGS
2.000


 


152
EPSAFSKLN
2.000


 


682
MVSPMVHVY
2.000


 


371
AGNIIHSLM
2.000


 


650
DNSPVHLQY
2.000


 


826
NLHAEPDYL
1.500


 


500
SNILDDLDL
1.500


 


148
ITVIEPSAF
1.500


 


520
DCSCDLVGL
1.500


 


221
ACNCDLLQL
1.500


 


561
NSEILCPGL
1.500


 


63
PSRPFQLSL
1.500


 


293
DSRMSTKTT
1.500


 


741
NQSTEFLSF
1.500


 


675
ASLYEQHMV
1.500


 


100
IADIEIGAF
1.350

















TABLE XVII







V3-HLA-B35-9mers-



158P1D7


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
MGAHEELKL
1.500



 


2
SLYEQHMGA
0.200


 


6
QHMGAHEEL
0.100


 


1
ASLYEQHMG
0.075


 


5
EQHMGAHEE
0.010


 


7
HMGAHEELK
0.010


 


4
YEQHMGAHE
0.001


 


3
LYEQHMGAH
0.000

















TABLE XVII







V4-HLA-B35-9mers-



158P1D7


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
HSLMKSILW
2.500



 


7
KSILWSKAS
1.000


 


1
IIHSLMKSI
0.400


 


11
WSKASGRGR
0.150


 


2
IHSLMKSIL
0.100


 


4
SLMKSILWS
0.100


 


13
KASGRGRRE
0.060


 


14
ASGRGRREE
0.050


 


5
LMKSILWSK
0.030


 


6
MKSILWSKA
0.010


 


9
ILWSKASGR
0.010


 


8
SILWSKASG
0.010


 


10
LWSKASGRG
0.001


 


12
SKASGRGRR
0.001

















TABLE XVIII







V1-HLA-B35-10mers-



158P1D7


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













319
KPSTQLPGPY
80.000



 


566
CPGLVNNPSM
40.000


 


728
SPLTGSNMKY
40.000


 


572
NPSMPTQTSY
40.000


 


652
SPVHLQYSMY
40.000


 


359
RPPPQNPRKL
40.000


 


456
NPMPKLKVLY
40.000


 


548
SPGHLDKKEL
30.000


 


790
YPGAHEELKL
30.000


 


329
CPIPCNCKVL
20.000


 


249
SPPFFKGSIL
20.000


 


605
VPLSVLILGL
20.000


 


497
LPVSNILDDL
20.000


 


156
FSKLNRLKVL
15.000


 


824
KANLHEAPDY
12.000


 


807
RPRKVLVEQT
12.000


 


747
LSFQDASSLY
10.000


 


691
RSPSFGPKHL
10.000


 


264
ESICPTPPVY
10.000


 


651
NSPVHLQYSM
10.000


 


69
LSLLNNGLTM
10.000


 


796
ELKLMETLMY
9.000


 


713
DAKHLQRSLL
9.000


 


198
LPYVGFLEHI
8.000


 


517
NPWDCSCDLV
8.000


 


499
VSNILDDLDL
7.500


 


126
ILKEDTFHGL
6.000


 


370
LAGNIIHSLM
6.000


 


458
MPKLKVLYLN
6.000


 


364
NPRKLILAGN
6.000


 


647
QMRDNSPVHL
6.000


 


446
AIKEILPGTF
6.000


 


535
LSKNTVTDDI
6.000


 


25
LSSRGSCDSL
5.000


 


9
YSSLLACISL
5.000


 


173
ESLPPNIFRF
5.000


 


16
ISLHSQTPVL
5.000


 


380
KSDLVEYFTL
4.500


 


220
WACNCDLLQL
4.500


 


435
HNLEYLYLEY
4.000


 


236
MPPQSIIGDV
4.000


 


382
DLVEYFTLEM
4.000


 


35
CNCEEKDGTM
4.000


 


575
MPTQTSYLMV
4.000


 


777
KNIAQLQPDM
4.000


 


191
RGNQLQTLPY
4.000


 


65
RPFQLSLLNN
4.000


 


811
VLVEQTKNEY
4.000


 


46
INCEAKGIKM
4.000


 


556
ELKALNSEIL
3.000


 


99
NIADIEIGAF
3.000


 


378
LMKSDLVEYF
3.000


 


751
DASSLYRNIL
3.000


 


423
LTKLSKGMFL
3.000


 


488
NLKTNQFTHL
3.000


 


377
SLMKSDLVEY
3.000


 


334
NCKVLSPSGL
3.000


 


603
DAVPLSVLIL
3.000


 


624
CAAGIVVLVL
3.000


 


217
DNKWACNCDL
3.000


 


297
STKTTSILKL
3.000


 


189
DLRGNQLQTL
3.000


 


170
NAIESLPPNI
2.400


 


475
PPHIFSGVPL
2.000


 


607
LSVLILGLLI
2.000


 


346
HCQERNIESL
2.000


 


295
RMSTKTTSIL
2.000


 


166
ILNDNAIESL
2.000


 


630
VLVLHRRRRY
2.000


 


765
ELQQLGITEY
2.000


 


115
KQLHINHNSL
2.000


 


61
VPPSRPFQLS
2.000


 


278
DPSGSLHLAA
2.000


 


432
LGLHNLEYLY
2.000


 


757
RNILEKEREL
2.000


 


227
LQLKTWLENM
2.000


 


91
ISIHLGFNNI
2.000


 


738
KTTNQSTEFL
2.000


 


176
PPNIFRFVPL
2.000


 


781
QLQPDMEAHY
2.000


 


592
NTADTILRSL
2.000


 


158
KLNRLKVLIL
2.000


 


84
FSGLTNAISI
2.000


 


668
HTTERPSASL
2.000


 


248
NSPPFFKGSI
2.000


 


287
ATSSINDSRM
2.000


 


428
KGMFLGLHNL
2.000


 


681
HMVSPMVHVY
2.000


 


22
TPVLSSRGSC
2.000


 


449
EILPGTFNPM
2.000


 


425
KLSKGMFLGL
2.000


 


408
MNLTRLQKLY
2.000


 


560
LNSEILCPGL
2.000


 


361
PPQNPRKLIL
2.000


 


62
PPSRPFQLSL
2.000


 


574
SMPTQTSYLM
2.000


 


482
VPLTKVNLKT
2.000


 


324
LPGPYCPIPC
2.000


 


462
KVLYLNNNLL
2.000


 


608
SVLILGLLIM
2.000

















TABLE XVIII







V3-HLA-B35-10mers



158P1D7


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













9
MGAHEELKLM
3.000



 


8
HMGAHEELKL
1.500


 


6
EQHMGAHEEL
1.000


 


2
ASLYEQHMGA
0.500


 


1
SASLYEQHMG
0.045


 


3
SLYEQHMGAH
0.020


 


5
YEQHMGAHEE
0.001


 


7
QHMGAHEELK
0.001


 


4
LYEQHMGAHE
0.000

















TABLE XVIII







V4-HLA-B35-10mers-



158P1D7


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













2
IIHSLMKSIL
1.000



 


4
HSLMKSILWS
0.500


 


1
NIIHSLMKSI
0.400


 


6
LMKSILWSKA
0.300


 


12
WSKASGRGRR
0.150


 


8
KSILWSKASG
0.100


 


14
KASGRGRREE
0.060


 


3
IHSLMKSILW
0.050


 


7
MKSILWSKAS
0.010


 


9
SILWSKASGR
0.010


 


10
ILWSKASGRG
0.010


 


5
SLMKSILWSK
0.010


 


13
SKASGRGRRE
0.001


 


11
LWSKASGRGR
0.001

















TABLE V







158P1D7 v.6-



HLA A1-9-mers


Each peptide is a portion of


SEQ ID NO: 13; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.









Pos
Subsequence
Score













7
LMNPSFGPK
1.000



 


5
HSLMNPSFG
0.015


 


1
GNIIHSLMN
0.013


 


4
IHSLMNPSF
0.010


 


3
IIHSLMNPS
0.010


 


8
MNPSFGPKH
0.005


 


6
SLMNPSFGP
0.005


 


2
NIIHSLMNP
0.005


 


15
KHLEEEEER
0.005


 


9
NPSFGPKHL
0.003


 


11
SFGPKHLEE
0.003


 


10
PSFGPKHLE
0.000


 


12
FGPKHLEEE
0.000


 


13
GPKHLEEEE
0.000


 


14
PKHLEEEEE
0.000

















TABLE VI







158P1D7 v.6-



HLA A1-10mers


Each peptide is a portion of


SEQ ID NO: 13; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.









Pos
Subsequence
Score













4
IIHSIMNPSF
0.200



 


7
SLMNpSFGPK
0.200


 


8
LMNPsFGPKH
0.100


 


1
AGNIiHSLMN
0.013


 


3
NIIHsLMNPS
0.010


 


6
HSLMnPSFGP
0.007


 


9
MNPSfGPKHL
0.003


 


2
GNIIhSLMNP
0.001


 


11
PSFGpKHLEE
0.001


 


15
PKHLeEEEER
0.001


 


5
IHSLmNPSFG
0.001


 


12
SFGPkHLEEE
0.001


 


10
NPSFgPKHLE
0.000


 


13
FGPKhLEEEE
0.000


 


14
GPKHIEEEEE
0.000

















TABLE VII







158P1D7 v.6-



HLA A0201-9-mers


Each peptide is a portion of


SEQ ID NO: 13; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.









Pos
Subsequence
Score













6
SLMNPSFGP
0.320



 


9
NPSFGPKHL
0.139


 


3
IIHSLMNPS
0.040


 


2
NIIHSLMNP
0.005


 


7
LMNPSFGPK
0.005


 


8
MNPSFGPKH
0.003


 


12
FGPKHLEEE
0.001


 


1
GNIIHSLMN
0.000


 


5
HSLMNPSFG
0.000


 


15
KHLEEEEER
0.000


 


4
IHSLMNPSF
0.000


 


11
SFGPKHLEE
0.000


 


10
PSFGPKHLE
0.000


 


13
GPKHLEEEE
0.000


 


14
PKHLEEEEE
0.000

















TABLE VIII







158P1D7 v.6-



HLA A0201-10-mers


Each peptide is a portion of


SEQ ID NO: 13; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.









Pos
Subsequence
Score













8
LMNPsFGPKH
0.348



 


9
MNPSfGPKHL
0.237


 


3
NIIHsLMNPS
0.024


 


4
IIHSIMNPSF
0.017


 


7
SLMNpSFGPK
0.014


 


1
AGNIiHSLMN
0.000


 


5
IHSLmNPSFG
0.000


 


2
GNIIhSLMNP
0.000


 


13
FGPKhLEEEE
0.000


 


10
NPSFgPKHLE
0.000


 


6
HSLMnPSFGP
0.000


 


12
SFGPkHLEEE
0.000


 


11
PSFGpKHLEE
0.000


 


14
GPKHIEEEEE
0.000


 


15
PKHLeEEEER
0.000

















TABLE IX







158P1D7 v.6-



HLA A3-9-mers


Each peptide is a portion of


SEQ ID NO: 13; each start


position is specified, the


length of peptide is 9 amino


acids and the end position


for each peptide is the start


position plus eight.









Pos
Subsequence
Score













7
LMNPSFGPK
27.000



 


6
SLMNPSFGP
0.135


 


15
KHLEEEEER
0.027


 


2
NIIHSLMNP
0.009


 


3
IIHSLMNPS
0.006


 


9
NPSFGPKHL
0.003


 


4
IHSLMNPSF
0.002


 


8
MNPSFGPKH
0.001


 


13
GPKHLEEEE
0.001


 


1
GNIIHSLMN
0.000


 


5
HSLMNPSFG
0.000


 


10
PSFGPKHLE
0.000


 


11
SFGPKHLEE
0.000


 


12
FGPKHLEEE
0.000


 


14
PKHLEEEEE
0.000

















TABLE X







158P1D7 v.6-



HLA A3-10-mers


Each peptide is a portion of


SEQ ID NO: 13; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.









Pos
Subsequence
Score













7
LMNPSFGPK
27.000



 


6
SLMNPSFGP
0.135


 


15
KHLEEEEER
0.027


 


2
NIIHSLMNP
0.009


 


3
IIHSLMNPS
0.006


 


9
NPSFGPKHL
0.003


 


4
IHSLMNPSF
0.002


 


8
MNPSFGPKH
0.001


 


13
GPKHLEEEE
0.001


 


1
GNIIHSLMN
0.000


 


5
HSLMNPSFG
0.000


 


10
PSFGPKHLE
0.000


 


11
SFGPKHLEE
0.000


 


12
FGPKHLEEE
0.000


 


14
PKHLEEEEE
0.000

















TABLE XI







158P1D7 v.6-



HLA A1101-9-mers


Each peptide is a portion of


SEQ ID NO: 13; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.









Pos
Subsequence
Score













7
LMNPSFGPK
0.400



 


15
KHLEEEEER
0.018


 


6
SLMNPSFGP
0.002


 


2
NIIHSLMNP
0.001


 


9
NPSFGPKHL
0.001


 


13
GPKHLEEEE
0.001


 


11
SFGPKHLEE
0.000


 


8
MNPSFGPKH
0.000


 


3
IIHSLMNPS
0.000


 


1
GNIIHSLMN
0.000


 


4
IHSLMNPSF
0.000


 


5
HSLMNPSFG
0.000


 


12
FGPKHLEEE
0.000


 


10
PSFGPKHLE
0.000


 


14
PKHLEEEEE
0.000

















TABLE XII







158P1D7 v.6-



HLA A1101-10-mers


Each peptide is a portion of


SEQ ID NO: 13; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.









Pos
Subsequence
Score













7
SLMNpSFGPK
0.800



 


4
IIHSIMNPSF
0.004


 


8
LMNPsFGPKH
0.004


 


3
NIIHsLMNPS
0.001


 


14
GPKHIEEEEE
0.001


 


15
PKHLeEEEER
0.000


 


2
GNIIhSLMNP
0.000


 


9
MNPSfGPKHL
0.000


 


10
NPSFgPKHLE
0.000


 


12
SFGPkHLEEE
0.000


 


6
HSLMnPSFGP
0.000


 


1
AGNIiHSLMN
0.000


 


13
FGPKhLEEEE
0.000


 


5
IHSLmNPSFG
0.000


 


11
PSFGpKHLEE
0.000

















TABLE XIII







158P1D7 v.6-



HLA A24-9-mers


Each peptide is a portion of


SEQ ID NO: 13; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.









Pos
Subsequence
Score













9
NPSFGPKHL
4.000



 


4
IHSLMNPSF
0.200


 


1
GNIIHSLMN
0.150


 


3
IIHSLMNPS
0.144


 


11
SFGPKHLEE
0.066


 


7
LMNPSFGPK
0.022


 


12
FGPKHLEEE
0.017


 


8
MNPSFGPKH
0.017


 


6
SLMNPSFGP
0.015


 


5
HSLMNPSFG
0.015


 


2
NIIHSLMNP
0.015


 


13
GPKHLEEEE
0.013


 


15
KHLEEEEER
0.004


 


10
PSFGPKHLE
0.001


 


14
PKHLEEEEE
0.000

















TABLE XIV







158P1D7 v.6-



HLA A24-10-mers


Each peptide is a portion of


SEQ ID NO; 13; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.









Pos
Subsequence
Score













9
MNPSfGPKHL
6.000



 


4
IIHSIMNPSF
2.000


 


3
NIIHsLMNPS
0.216


 


1
AGNIiHSLMN
0.150


 


12
SFGPkHLEEE
0.666


 


13
FGPKhLEEEE
0.020


 


8
LMNPsFGPKH
0.020


 


7
SLMNpSFGPK
0.018


 


2
GNIIhSLMNP
0.015


 


6
HSLMnPSFGP
0.015


 


14
GPKHIEEEEE
0.011


 


10
NPSFgPKHLE
0.010


 


11
PSFGpKHLEE
0.001


 


5
IHSLmNPSFG
0.001


 


15
PKHLeEEEER
0.000

















TABLE XV







158P1D7 v.6-



HLA B7-9-mers


Each peptide is a portion of


SEQ ID NO: 13; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position plus eight.









Pos
Subsequence
Score













9
NPSFGPKHL
80.000



 


13
GPKHLEEEE
0.200


 


6
SLMNPSFGP
0.045


 


3
IIHSLMNPS
0.020


 


1
GNIIHSLMN
0.020


 


5
HSLMNPSFG
0.010


 


7
LMNPSFGPK
0.010


 


8
MNPSFGPKH
0.010


 


2
NIIHSLMNP
0.010


 


12
FGPKHLEEE
0.010


 


4
IHSLMNPSF
0.002


 


10
PSFGPKHLE
0.002


 


15
KHLEEEEER
0.001


 


11
SFGPKHLEE
0.001


 


14
PKHLEEEEE
0.000

















TABLE XVI







158P1D7 v.6-



HLA B7-10-mers


Each peptide is a portion of


SEQ ID NO: 13; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.









Pos
Subsequence
Score













9
MNPSfGPKHL
4.000



 


10
NPSFgPKHLE
0.300


 


14
GPKHIEEEEE
0.200


 


1
AGNIiHSLMN
0.060


 


7
SLMNpSFGPK
0.030


 


4
IIHSIMNPSF
0.020


 


3
NIIHsLMNPS
0.020


 


6
HSLMnPSFGP
0.015


 


13
FGPKhLEEEE
0.010


 


8
LMNPsFGPKH
0.010


 


2
GNIIhSLMNP
0.010


 


12
SFGPkHLEEE
0.001


 


11
SFGPkHLEEE
0.001


 


5
IHSLmNPSFG
0.001


 


15
PKHLeEEEER
0.000

















TABLE XVII







158P1D7 v.6-



HLA B3501-9-mers


Each peptide is a portion of


SEQ ID NO: 13; each start


position is specified, the


length of peptide is 9 amino


acids, and the end position


for each peptide is the start


position pius eight.









Pos
Subsequence
Score













9
NPSFGPKHL
20.000



 


13
GPKHLEEEE
0.600


 


1
GNIIHSLMN
0.100


 


4
IHSLMNPSF
0.100


 


3
IIHSLMNPS
0.100


 


5
HSLMNPSFG
0.050


 


7
LMNPSFGPK
0.010


 


8
MNPSFGPKH
0.010


 


6
SLMNPSFGP
0.010


 


2
NIIHSLMNP
0.010


 


12
FGPKHLEEE
0.010


 


15
KHLEEEEER
0.006


 


10
PSFGPKHLE
0.005


 


11
SFGPKHLEE
0.001


 


14
PKHLEEEEE
0.000

















TABLE XVIII







158P1D7 v.6-



HLA B3501-10-mers


Each peptide is a portion of


SEQ ID NO: 13; each start


position is specified, the


length of peptide is 10 amino


acids, and the end position


for each peptide is the start


position plus nine.









Pos
Subsequence
Score













9
MNPSfGPKHL
1.000



 


4
IIHSIMNPSF
1.000


 


14
GPKHIEEEEE
0.900


 


10
NPSFgPKHLE
0.200


 


1
AGNIiHSLMN
0.100


 


3
NIIHsLMNPS
0.100


 


6
HSLMnPSFGP
0.050


 


2
GNIIhSLMNP
0.010


 


8
LMNPsFGPKH
0.010


 


13
FGPKhLEEEE
0.010


 


7
SLMNpSFGPK
0.010


 


11
PSFGpKHLEE
0.005


 


12
SFGPkHLEEE
0.001


 


5
IHSLmNPSFG
0.001


 


15
PKHLeEEEER
0.000

















TABLE XIX







Motif-bearing Subsequences of the



158P1D7 Protein







Protein Motifs of 158P1D7











N-glycosyation site



Number of matches: 3


1 292-295 NDSR (SEQ ID NO: 45)


2 409-412 NLTR (SEQ ID NO: 46)


3 741-744 NQST (SEQ ID NO: 47)


 


cAMP- and cGMP-dependent protein kinase


phosphorylation site


262-265 KKES (SEQ ID NO: 48)


 


Protein kinase C phosphorylation site


Number of matches: 3


1 26-28 SSR


2 297-299 STK


3 670-672 TER


 


Casein kinase II phosphorylation site


Number of matches: 12


 1 149-152 TVIE (SEQ ID NO: 49)


 2 186-189 THLD (SEQ ID NO: 50)


 3 231-234 TWLE (SEQ ID NO: 51)


 4 290-293 SIND (SEQ ID NO: 52)


 5 354-357 SLSD (SEQ ID NO: 53)


 6 510-513 TQID (SEQ ID NO: 54)


 7 539-542 TVTD (SEQ ID NO: 55)


 8 600-603 SLTD (SEQ ID NO: 56)


 9 676-679 SLYE (SEQ ID NO: 57)


10 720-723 SLLE (SEQ ID NO: 58)


11 748-751 SFQD (SEQ ID NO: 59)


12 816-819 TKNE (SEQ ID NO: 60)


 


Tyrosine kinase phosphorylation site


798-805 KLMETLMY (SEQ ID NO: 61)


 


N-myristoylation site


Number of matches: 8


1 29-34 GSCDSL(SEQ ID NO: 62)


2 86-91 GLTNAI(SEQ ID NO: 63)


3 106-111 GAFNGL(SEQ ID NO: 64)


4 255-260 GSILSR(SEQ ID NO: 65)


5 405-410 GSFMNL(SEQ ID NO: 66)


6 420-425 GNHLTK(SEQ ID NO: 67)


7 429-434 GMFLGL(SEQ ID NO: 68)


8 481-486 GVPLTK(SEQ ID NO: 69)


 


Two Protein Motifs were predicted by Pfam


1-Archaeal-ATPase at aa 441-451


2-Leucine rich repeat C-terminal at aa 218-268


and aa 517-567
















TABLE XX







Frequently Occurring Motifs











avrg. %




Name
identity
Description
Potential Function





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





transcription factor, nuclear location probable


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




terminal)/b6/petB


ig
19%
Immunoglobulin domain
domains are one hundred amino acids long and





include a conserved intradomain disulfide bond.


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





containing a Trp-Asp motif. Function in signal





transduction and protein interaction


PDZ
23%
PDZ domain
may function in targeting signaling molecules to





sub-membranous sites


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





interactions


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





serine/threonine and tyrosine protein kinases





containing an ATP binding site and a catalytic





site


PH
16%
PH domain
pleckstrin homology involved in intracellular





signaling or as constituents of the cytoskeleton


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





domain of membrane-bound proteins or in





secreted proteins


rvt
49%
Reverse transcriptase (RNA-




dependent DNA polymerase)


ank
25%
Ank repeat
Cytoplasmic protein, associates integral





membrane proteins to the cytoskeleton


oxidored q1
32%
NADH-
membrane associated. Involved in proton




Ubiquinone/plastoquinone
translocation across the membrane




(complex I), various chains


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





loop flanked on both sides by a 12 residue alpha-





helical domain


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





catalytic aspartyl residue


Collagen
42%
Collagen triple helix repeat (20
extracellular structural proteins involved in




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, with




(rhodopsin family)
the N-terminus located extracellularly while the





C-terminus is cytoplasmic. Signal through G





proteins
















TABLE XXI





TNM CLASSIFICATION OF BLADDER TUMORS







Primary tumor (T)







The suffix (m) should be added to the appropriate T category to


indicate multiple tumors. The suffix (is) may be added to any T


to indicate the presence of associated carcinoma in situ.








TX
Primary tumor cannot be assessed


TO
No evidence of primary tumor


Ta
Noninvasive papillary carcinoma


Tis
Carcinoma in situ: “flat tumor”


T1
Tumor invades sub-epithelial connective tissue


T2
Tumor invades superficial muscle (inner half)


T3
Tumor invades deep muscle or perivesical fat










T3a
Tumor invades deep muscle (outer half)



T3b
Tumor invades perivesical fat










i.
microscopically



ii.
macroscopically (extravesical mass)








T4
Tumor invades any of the following: prostate, uterus,



vagina, pelvic wall, or abdominal wall










T4a
Tumor invades the prostate, uterus, vagina



T4b
Tumor invades the pelvic wall or abdominal wall or both







Regional lymph nodes (N)







Regional lymph nodes are those within the true pelvis: all


others are distant nodes








NX
Regional lymph nodes cannot be assessed


N0
No regional lymph node metastasis


N1
Metastasis in a single lymph node, 2 cm or less in greatest



dimension


N2
Metastasis in a single lymph node, more than 2 cm but not



more than 5 cm in greatest dimension, or multiple lymph



nodes, none more than 5 cm in greatest dimension


N3
Metastasis in a lymph node more than 5 cm in greatest



dimension







Distant metastasis (M)








MX
Presence of distant metastasis cannot be assessed


M0
No distant metastasis


M1
Distant metastasis







Stage grouping













Stage
0a
Ta
N0
M0




0is
Tis
N0
M0




I
T1
N0
M0




II
T2
N0
M0





T3a
N0
M0




III
T3b
N0
M0





T4a
N0
M0




IV
T4b
N0
M0





Any T
N1-3
M0





Any T
Any N
M1


















TABLE XXII







V1-HLA-A1-



9mers-158P1D7


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













436
NLEYLYLEY
32



 


650
DNSPVHLQY
27


 


308
TKAPGLIPY
25


 


812
LVEQTKNEY
25


 


431
FLGLHNLEY
24


 


601
LTDAVPLSV
24


 


192
GNQLQTLPY
23


 


573
PSMPTQTSY
23


 


265
SICPTPPVY
22


 


797
LKLMETLMY
22


 


1
MKLWIHLFY
21


 


522
SCDLVGLQQ
21


 


670
TERPSASLY
21


 


682
MVSPMVHVY
21


 


711
GSDAKHLQR
20


 


729
PLTGSNMKY
20


 


828
HAEPDYLEV
20


 


320
PSTQLPGPY
19


 


441
YLEYNAIKE
19


 


502
ILDDLDLLT
19


 


551
HLDKKELKA
19


 


748
SFQDASSLY
19


 


223
NCDLLQLKT
18


 


409
NLTRLQKLY
18


 


433
GLHNLEYLY
18


 


546
CTSPGHLDK
18


 


653
PVHLQYSMY
18


 


743
STEFLSFQD
18


 


763
ERELQQLGI
18


 


793
AHEELKLME
18


 


817
KNEYFELKA
18


 


39
EKDGTMLIN
17


 


47
NCEAKGIKM
17


 


81
TNDFSGLTN
17


 


142
QADNNFITV
17


 


276
HEDPSGSLH
17


 


388
TLEMLHLGN
17


 


457
PMPKLKVLY
17


 


540
VTDDILCTS
17


 


669
TTERPSASL
17


 


749
FQDASSLYR
17


 


766
LQQLGITEY
17


 


771
ITEYLRKNI
17


 


56
VSEISVPPS
16


 


380
KSDLVEYFT
16


 


383
LVEYFTLEM
16


 


503
LDDLDLLTQ
16


 


554
KKELKALNS
16


 


631
LVLHRRRRY
16


 


825
ANLHAEPDY
16


 


150
VIEPSAFSK
15


 


337
VLSPSGLLI
15


 


378
LMKSDLVEY
15


 


401
VLEEGSFMN
15


 


782
LQPDMEAHY
15

















TABLE XXII







V3-HLA-A1-



9mers-158P1D7


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













3
LYEQHMGAH
10



 


8
MGAHEELKL
8


 


1
ASLYEQHMG
6


 


2
SLYEQHMGA
5

















TABLE XXII







V4-HLA-A1-



9mers-158P1D7


Each peptide is a portion


of SEQ ID NO: 9; each


start position is specified,


the length of peptide is 9


amino acids, and the end


position for each peptide


is the start position plus


eight.









Pos
123456789
score













3
HSLMKSILW
10



 


4
SLMKSILWS
9


 


14
ASGRGRREE
8


 


11
WSKASGRGR
5


 


12
SKASGRGRR
5


 


7
KSILWSKAS
4

















TABLE XXIII







V1-HLA-A2-



9mers-158P1D7


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













71
LLNNGLTML
29



 


614
LLIMFITIV
29


 


465
YLNNNLLQV
28


 


774
YLRKNIAQL
28


 


429
GMFLGLHNL
27


 


527
GLQQWIQKL
27


 


597
ILRSLTDAV
26


 


17
SLHSQTPVL
25


 


501
NILDDLDLL
25


 


611
ILGLLIMFI
25


 


758
NILEKEREL
25


 


305
KLPTKAPGL
24


 


606
PLSVLILGL
24


 


609
VLILGLLIM
24


 


624
CAAGIVVLV
24


 


68
QLSLLNNGL
23


 


116
QLHINHNSL
23


 


154
SAFSKLNRL
23


 


158
KLNRLKVLI
23


 


164
VLILNDNAI
23


 


196
QTLPYVGFL
23


 


370
LAGNIIHSL
23


 


415
KLYLNGNHL
23


 


439
YLYLEYNAI
23


 


463
VLYLNNNLL
23


 


613
GLLIMFITI
23


 


803
LMYSRPRKV
23


 


106
GAFNGLGLL
22


 


225
DLLQLKTWL
22


 


312
GLIPYITKP
22


 


337
VLSPSGLLI
22


 


367
KLILAGNII
22


 


393
HLGNNRIEV
22


 


470
LLQVLPPHI
22


 


544
ILCTSPGHL
22


 


564
ILCPGLVNN
22


 


574
SMPTQTSYL
22


 


4
WIHLFYSSL
21


 


70
SLLNNGLTM
21


 


92
SIHLGFNNI
21


 


187
HLDLRGNQL
21


 


295
RMSTKTTSI
21


 


309
KAPGLIPYI
21


 


323
QLPGPYCPI
21


 


391
MLHLGNNRI
21


 


446
AIKEILPGT
21


 


581
YLMVTTPAT
21


 


604
AVPLSVLIL
21


 


623
FCAAGIVVL
21


 


625
AAGIVVLVL
21


 


681
HMVSPMVHV
21


 


118
HINHNSLEI
20


 


130
DTFHGLENL
20


 


140
FLQADNNFI
20


 


203
FLEHIGRIL
20


 


240
SIIGDVVCN
20


 


316
YITKPSTQL
20


 


369
ILAGNIIHS
20


 


453
GTFNPMPKL
20


 


477
HIFSGVPLT
20


 


524
DLVGLQQWI
20


 


593
TADTILRSL
20


 


754
SLYRNILEK
20


 


826
NLHAEPDYL
20


 


45
LINCEAKGI
19


 


171
AIESLPPNI
19


 


178
NIFRFVPLT
19


 


302
SILKLPTKA
19


 


450
ILPGTFNPM
19


 


473
VLPPHIFSG
19


 


502
ILDDLDLLT
19


 


601
LTDAVPLSV
19


 


610
LILGLLIMF
19


 


11
SLLACISLH
18


 


103
IEIGAFNGL
18


 


109
NGLGLLKQL
18


 


112
GLLKQLHIN
18


 


133
HGLENLEFL
18


 


159
LNRLKVLIL
18


 


167
LNDNAIESL
18


 


174
SLPPNIFRF
18


 


190
LRGNQLQTL
18


 


221
ACNCDLLQL
18


 


290
SINDSRMST
18


 


336
KVLSPSGLL
18


 


344
LIHCQERNI
18


 


350
RNIESLSDL
18


 


408
MNLTRLQKL
18


 


417
YLNGNHLTK
18


 


418
LNGNHLTKL
18


 


432
LGLHNLEYL
18


 


462
KVLYLNNNL
18


 


466
LNNNLLQVL
18


 


479
FSGVPLTKV
18


 


494
FTHLPVSNI
18


 


551
HLDKKELKA
18


 


559
ALNSEILCP
18


 


582
LMVTTPATT
18


 


596
TILRSLTDA
18


 


608
SVLILGLLI
18


 


620
TIVFCAAGI
18


 


669
TTERPSASL
18


 


798
KLMETLMYS
18


 


828
HAEPDYLEV
18


 


829
AEPDYLEVL
18


 


48
CEAKGIKMV
17


 


51
KGIKMVSEI
17


 


87
LTNAISIHL
17


 


95
LGFNNIADI
17


 


157
SKLNRLKVL
17


 


180
FRFVPLTHL
17


 


193
NQLQTLPYV
17


 


202
GFLEHIGRI
17


 


228
QLKTWLENM
17


 


256
SILSRLKKE
17


 


378
LMKSDLVEY
17


 


394
LGNNRIEVL
17


 


410
LTRLQKLYL
17


 


456
NPMPKLKVL
17


 


469
NLLQVLPPH
17


 


481
GVPLTKVNL
17


 


534
KLSKNTVTD
17


 


556
ELKALNSEI
17


 


600
SLTDAVPLS
17


 


602
TDAVPLSVL
17


 


616
IMFITIVFC
17


 


621
IVFCAAGIV
17


 


716
HLQRSLLEQ
17


 


720
SLLEQENHS
17


 


739
TTNQSTEFL
17


 


770
GITEYLRKN
17


 


2
KLWIHLFYS
16


 


8
FYSSLLACI
16


 


10
SSLLACISL
16


 


26
SSRGSCDSL
16


 


44
MLINCEAKG
16


 


99
NIADIEIGA
16


 


119
INHNSLEIL
16


 


123
SLEILKEDT
16


 


142
QADNNFITV
16


 


143
ADNNFITVI
16


 


166
ILNDNAIES
16


 


182
FVPLTHLDL
16


 


189
DLRGNQLQT
16


 


205
EHIGRILDL
16


 


210
ILDLQLEDN
16


 


283
LHLAATSSI
16


 


298
TKTTSILKL
16


 


329
CPIPCNCKV
16


 


373
NIIHSLMKS
16


 


381
SDLVEYFTL
16


 


405
GSFMNLTRL
16


 


442
LEYNAIKEI
16


 


520
DCSCDLVGL
16


 


603
DAVPLSVLI
16


 


607
LSVLILGLL
16


 


767
QQLGITEYL
16


 


778
NIAQLQPDM
16


 


805
YSRPRKVLV
16


 


833
YLEVLEQQT
16


 


6
HLFYSSLLA
15


 


12
LLACISLHS
15


 


53
IKMVSEISV
15


 


64
SRPFQLSLL
15


 


105
IGAFNGLGL
15


 


126
ILKEDTFHG
15


 


147
FITVIEPSA
15


 


161
RLKVLILND
15


 


209
RILDLQLED
15


 


226
LLQLKTWLE
15


 


241
IIGDVVCNS
15


 


253
FKGSILSRL
15


 


342
GLLIHCQER
15


 


347
CQERNIESL
15


 


354
SLSDLRPPP
15


 


384
VEYFTLEML
15


 


426
LSKGMFLGL
15


 


455
FNPMPKLKV
15


 


458
MPKLKVLYL
15


 


495
THLPVSNIL
15


 


498
PVSNILDDL
15


 


500
SNILDDLDL
15


 


504
DDLDLLTQI
15


 


507
DLLTQIDLE
15


 


552
LDKKELKAL
15


 


590
TTNTADTIL
15


 


627
GIVVLVLHR
15


 


659
SMYGHKTTH
15


 


676
SLYEQHMVS
15


 


713
DAKHLQRSL
15


 


747
LSFQDASSL
15


 


815
QTKNEYFEL
15


 


5
IHLFYSSLL
14


 


16
ISLHSQTPV
14


 


33
SLCNCEEKD
14


 


83
DFSGLTNAI
14


 


85
SGLTNAISI
14


 


86
GLTNAISIH
14


 


90
AISIHLGFN
14


 


111
LGLLKQLHI
14


 


127
LKEDTFHGL
14


 


151
IEPSAFSKL
14


 


165
LILNDNAIE
14


 


207
IGRILDLQL
14


 


233
LENMPPQSI
14


 


257
ILSRLKKES
14


 


282
SLHLAATSS
14


 


303
ILKLPTKAP
14


 


330
PIPCNCKVL
14


 


343
LLIHCQERN
14


 


368
LILAGNIIH
14


 


377
SLMKSDLVE
14


 


383
LVEYFTLEM
14


 


387
FTLEMLHLG
14


 


401
VLEEGSFMN
14


 


422
HLTKLSKGM
14


 


431
FLGLHNLEY
14


 


434
LHNLEYLYL
14


 


506
LDLLTQIDL
14


 


508
LLTQIDLED
14


 


532
IQKLSKNTV
14


 


557
LKALNSEIL
14


 


562
SEILCPGLV
14


 


599
RSLTDAVPL
14


 


675
ASLYEQHMV
14


 


721
LLEQENHSP
14


 


722
LEQENHSPL
14


 


746
FLSFQDASS
14


 


752
ASSLYRNIL
14


 


789
HYPGAHEEL
14


 


792
GAHEELKLM
14


 


811
VLVEQTKNE
14

















TABLE XXIII







3-HLA-A2-



9mers-158P1D7


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
SLYEQHMGA
20



 


6
QHMGAHEEL
15


 


8
MGAHEELKL
15

















TABLE XXIII







4-HLA-A2-



9mers-158P1D7


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
IIHSLMKSI
20



 


4
SLMKSILWS
18


 


8
SILWSKASG
16


 


5
LMKSILWSK
15


 


9
ILWSKASGR
15


 


2
IHSLMKSIL
12

















TABLE XXIV







V1-HLA-



A0203-9mers-158P1D7









Pos
123456789
score










NoResultsFound.











V3-HLA-



A0203-9mers-158P1D7







NoResultsFound.

















TABLE XXIV







V4-HLA-



A0203-9mers-158P1D7









Pos
123456789
score










NoResultsFound.

















TABLE XXV







V1-HLA-A3-



9mers-158P1D7


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













754
SLYRNILEK
31



 


417
YLNGNHLTK
29


 


150
VIEPSAFSK
26


 


632
VLHRRRRYK
26


 


70
SLLNNGLTM
24


 


265
SICPTPPVY
23


 


478
IFSGVPLTK
23


 


682
MVSPMVHVY
23


 


11
SLLACISLH
22


 


486
KVNLKTNQF
22


 


107
AFNGLGLLK
21


 


189
DLRGNQLQT
21


 


291
INDSRMSTK
21


 


415
KLYLNGNHL
21


 


534
KLSKNTVTD
21


 


564
ILCPGLVNN
21


 


631
LVLHRRRRY
21


 


653
PVHLQYSMY
21


 


676
SLYEQHMVS
21


 


688
HVYRSPSFG
21


 


802
TLMYSRPRK
21


 


158
KLNRLKVLI
20


 


367
KLILAGNII
20


 


431
FLGLHNLEY
20


 


563
EILCPGLVN
20


 


608
SVLILGLLI
20


 


781
QLQPDMEAH
20


 


809
RKVLVEQTK
20


 


187
HLDLRGNQL
19


 


301
TSILKLPTK
19


 


337
VLSPSGLLI
19


 


400
EVLEEGSFM
19


 


409
NLTRLQKLY
19


 


436
NLEYLYLEY
19


 


488
NLKTNQFTH
19


 


609
VLILGLLIM
19


 


633
LHRRRRYKK
19


 


729
PLTGSNMKY
19


 


774
YLRKNIAQL
19


 


24
VLSSRGSCD
18


 


86
GLTNAISIH
18


 


161
RLKVLILND
18


 


174
SLPPNIFRF
18


 


179
IFRFVPLTH
18


 


209
RILDLQLED
18


 


240
SIIGDVVCN
18


 


255
GSILSRLKK
18


 


282
SLHLAATSS
18


 


368
LILAGNIIH
18


 


372
GNIIHSLMK
18


 


377
SLMKSDLVE
18


 


407
FMNLTRLQK
18


 


529
QQWIQKLSK
18


 


546
CTSPGHLDK
18


 


583
MVTTPATTT
18


 


628
IVVLVLHRR
18


 


634
HRRRRYKKK
18


 


670
TERPSASLY
18


 


44
MLINCEAKG
17


 


149
TVIEPSAFS
17


 


194
QLQTLPYVG
17


 


305
KLPTKAPGL
17


 


311
PGLIPYITK
17


 


312
GLIPYITKP
17


 


342
GLLIHCQER
17


 


357
DLRPPPQNP
17


 


359
RPPPQNPRK
17


 


412
RLQKLYLNG
17


 


433
GLHNLEYLY
17


 


460
KLKVLYLNN
17


 


465
YLNNNLLQV
17


 


469
NLLQVLPPH
17


 


472
QVLPPHIFS
17


 


604
AVPLSVLIL
17


 


610
LILGLLIMF
17


 


613
GLLIMFITI
17


 


765
ELQQLGITE
17


 


768
QLGITEYLR
17


 


23
PVLSSRGSC
16


 


163
KVLILNDNA
16


 


166
ILNDNAIES
16


 


239
QSIIGDVVC
16


 


245
VVCNSPPFF
16


 


284
HLAATSSIN
16


 


336
KVLSPSGLL
16


 


420
GNHLTKLSK
16


 


439
YLYLEYNAI
16


 


440
LYLEYNAIK
16


 


502
ILDDLDLLT
16


 


556
ELKALNSEI
16


 


559
ALNSEILCP
16


 


568
GLVNNPSMP
16


 


597
ILRSLTDAV
16


 


615
LIMFITIVF
16


 


621
IVFCAAGIV
16


 


629
VVLVLHRRR
16


 


630
VLVLHRRRR
16


 


650
DNSPVHLQY
16


 


659
SMYGHKTTH
16


 


716
HLQRSLLEQ
16


 


728
SPLTGSNMK
16


 


769
LGITEYLRK
16


 


810
KVLVEQTKN
16


 


812
LVEQTKNEY
16


 


17
SLHSQTPVL
15


 


55
MVSEISVPP
15


 


60
SVPPSRPFQ
15


 


71
LLNNGLTML
15


 


110
GLGLLKQLH
15


 


113
LLKQLHINH
15


 


116
QLHINHNSL
15


 


125
EILKEDTFH
15


 


164
VLILNDNAI
15


 


232
WLENMPPQS
15


 


257
ILSRLKKES
15


 


260
RLKKESICP
15


 


271
PVYEEHEDP
15


 


303
ILKLPTKAP
15


 


369
ILAGNIIHS
15


 


425
KLSKGMFLG
15


 


449
EILPGTFNP
15


 


462
KVLYLNNNL
15


 


463
VLYLNNNLL
15


 


473
VLPPHIFSG
15


 


481
GVPLTKVNL
15


 


526
VGLQQWIQK
15


 


626
AGIVVLVLH
15


 


627
GIVVLVLHR
15


 


656
LQYSMYGHK
15


 


707
NEKEGSDAK
15


 


746
FLSFQDASS
15


 


788
AHYPGAHEE
15


 


798
KLMETLMYS
15

















TABLE XXV







V3-HLA-A3-



9mers-158P1D7


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
SLYEQHMGA
17



 


7
HMGAHEELK
12

















TABLE XXV







V4-HLA-A3-



9mers-158P1D7


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
ILWSKASGR
23



 


8
SILWSKASG
16


 


4
SLMKSILWS
15


 


5
LMKSILWSK
13


 


1
IIHSLMKSI
12

















TABLE XXVI







V1-HLA-A26-



9mers-158P1D7


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













130
DTFHGLENL
32



 


244
DVVCNSPPF
31


 


205
EHIGRILDL
27


 


782
MVSPMVHVY
25


 


819
EYFELKANL
25


 


400
EVLEEGSFM
24


 


498
PVSNILDDL
24


 


604
AVPLSVLIL
23


 


761
EKERELQQL
23


 


148
ITVIEPSAF
22


 


196
QTLPYVGFL
22


 


595
DTILRSLTD
22


 


653
PVHLQYSMY
22


 


275
EHEDPSGSL
21


 


453
GTFNPMPKL
21


 


650
DNSPVHLQY
21


 


277
EDPSGSLHL
20


 


336
KVLSPSGLL
20


 


443
EYNAIDEIL
20


 


486
KVNLKTNQF
20


 


520
DCSCDLVGL
20


 


631
LVLHRRRRY
20


 


795
EELKLMETL
20


 


812
LVEQTKNEY
20


 


87
LTNAISIHL
19


 


154
SAFSKLNRL
19


 


182
FVPLTHLDL
19


 


350
RNIESLSDL
19


 


462
KVLYLNNNL
19


 


607
LSVLILGLL
19


 


610
LILGLLIMF
19


 


139
EFLQADNNF
19


 


245
VVCNSPPFF
18


 


423
LTKLSKGMF
18


 


481
GVPLTKVNL
18


 


539
TVTDDILCT
18


 


628
IVVLVLHRR
18


 


669
TTERPSASL
18


 


713
DAKHLQRSL
18


 


801
ETLMYSRPR
18


 


106
GAFNGLGLL
17


 


136
ENLEFLQAD
17


 


149
TVIEPSAFS
17


 


225
DLLQLKTWL
17


 


308
TKAPGLIPY
17


 


405
GSFMNLTRL
17


 


410
LTRLQKLYL
17


 


501
NILDDLDLL
17


 


590
TTNTADTIL
17


 


738
KTTNQSTEF
17


 


739
TTNQSTEFL
17


 


76
LTMLHTNDF
16


 


89
NAISIHLGF
16


 


180
FRFVPLTHL
16


 


253
FKGSILSRL
16


 


265
SICPTPPVY
16


 


298
TKTTSILKL
16


 


299
KTTSILKLP
16


 


429
GMFLGLHNL
16


 


540
VTDDILCTS
16


 


563
EILCPGLVN
16


 


593
TADTILRSL
16


 


815
QTKNEYFEL
16


 


822
ELKANLHAE
16


 


58
EISVPPSRP
15


 


104
EIGAFNGLG
15


 


133
HGLENLEFL
15


 


174
SLPPNIFRF
15


 


250
PPFFKGSIL
15


 


353
ESLSDLRPP
15


 


370
LAGNIIHSL
15


 


378
LMKSDLVEY
15


 


385
EYFTLEMLH
15


 


449
EILPGTFNP
15


 


504
DDLDLLTQI
15


 


615
LIMFITIVF
15


 


621
IVFCAAGIV
15


 


705
ERNEKEGSD
15


 


725
ENHSPLTGS
15


 


758
NILEKEREL
15


 


 832
DYLEVLEQQ
15

















TABLE XXVI







V3-HLA-A26-9mers-158P1D7



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


start position is specified, the length of pep-


tide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Pos
123456789
score





 










5
EQHMGAHEE
10



 


8
MGAHEELKL
10


 


6
QHMGAHEEL
8





 








V4-HLA-A26-9mers-158P1D7



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


start position is specified, the length of pep-


tide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Pos
123456789
score













2
IHSLMKSIL
9



 


5
LMKSILWSK
8


 


1
IIHSLMKSI
7


 


4
SLMKSILWS
6


 


8
SILWSKASG
6


 


7
KSILWSKAS
5

















TABLE XXVII







V1-HLA-B0702-9mers-158P1D7



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


start position is specified, the length of pep-


tide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Pos
123456789
score













456
NPMPKLKVL
23



 


458
MPKLKVLYL
23


 


692
SPSFGPKHL
22


 


250
PPFFKGSIL
21


 


61
VPPSRPFQL
20


 


278
DPSGSLHLA
20


 


360
PPPQNPRKL
20


 


361
PPQNPRKLI
20


 


517
NPWDCSCDL
20


 


310
APGLIPYIT
19


 


175
LPPNIFRFV
18


 


314
IPYITKPST
18


 


586
TPATTTNTA
18


 


306
LPTKAPGLI
17


 


329
CPIPCNCKV
17


 


474
LPPHIFSGV
17


 


625
AAGIVVLVL
17


 


804
MYSRPRKVL
17


 


62
PPSRPFQLS
16


 


237
PPQSIIGDV
16


 


249
SPPFFKGSI
16


 


364
NPRKLILAG
16


 


572
NPSMPTQTS
16


 


575
MPTQTSYLM
16


 


652
SPVHLQYSM
16


 


807
RPRKVLVEQ
16


 


63
PSRPFQLSL
15


 


105
IGAFNGLGL
15


 


159
LNRLKVLIL
15


 


205
EHIGRILOL
15


 


207
IGRILDLQL
15


 


267
CPTPPVYEE
15


 


316
YITKPSTQL
15


 


426
LSKGMFLGL
15


 


602
TDAVPLSVL
15


 


604
AVPLSVLIL
15


 


623
FCAAGIVVL
15


 


752
ASSLYRNIL
15


 


26
SSRGSCDSL
14


 


103
IEIGAFNGL
14


 


152
EPSAFSKLN
14


 


177
PNIFRFVPL
14


 


180
FRFVPLTHL
14


 


221
ACNCDLLQL
14


 


275
EHEDPSGSL
14


 


319
KPSTQLPGP
14


 


326
GPYCPIPCN
14


 


336
KVLSPSGLL
14


 


339
SPSGLLIHC
14


 


410
LTRLQKLYL
14


 


453
GTFNPMPKL
14


 


476
PHIFSGVPL
14


 


520
DCSCDLVGL
14


 


599
RSLTDAVPL
14


 


606
PLSVLILGL
14


 


669
TTERPSASL
14


 


672
RPSASLYEQ
14


 


774
YLRKNIAQL
14


 


830
EPDYLEVLE
14


 


17
SLHSQTPVL
13


 


37
CEEKDGTML
13


 


65
RPFQLSLLN
13


 


196
QTLPYVGFL
13


 


198
LPYVGFLEH
13


 


264
ESICPTPPV
13


 


277
EDPSGSLHL
13


 


324
LPGPYCPIP
13


 


331
IPCNCKVLS
13


 


359
RPPPQNPRK
13


 


362
PQNPRKLIL
13


 


375
IHSLMKSDL
13


 


402
LEEGSFMNL
13


 


648
MRDNSPVHL
13


 


714
AKHLQRSLL
13


 


767
QQLGITEYL
13


 


791
PGAHEELKL
13


 


829
AEPDYLEVL
13


 


59
ISVPPSRPF
12


 


68
QLSLLNNGL
12


 


83
DFSGLTNAI
12


 


109
NGLGLLKQL
12


 


151
IEPSAFSKL
12


 


172
IESLPPNIF
12


 


176
PPNIFRFVP
12


 


182
FVPLTHLDL
12


 


187
HLDLRGNQL
12


 


189
DLRGNQLQT
12


 


219
KWACNCDLL
12


 


234
ENMPPQSII
12


 


296
MSTKTTSIL
12


 


298
TKTTSILKL
12


 


305
KLPTKAPGL
12


 


323
QLPGPYCPI
12


 


337
VLSPSGLLI
12


 


386
YFTLEMLHL
12


 


415
KLYLNGNHL
12


 


418
LNGNHLTKL
12


 


424
TKLSKGMFL
12


 


434
LHNLEYLYL
12


 


443
EYNAIKEIL
12


 


451
LPGTFNPMP
12


 


481
GVPLTKVNL
12


 


489
LKTNQFTHL
12


 


497
LPVSNILDD
12


 


498
PVSNILDDL
12


 


500
SNILDDLDL
12


 


552
LDKKELKAL
12


 


566
CPGLVNNPS
12


 


624
CAAGIVVLV
12


 


684
SPMVHVYRS
12


 


709
KEGSDAKHL
12


 


739
TTNQSTEFL
12


 


789
HYPGAHEEL
12


 


790
YPGAHEELK
12


 


795
EELKLMETL
12


 


819
EYFELKANL
12


 


5
IHLFYSSLL
11


 


71
LLNNGLTML
11


 


79
LHTNDFSGL
11


 


87
LTNAISIHL
11


 


119
INHNSLEIL
11


 


127
LKEDTFHGL
11


 


133
HGLENLEFL
11


 


157
SKLNRLKVL
11


 


167
LNDNAIESL
11


 


190
LRGNQLQTL
11


 


195
LQTLPYVGF
11


 


203
FLEHIGRIL
11


 


218
NKWACNCDL
11


 


225
DLLQLKTWL
11


 


253
FKGSILSRL
11


 


295
RMSTKTTSI
11


 


300
TTSILKLPT
11


 


330
PIPCNCKVL
11


 


350
RNIESLSDL
11


 


370
LAGNIIHSL
11


 


394
LGNNRIEVL
11


 


405
GSFMNLTRL
11


 


450
ILPGTFNPM
11


 


455
FNPMPKLKV
11


 


462
KVLYLNNNL
11


 


466
LNNNLLQVL
11


 


475
PPHIFSGVP
11


 


479
FSGVPLTKV
11


 


482
VPLTKVNLK
11


 


495
THLPVSNIL
11


 


537
KNTVTDDIL
11


 


544
ILCTSPGHL
11


 


548
SPGHLDKKE
11


 


557
LKALNSEIL
11


 


561
NSEILCPGL
11


 


574
SMPTQTSYL
11


 


590
TTNTADTIL
11


 


593
TADTILRSL
11


 


597
ILRSLTDAV
11


 


681
HMVSPMVHV
11


 


722
LEQENHSPL
11


 


741
NQSTEFLSF
11


 


761
EKERELQQL
11


 


780
AQLQPDMEA
11


 


783
QPDMEAHYP
11


 


805
YSRPRKVLV
11


 


826
NLHAEPDYL
11

















TABLE XXVII







V3-HLA-B0702-9mers-158P1D7



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


start position is specified, the length of pep-


tide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Pos
123456789
score













6
QHMGAHEEL
13



 


8
MGAHEELKL
13


 


2
SLYEQHMGA
6

















TABLE XXVII







V4-HLA-B0702-9mers-158P1D7



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


start position is specified, the length of pep-


tide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Pos
123456789
score













2
IHSLMKSIL
13



 


6
MKSILWSKA
8


 


1
IIHSLMKSI
7


 


13
KASGRGRRE
6

















TABLE XXVII







V1-HLA-B08-9mers-158P1D7



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


start position is specified, the length of pep-


tide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Pos
123456789
score













458
MPKLKVLYL
38



 


159
LNRLKVLIL
28


 


456
NPMPKLKVL
27


 


758
NILEKEREL
27


 


154
SAFSKLNRL
26


 


187
HLDLRGNQL
26


 


250
PPFFKGSIL
26


 


305
KLPTKAPGL
26


 


556
ELKALNSEI
26


 


61
VPPSRPFQL
25


 


713
DAKHLQRSL
24


 


258
LSRLKKESI
23


 


774
YLRKNIAQL
23


 


552
LDKKELKAL
22


 


157
SKLNRLKVL
21


 


205
EHIGRILDL
21


 


638
RYKKKQVDE
21


 


734
NMKYKTTNQ
21


 


815
QTKNEYFEL
21


 


303
ILKLPTKAP
20


 


424
TKLSKGMFL
20


 


426
LSKGMFLGL
20


 


760
LEKERELQQ
20


 


126
ILKEDTFHG
19


 


177
PNIFRFVPL
19


 


394
LGNNRIEVL
19


 


463
VLYLNNNLL
19


 


692
SPSFGPKHL
19


 


796
ELKLMETLM
19


 


822
ELKANLHAE
19


 


17
SLHSQTPVL
18


 


26
SSRGSCDSL
18


 


38
EEKDGTMLI
18


 


68
QLSLLNNGL
18


 


161
RLKVLILND
18


 


362
PQNPRKLIL
18


 


408
MNLTRLQKL
18


 


482
VPLTKVNLK
18


 


527
GLQQWIQKL
18


 


606
PLSVLILGL
18


 


636
RRRYKKKQV
18


 


696
GPKHLEEEE
18


 


813
VEQTKNEYF
18

















TABLE XXVIII







V3-HLA-B08-9mers-158P1D7



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


start position is specified, the length of pep-


tide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Pos
123456789
score













6
QHMGAHEEL
11



 


2
SLYEQHMGA
10


 


8
MGAHEELKL
10

















TABLE XXVIII







V4-HLA-B08-9mers-158P1D7



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


start position is specified, the length of pep-


tide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Pos
123456789
score













9
ILWSKASGR
17



 


1
IIHSLMKSI
12


 


2
IHSLMKSIL
12


 


13
KASGRGRRE
12


 


3
HSLMKSILW
11


 


5
LMKSILWSK
10


 


11
WSKASGRGR
10


 


4
SLMKSILWS
9

















TABLE XXIX







V1-HLA-B1510-9mers-158P1D7



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


start position is specified, the length of pep-


tide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Pos
123456789
score













275
EHEDPSGSL
24



 


375
IHSLMKSDL
24


 


205
EHIGRILOL
23


 


495
THLPVSNIL
23


 


5
IHLFYSSLL
22


 


476
PHIFSGVPL
22


 


79
LHTNDFSGL
20


 


434
LHNLEYLYL
20


 


132
FHGLENLEF
17


 


623
FCAAGIVVL
17


 


687
VHVYRSPSF
17


 


602
TDAVPLSVL
16


 


18
LHSQTPVLS
15


 


360
PPPQNPRKL
15


 


804
MYSRPRKVL
15


 


105
IGAFNGLGL
14


 


345
IHOQERNIE
14


 


392
LHLGNNRIE
14


 


405
GSFMNLTRL
14


 


453
GTFNPMPKL
14


 


456
NPMPKLKVL
14


 


481
GVPLTKVNL
14


 


680
QHMVSPMVH
14


 


758
NILEKEREL
14


 


774
YLRKNIAQL
14


 


788
AHYPGAHEE
14


 


795
EELKLMETL
14


 


17
SLHSQTPVL
13


 


59
ISVPPSRPF
13


 


93
IHLGFNNIA
13


 


103
IEIGAFNGL
13


 


186
THLDLRGNQ
13


 


196
QTLPYVGFL
13


 


203
FLEHIGRIL
13


 


316
YITKPSTQL
13


 


330
PIPCNCKVL
13


 


347
CQERNIESL
13


 


362
PQNPRKLIL
13


 


394
LGNNRIEVL
13


 


520
DCSCDLVGL
13


 


527
GLQQWIQKL
13


 


544
ILCTSPGHL
13


 


550
GHLDKKELK
13


 


593
TADTILRSL
13


 


606
PLSVLILGL
13


 


625
AAGIVVLVL
13


 


648
MRDNSPVHL
13


 


666
THHTTERPS
13


 


669
TTERPSASL
13


 


692
SPSFGPKHL
13


 


726
NHSPLTGSN
13


 


793
AHEELKLME
13


 


819
EYFELKANL
13


 


827
LHAEPDYLE
13


 


829
AEPDYLEVL
13


 


37
CEEKDGTML
12


 


63
PSRPFQLSL
12


 


106
GAFNGLGLL
12


 


119
INHNSLEIL
12


 


127
LKEDTFHGL
12


 


133
HGLENLEFL
12


 


151
IEPSAFSKL
12


 


154
SAFSKLNRL
12


 


157
SKLNRLKVL
12


 


174
SLPPNIFRF
12


 


177
PNIFRFVPL
12


 


180
FRFVPLTHL
12


 


207
IGRILDLQL
12


 


219
KWACNCDLL
12


 


225
DLLQLKTWL
12


 


253
FKGSILSRL
12


 


277
EDPSGSLHL
12


 


298
TKTTSILKL
12


 


381
SDLVEYFTL
12


 


386
YFTLEMLHL
12


 


402
LEEGSFMNL
12


 


429
GMFLGLHNL
12


 


443
EYNAIKEIL
12


 


466
LNNNLLQVL
12


 


549
PGHLDKKEL
12


 


552
LDKKELKAL
12


 


557
LKALNSEIL
12


 


561
NSEILCPGL
12


 


599
RSLTDAVPL
12


 


662
GHKTTHHTT
12


 


667
HHTTERPSA
12


 


698
KHLEEEEER
12


 


713
DAKHLQRSL
12


 


722
LEQENHSPL
12


 


739
TTNQSTEFL
12


 


752
ASSLYRNIL
12


 


761
EKERELQQL
12


 


789
HYPGAHEEL
12


 


26
SSRGSCDSL
11


 


61
VPPSRPFQL
11


 


68
QLSLLNNGL
11


 


71
LLNNGLTML
11


 


109
NGLGLLKQL
11


 


116
QLHINHNSL
11


 


130
DTFHGLENL
11


 


159
LNRLKVLIL
11


 


167
LNDNAIESL
11


 


172
IESLPPNIF
11


 


190
LRGNQLQTL
11


 


288
TSSINDSRM
11


 


296
MSTKTTSIL
11


 


305
KLPTKAPGL
11


 


335
CKVLSPSGL
11


 


336
KVLSPSGLL
11


 


350
RNIESLSDL
11


 


370
LAGNIIHSL
11


 


410
LTRLQKLYL
11


 


415
KLYLNGNHL
11


 


424
TKLSKGMFL
11


 


426
LSKGMFLGL
11


 


432
LGLHNLEYL
11


 


447
IKEILPGTF
11


 


458
MPKLKVLYL
11


 


463
VLYLNNNLL
11


 


498
PVSNILDDL
11


 


501
NILDDLDLL
11


 


517
NPWDCSCDL
11


 


537
KNTVTDDIL
11


 


590
TTNTADTIL
11


 


604
AVPLSVLIL
11


 


633
LHRRRRYKK
11


 


654
VHLQYSMYG
11


 


714
AKHLQRSLL
11


 


715
KHLQRSLLE
11


 


747
LSFQDASSL
11


 


767
QQLGITEYL
11


 


791
PGAHEELKL
11


 


815
QTKNEYFEL
11


 


826
NLHAEPDYL
11

















TABLE XXIX







V3-HLA-B1510-9mers-158P1D7



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


start position is specified, the length of pep-


tide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Pos
123456789
score













6
QHMGAHEEL
22



 


8
MGAHEELKL
11

















TABLE XXIX







V4-HLA-B1510-9mers-158P1D7



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


start position is specified, the length of pep-


tide is 9 amino acids, and the end position for


each peptide is the start position plus eight.









Pos
123456789
score





2
IHSLMKSIL
24

















TABLE XXX







V1-HLA-B2705-



9mers-158P1D7


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













180
FRFVPLTHL
27



 


358
LRPPPQNPR
25


 


64
SRPFQLSLL
22


 


190
LRGNQLQTL
22


 


429
GMFLGLHNL
22


 


634
HRRRRYKKK
22


 


648
MRDNSPVHL
22


 


690
YRSPSFGPK
22


 


756
YRNILEKER
22


 


405
GSFMNLTRL
21


 


637
RRYKKKQVD
21


 


255
GSILSRLKK
20


 


350
RNIESLSDL
20


 


453
GTFNPMPKL
20


 


527
GLQQWIQKL
20


 


719
RSLLEQENH
20


 


763
ERELQQLGI
20


 


106
GAFNGLGLL
19


 


359
RPPPQNPRK
19


 


462
KVLYLNNNL
19


 


819
EYFELKANL
19


 


130
DTFHGLENL
18


 


139
EFLQADNNF
18


 


154
SAFSKLNRL
18


 


205
EHIGRILDL
18


 


225
DLLQLKTWL
18


 


252
FFKGSILSR
18


 


481
GVPLTKVNL
18


 


599
RSLTDAVPL
18


 


747
LSFQDASSL
18


 


809
RKVLVEQTK
18


 


109
NGLGLLKQL
17


 


160
NRLKVLILN
17


 


202
GELEHIGRI
17


 


208
GRILDLQLE
17


 


211
LDLQLEDNK
17


 


298
TKTTSILKL
17


 


301
TSILKLPTK
17


 


316
YITKPSTQL
17


 


372
GNIIHSLMK
17


 


411
TRLQKLYLN
17


 


420
GNHLTKLSK
17


 


550
GHLDKKELK
17


 


610
LILGLLIMF
17


 


623
FCAAGIVVL
17


 


627
GIVVLVLHR
17


 


628
IVVLVLHRR
17


 


635
RRRRYKKKQ
17


 


636
RRRYKKKQV
17


 


698
KHLEEEEER
17


 


754
SLYRNILEK
17


 


766
LQQLGITEY
17


 


774
YLRKNIAQL
17


 


103
IEIGAFNGL
16


 


125
EILKEDTFH
16


 


173
ESLPPNIFR
16


 


174
SLPPNIFRF
16


 


201
VGFLEHIGR
16


 


259
SRLKKESIC
16


 


336
KVLSPSGLL
16


 


342
GLLIHCQER
16


 


366
RKLILAGNI
16


 


390
EMLHLGNNR
16


 


397
NRIEVLEEG
16


 


402
LEEGSFMNL
16


 


415
KLYLNGNHL
16


 


478
IFSGVPLTK
16


 


486
KVNLKTNQF
16


 


495
THLPVSNIL
16


 


506
LDLLTQIDL
16


 


526
VGLQQWIQK
16


 


659
SMYGHKTTH
16


 


711
GSDAKHLQR
16


 


728
SPLTGSNMK
16


 


738
KTTNQSTEF
16


 


769
LGITEYLRK
16


 


795
EELKLMETL
16


 


5
IHLFYSSLL
15


 


10
SSLLACISL
15


 


20
SQTPVLSSR
15


 


51
KGIKMVSEI
15


 


57
SEISVPPSR
15


 


59
ISVPPSRPF
15


 


63
PSRPFQLSL
15


 


71
LLNNGLTML
15


 


86
GLTNAISIH
15


 


100
IADIEIGAF
15


 


107
AFNGLGLLK
15


 


124
LEILKEDTE
15


 


132
FHGLENLEF
15


 


153
PSAFSKLNR
15


 


155
AFSKLNRLK
15


 


207
IGRILOLQL
15


 


250
PPFFKGSIL
15


 


253
FKGSILSRL
15


 


305
KLPTKAPGL
15


 


309
KAPGLIPYI
15


 


311
PGLIPYITK
15


 


370
LAGNIIHSL
15


 


399
IEVLEEGSF
15


 


408
MNLTRLQKL
15


 


418
LNGNHLTKL
15


 


440
LYLEYNAIK
15


 


463
VLYLNNNLL
15


 


469
NLLQVLPPH
15


 


482
VPLTKVNLK
15


 


500
SNILDOLOL
15


 


547
TSPGHLDKK
15


 


604
AVPLSVLIL
15


 


606
PLSVLILGL
15


 


609
VLILGLLIM
15


 


625
AAGIVVLVL
15


 


629
VVLVLHRRR
15


 


640
KKKQVDEQM
15


 


664
KTTHHTTER
15


 


691
RSPSFGPKH
15


 


708
EKEGSDAKH
15


 


729
PLTGSNMKY
15


 


758
NILEKEREL
15


 


767
QQLGITEYL
15


 


11
SLLACISLH
14


 


26
SSRGSCDSL
14


 


37
CEEKDGTML
14


 


68
QLSLLNNGL
14


 


89
NAISIHLGF
14


 


110
GLGLLKQLH
14


 


113
LLKQLHINH
14


 


133
HGLENLEFL
14


 


148
ITVIEPSAF
14


 


150
VIEPSAFSK
14


 


151
IEPSAFSKL
14


 


157
SKLNRLKVL
14


 


159
LNRLKVLIL
14


 


167
LNDNAIESL
14


 


172
IESLPPNIF
14


 


196
QTLPYVGFL
14


 


198
LPYVGFLEH
14


 


221
ACNCDLLQL
14


 


254
KGSILSRLK
14


 


277
EDPSGSLHL
14


 


287
ATSSINDSR
14


 


294
SRMSTKTTS
14


 


295
RMSTKTTSI
14


 


335
CKVLSPSGL
14


 


347
CQERNIESL
14


 


349
ERNIESLSD
14


 


360
PPPQNPRKL
14


 


365
PRKLILAGN
14


 


368
LILAGNIIH
14


 


375
IHSLMKSDL
14


 


381
SDLVEYFTL
14


 


394
LGNNRIEVL
14


 


414
QKLYLNGNH
14


 


417
YLNGNHLTK
14


 


424
TKLSKGMFL
14


 


456
NPMPKLKVL
14


 


458
MPKLKVLYL
14


 


476
PHIFSGVPL
14


 


546
CTSPGHLDK
14


 


552
LDKKELKAL
14


 


573
PSMPTQTSY
14


 


598
LRSLTDAVP
14


 


602
TDAVPLSVL
14


 


607
LSVLILGLL
14


 


626
AGIVVLVLH
14


 


630
VLVLHRRRR
14


 


652
SPVHLQYSM
14


 


669
TTERPSASL
14


 


687
VHVYRSPSF
14


 


701
EEEEERNEK
14


 


707
NEKEGSDAK
14


 


713
DAKHLQRSL
14


 


778
NIAQLQPDM
14


 


791
PGAHEELKL
14


 


792
GAHEELKLM
14


 


802
TLMYSRPRK
14


 


806
SRPRKVLVE
14


 


4
WIHLFYSSL
13


 


32
DSLCNCEEK
13


 


46
INCEAKGIK
13


 


87
LTNAISIHL
13


 


95
LGFNNIADI
13


 


111
LGLLKQLHI
13


 


119
INHNSLEIL
13


 


143
ADNNFITVI
13


 


177
PNIFRFVPL
13


 


183
VPLTHLDLR
13


 


187
HLDLRGNQL
13


 


192
GNQLQTLPY
13


 


195
LQTLPYVGF
13


 


244
DVVCNSPPF
13


 


275
EHEDPSGSL
13


 


291
INDSRMSTK
13


 


296
MSTKTTSIL
13


 


308
TKAPGLIPY
13


 


312
GLIPYITKP
13


 


362
PQNPRKLIL
13


 


384
VEYFTLEML
13


 


385
EYFTLEMLH
13


 


386
YFTLEMLHL
13


 


391
MLHLGNNRI
13


 


400
EVLEEGSFM
13


 


404
EGSFMNLTR
13


 


407
FMNLTRLQK
13


 


410
LTRLQKLYL
13


 


423
LTKLSKGMF
13


 


426
LSKGMFLGL
13


 


432
LGLHNLEYL
13


 


433
GLHNLEYLY
13


 


434
LHNLEYLYL
13


 


447
IKEILPGTF
13


 


457
PMPKLKVLY
13


 


466
LNNNLLQVL
13


 


471
LQVLPPHIF
13


 


501
NILDDLDLL
13


 


504
DDLDLLTQI
13


 


529
QQWIQKLSK
13


 


537
KNTVTDDIL
13


 


549
PGHLDKKEL
13


 


567
PGLVNNPSM
13


 


590
TTNTADTIL
13


 


593
TADTILRSL
13


 


611
ILGLLIMFI
13


 


613
GLLIMFITI
13


 


615
LIMFITIVF
13


 


633
LHRRRRYKK
13


 


705
ERNEKEGSD
13


 


709
KEGSDAKHL
13


 


714
AKHLQRSLL
13


 


718
QRSLLEQEN
13


 


739
TTNQSTEFL
13


 


741
NQSTEFLSF
13


 


749
FQDASSLYR
13


 


752
ASSLYRNIL
13


 


761
EKERELQQL
13


 


789
HYPGAHEEL
13


 


799
LMETLMYSR
13


 


801
ETLMYSRPR
13


 


808
PRKVLVEQT
13


 


812
LVEQTKNEY
13


 


829
AEPDYLEVL
13

















TABLE XXX







V3-HLA-B2705-



9mers-158P1D7


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
MGAHEELKL
14



 


6
QHMGAHEEL
13


 


3
LYEQHMGAH
10


 


7
HMGAHEELK
10


 


1
ASLYEQHMG
6

















TABLE XXX







V4-HLA-B2705-



9mers-158P1D7


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
IHSLMKSIL
14



 


5
LMKSILWSK
14


 


9
ILWSKASGR
14


 


12
SKASGRGRR
14


 


11
WSKASGRGR
11


 


1
IIHSLMKSI
9


 


4
SLMKSILWS
7


 


7
KSILWSKAS
6


 


8
SILWSKASG
6


 


13
KASGRGRRE
6

















TABLE XXXI







V1-HLA-B2709-



9mers-158P1D7


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













636
RRRYKKKQV
23



 


180
FRFVPLTHL
22


 


648
MRDNSPVHL
21


 


64
SRPFQLSLL
20


 


190
LRGNQLQTL
20


 


599
RSLTDAVPL
19


 


763
ERELQQLGI
19


 


366
RKLILAGNI
16


 


405
GSFMNLTRL
16


 


429
GMFLGLHNL
16


 


453
GTFNPMPKL
16


 


637
RRYKKKQVD
16


 


106
GAFNGLGLL
15


 


208
GRILDLQLE
15


 


336
KVLSPSGLL
15


 


350
RNIESLSDL
15


 


462
KVLYLNNNL
15


 


481
GVPLTKVNL
15


 


709
KEGSDAKHL
15


 


154
SAFSKLNRL
14


 


196
QTLPYVGFL
14


 


202
GELEHIGRI
14


 


221
ACNCDLLQL
14


 


415
KLYLNGNHL
14


 


635
RRRRYKKKQ
14


 


747
LSFQDASSL
14


 


5
IHLFYSSLL
13


 


109
NGLGLLKQL
13


 


130
DTFHGLENL
13


 


207
IGRILDLQL
13


 


253
FKGSILSRL
13


 


411
TRLQKLYLN
13


 


424
TKLSKGMFL
13


 


495
THLPVSNIL
13


 


500
SNILDDLDL
13


 


501
NILDDLDLL
13


 


527
GLQQWIQKL
13


 


537
KNTVTDDIL
13


 


604
AVPLSVLIL
13


 


613
GLLIMFITI
13


 


625
AAGIVVLVL
13


 


767
QQLGITEYL
13


 


819
EYFELKANL
13


 


10
SSLLACISL
12


 


17
SLHSQTPVL
12


 


51
KGIKMVSEI
12


 


61
VPPSRPFQL
12


 


63
PSRPFQLSL
12


 


79
LHTNDFSGL
12


 


89
NAISIHLGF
12


 


103
IEIGAFNGL
12


 


105
IGAFNGLGL
12


 


133
HGLENLEFL
12


 


151
IEPSAFSKL
12


 


157
SKLNRLKVL
12


 


159
LNRLKVLIL
12


 


160
NRLKVLILN
12


 


171
AIESLPPNI
12


 


177
PNIFRFVPL
12


 


205
EHIGRILDL
12


 


219
KWACNCDLL
12


 


225
DLLQLKTWL
12


 


250
PPFFKGSIL
12


 


259
SRLKKESIC
12


 


277
EDPSGSLHL
12


 


295
RMSTKTTSI
12


 


298
TKTTSILKL
12


 


316
YITKPSTQL
12


 


362
PQNPRKLIL
12


 


381
SDLVEYFTL
12


 


384
VEYFTLEML
12


 


386
YFTLEMLHL
12


 


408
MNLTRLQKL
12


 


432
LGLHNLEYL
12


 


458
MPKLKVLYL
12


 


463
VLYLNNNLL
12


 


476
PHIFSGVPL
12


 


506
LDLLTQIDL
12


 


520
DCSCDLVGL
12


 


607
LSVLILGLL
12


 


621
IVFCAAGIV
12


 


671
ERPSASLYE
12


 


758
NILEKEREL
12


 


775
LRKNIAQLQ
12


 


795
EELKLMETL
12


 


806
SRPRKVLVE
12


 


808
PRKVLVEQT
12


 


16
ISLHSQTPV
11


 


27
SRGSCDSLC
11


 


37
CEEKDGTML
11


 


59
ISVPPSRPF
11


 


70
SLLNNGLTM
11


 


85
SGLTNAISI
11


 


87
LTNAISIHL
11


 


111
LGLLKQLHI
11


 


119
INHNSLEIL
11


 


139
EFLQADNNF
11


 


158
KLNRLKVLI
11


 


182
FVPLTHLDL
11


 


187
HLDLRGNQL
11


 


193
NQLQTLPYV
11


 


203
FLEHIGRIL
11


 


294
SRMSTKTTS
11


 


296
MSTKTTSIL
11


 


309
KAPGLIPYI
11


 


335
CKVLSPSGL
11


 


349
ERNIESLSD
11


 


358
LRPPPQNPR
11


 


365
PRKLILAGN
11


 


367
KLILAGNII
11


 


370
LAGNIIHSL
11


 


375
IHSLMKSDL
11


 


397
NRIEVLEEG
11


 


402
LEEGSFMNL
11


 


410
LTRLQKLYL
11


 


426
LSKGMFLGL
11


 


434
LHNLEYLYL
11


 


443
EYNAIKEIL
11


 


456
NPMPKLKVL
11


 


486
KVNLKTNQF
11


 


489
LKTNQFTHL
11


 


498
PVSNILDDL
11


 


504
DDLDLLTQI
11


 


544
ILCTSPGHL
11


 


549
PGHLDKKEL
11


 


561
NSEILCPGL
11


 


567
PGLVNNPSM
11


 


593
TADTILRSL
11


 


603
DAVPLSVLI
11


 


606
PLSVLILGL
11


 


608
SVLILGLLI
11


 


623
FCAAGIVVL
11


 


624
CAAGIVVLV
11


 


640
KKKQVDEQM
11


 


675
ASLYEQHMV
11


 


681
HMVSPMVHV
11


 


690
YRSPSFGPK
11


 


714
AKHLQRSLL
11


 


738
KTTNQSTEF
11


 


752
ASSLYRNIL
11


 


761
EKERELQQL
11


 


774
YLRKNIAQL
11


 


791
PGAHEELKL
11


 


792
GAHEELKLM
11


 


803
LMYSRPRKV
11


 


828
HAEPDYLEV
11


 


829
AEPDYLEVL
11

















TABLE XXXI







V3-HLA-B2709-



9mers-158P1D7


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
MGAHEELKL
11



 


6
QHMGAHEEL
10

















TABLE XXXI







V4-HLA-B2709-



9mers-158P1D7


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
IHSLMKSIL
11



 


1
IIHSLMKSI
10

















TABLE XXXII







V1-HLA-B4402-



9mers-158P1D7


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













829
AEPDYLEVL
27



 


103
IEIGAFNGL
26


 


124
LEILKEDTF
25


 


670
TERPSASLY
25


 


172
IESLPPNIF
24


 


442
LEYNAIKEI
24


 


709
KEGSDAKHL
24


 


795
EELKLMETL
24


 


38
EEKDGTMLI
23


 


151
IEPSAFSKL
23


 


402
LEEGSFMNL
22


 


205
EHIGRILDL
21


 


384
VEYFTLEML
21


 


399
IEVLEEGSF
21


 


722
LEQENHSPL
21


 


813
VEQTKNEYF
21


 


37
CEEKDGTML
20


 


174
SLPPNIFRF
19


 


233
LENMPPQSI
19


 


456
NPMPKLKVL
19


 


157
SKLNRLKVL
18


 


109
NGLGLLKQL
17


 


562
SEILCPGLV
17


 


604
AVPLSVLIL
17


 


682
MVSPMVHVY
17


 


752
ASSLYRNIL
17


 


89
NAISIHLGF
16


 


100
IADIEIGAF
16


 


143
ADNNFITVI
16


 


164
VLILNDNAI
16


 


177
PNIFRFVPL
16


 


221
ACNCDLLQL
16


 


224
CDLLQLKTW
16


 


265
SICPTPPVY
16


 


298
TKTTSILKL
16


 


370
LAGNIIHSL
16


 


394
LGNNRIEVL
16


 


500
SNILDDLDL
16


 


625
AAGIVVLVL
16


 


650
DNSPVHLQY
16


 


703
EEERNEKEG
16


 


714
AKHLQRSLL
16


 


804
MYSRPRKVL
16


 


818
NEYFELKAN
16


 


48
CEAKGIKMV
15


 


57
SEISVPPSR
15


 


95
LGFNNIADI
15


 


106
GAFNGLGLL
15


 


154
SAFSKLNRL
15


 


167
LNDNAIESL
15


 


187
HLDLRGNQL
15


 


196
QTLPYVGFL
15


 


276
HEDPSGSLH
15


 


308
TKAPGLIPY
15


 


330
PIPCNCKVL
15


 


347
CQERNIESL
15


 


360
PPPQNPRKL
15


 


362
PQNPRKLIL
15


 


408
MNLTRLQKL
15


 


409
NLTRLQKLY
15


 


429
GMFLGLHNL
15


 


448
KEILPGTFN
15


 


486
KVNLKTNQF
15


 


495
THLPVSNIL
15


 


501
NILDDLDLL
15


 


552
LDKKELKAL
15


 


593
TADTILRSL
15


 


606
PLSVLILGL
15


 


615
LIMFITIVF
15


 


623
FCAAGIVVL
15


 


692
SPSFGPKHL
15


 


741
NQSTEFLSF
15


 


761
EKERELQQL
15


 


774
YLRKNIAQL
15


 


10
SSLLACISL
14


 


59
ISVPPSRPF
14


 


61
VPPSRPFQL
14


 


63
PSRPFQLSL
14


 


64
SRPFQLSLL
14


 


76
LTMLHTNDF
14


 


83
DFSGLTNAI
14


 


85
SGLTNAISI
14


 


128
KEDTFHGLE
14


 


135
LENLEFLQA
14


 


138
LEFLQADNN
14


 


139
EFLQADNNF
14


 


234
ENMPPQSII
14


 


277
EDPSGSLHL
14


 


305
KLPTKAPGL
14


 


309
KAPGLIPYI
14


 


337
VLSPSGLLI
14


 


350
RNIESLSDL
14


 


367
KLILAGNII
14


 


403
EEGSFMNLT
14


 


405
GSFMNLTRL
14


 


415
KLYLNGNHL
14


 


453
GTFNPMPKL
14


 


463
VLYLNNNLL
14


 


476
PHIFSGVPL
14


 


498
PVSNILDDL
14


 


527
GLQQWIQKL
14


 


555
KELKALNSE
14


 


573
PSMPTQTSY
14


 


574
SMPTQTSYL
14


 


599
RSLTDAVPL
14


 


607
LSVLILGLL
14


 


610
LILGLLIMF
14


 


631
LVLHRRRRY
14


 


648
MRDNSPVHL
14


 


701
EEEEERNEK
14


 


702
EEEERNEKE
14


 


744
TEFLSFQDA
14


 


766
LQQLGITEY
14


 


767
QQLGITEYL
14


 


819
EYFELKANL
14


 


825
ANLHAEPDY
14


 


1
MKLWIHLFY
13


 


17
SLHSQTPVL
13


 


51
KGIKMVSEI
13


 


68
QLSLLNNGL
13


 


127
LKEDTFHGL
13


 


130
DTFHGLENL
13


 


133
HGLENLEFL
13


 


148
ITVIEPSAF
13


 


159
LNRLKVLIL
13


 


180
FRFVPLTHL
13


 


182
FVPLTHLDL
13


 


190
LRNGQLQTL
13


 


192
GNQLQTLPY
13


 


204
LEHIGRILD
13


 


212
DLQLEDNKW
13


 


219
KWACNCDLL
13


 


263
KESICPTPP
13


 


274
EEHEDPSGS
13


 


275
EHEDPSGSL
13


 


336
KVLSPSGLL
13


 


348
QERNIESLS
13


 


352
IESLSDLRP
13


 


361
PPQNPRKLI
13


 


379
MKSDLVEYF
13


 


381
SDLVEYFTL
13


 


389
LEMLHLGNN
13


 


418
LNGNHLTKL
13


 


426
LSKGMFLGL
13


 


432
LGLHNLEYL
13


 


443
EYNAIKEIL
13


 


457
PMPKLKVLY
13


 


458
MPKLKVLYL
13


 


462
KVLYLNNNL
13


 


466
LNNNLLQVL
13


 


471
LQVLPPHIF
13


 


481
GVPLTKVNL
13


 


506
LDLLTQIDL
13


 


511
QIDLEDNPW
13


 


520
DCSCDLVGL
13


 


523
CDLVGLQQW
13


 


549
PGHLDKKEL
13


 


603
DAVPLSVLI
13


 


704
EERNEKEGS
13


 


707
NEKEGSDAK
13


 


724
QENHSPLTG
13


 


747
LSFQDASSL
13


 


748
SFQDASSLY
13


 


758
NILEKEREL
13


 


760
LEKERELQQ
13


 


772
TEYLRKNIA
13


 


786
MEAHYPGAH
13


 


797
LKLMETLMY
13

















TABLE XXXII







V3-HLA-B4402-



9mers-158P1D7


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













6
QHMGAHEEL
12



 


8
MGAHEELKL
12


 


4
YEQHMGAHE
10


 


1
ASLYEQHMG
5

















TABLE XXXII







V4-HLA-B4402.



9mers-158P1D7


Each peptide is a portion of


SEQ ID NO: 9; each start


position is specified, the length


of peptide is 9 amino acids,


and the end position for each


peptide is the start position


plus eight.









Pos
123456789
score













3
HSLMKSILW
13



 


2
IHSLMKSIL
12


 


1
IIHSLMKSI
10


 


7
KSILWSKAS
9


 


4
SLMKSILWS
6


 


14
ASGRGRREE
6

















TABLE XXXIII







V1-HLA-B5101-



9mers-158P1D7


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













603
DAVPLSVLI
25



 


751
DASSLYRNI
25


 


306
LPTKAPGLI
24


 


625
AAGIVVLVL
24


 


111
LGLLKQLHI
23


 


175
LPPNIFRFV
23


 


309
KAPGLIPYI
23


 


456
NPMPKLKVL
23


 


142
QADNNFITV
22


 


474
LPPHIFSGV
22


 


624
CAAGIVVLV
22


 


85
SGLTNAISI
21


 


154
SAFSKLNRL
21


 


249
SPPFFKGSI
21


 


329
CPIPCNCKV
21


 


360
PPPQNPRKL
21


 


361
PPQNPRKLI
21


 


458
MPKLKVLYL
21


 


713
DAKHLQRSL
21


 


51
KGIKMVSEI
20


 


95
LGFNNIADI
20


 


593
TADTILRSL
20


 


61
VPPSRPFQL
19


 


237
PPQSIIGDV
19


 


370
LAGNIIHSL
19


 


504
DDLDLLTQI
19


 


517
NPWDCSCDL
19


 


692
SPSFGPKHL
19


 


828
HAEPDYLEV
19


 


106
GAFNGLGLL
18


 


109
NGLGLLKQL
18


 


198
LPYVGFLEH
18


 


250
PPFFKGSIL
18


 


394
LGNNRIEVL
18


 


442
LEYNAIKEI
18


 


482
VPLTKVNLK
18


 


803
LMYSRPRKV
18


 


133
HGLENLEFL
17


 


278
DPSGSLHLA
17


 


314
IPYITKPST
17


 


432
LGLHNLEYL
17


 


439
YLYLEYNAI
17


 


605
VPLSVLILG
17


 


613
GLLIMFITI
17


 


83
DFSGLTNAI
16


 


202
GELEHIGRI
16


 


586
TPATTTNTA
16


 


105
IGAFNGLGL
15


 


143
ADNNFITVI
15


 


170
NAIESLPPN
15


 


183
VPLTHLDLR
15


 


207
IGRILDLQL
15


 


236
MPPQSIIGD
15


 


283
LHLAATSSI
15


 


285
LAATSSIND
15


 


326
GPYCPIPCN
15


 


524
DLVGLQQWI
15


 


589
TTTNTADTI
15


 


601
LTDAVPLSV
15


 


791
PGAHEELKL
15


 


807
RPRKVLVEQ
15


 


13
LACISLHSQ
14


 


16
ISLHSQTPV
14


 


45
LINCEAKGI
14


 


49
EAKGIKMVS
14


 


74
NGLTMLHTN
14


 


140
FLQADNNFI
14


 


269
TPPVYEEHE
14


 


339
SPSGLLIHC
14


 


364
NPRKLILAG
14


 


391
MLHLGNNRI
14


 


445
NAIKEILPG
14


 


451
LPGTFNPMP
14


 


470
LLQVLPPHI
14


 


497
LPVSNILDD
14


 


532
IQKLSKNTV
14


 


558
KALNSEILC
14


 


566
CPGLVNNPS
14


 


587
PATTTNTAD
14


 


622
VFCAAGIVV
14


 


728
SPLTGSNMK
14


 


792
GAHEELKLM
14


 


22
TPVLSSRGS
13


 


100
IADIEIGAF
13


 


157
SKLNRLKVL
13


 


176
PPNIFRFVP
13


 


193
NQLQTLPYV
13


 


199
PYVGFLEHI
13


 


225
DLLQLKTWL
13


 


233
LENMPPQSI
13


 


258
LSRLKKESI
13


 


286
AATSSINDS
13


 


295
RMSTKTTSI
13


 


298
TKTTSILKL
13


 


324
LPGPYCPIP
13


 


331
IPCNCKVLS
13


 


337
VLSPSGLLI
13


 


344
LIHCQERNI
13


 


359
RPPPQNPRK
13


 


366
RKLILAGNI
13


 


384
VEYFTLEML
13


 


408
MNLTRLQKL
13


 


455
FNPMPKLKV
13


 


463
VLYLNNNLL
13


 


475
PPHIFSGVP
13


 


479
FSGVPLTKV
13


 


494
FTHLPVSNI
13


 


536
SKNTVTDDI
13


 


548
SPGHLDKKE
13


 


549
PGHLDKKEL
13


 


572
NPSMPTQTS
13


 


608
SVLILGLLI
13


 


611
ILGLLIMFI
13


 


623
FCAAGIVVL
13


 


672
RPSASLYEQ
13


 


684
SPMVHVYRS
13


 


758
NILEKEREL
13


 


771
ITEYLRKNI
13


 


779
IAQLQPDME
13


 


790
YPGAHEELK
13


 


829
AEPDYLEVL
13


 


8
FYSSLLACI
12


 


41
DGTMLINCE
12


 


53
IKMVSEISV
12


 


65
RPFQLSLLN
12


 


89
NAISIHLGF
12


 


92
SIHLGFNNI
12


 


97
FNNIADIEI
12


 


130
DTFHGLENL
12


 


151
IEPSAFSKL
12


 


152
EPSAFSKLN
12


 


156
FSKLNRLKV
12


 


159
LNRLKVLIL
12


 


164
VLILNDNAI
12


 


267
CPTPPVYEE
12


 


319
KPSTQLPGP
12


 


323
QLPGPYCPI
12


 


415
KLYLNGNHL
12


 


418
LNGNHLTKL
12


 


426
LSKGMFLGL
12


 


465
YLNNNLLQV
12


 


466
LNNNLLQVL
12


 


495
THLPVSNIL
12


 


506
LDLLTQIDL
12


 


520
DCSCDLVGL
12


 


544
ILCTSPGHL
12


 


556
ELKALNSEI
12


 


575
MPTQTSYLM
12


 


614
LLIMFITIV
12


 


620
TIVFCAAGI
12


 


621
IVFCAAGIV
12


 


674
SASLYEQHM
12

















TABLE XXXIII







V3-HLA-B5101-



9mers-158P1D7


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
MGAHEELKL
16



 


6
QHMGAHEEL
7

















TABLE XXXIII







V4-HLA-B5101-



9mers-158P1D7


Each peptide is a portion of


SEQ ID NO: 9; each start


position is specified, the length


of peptideis 9 amino acids,


and the end position for each


peptide is the start position


plus eight.









Pos
123456789
score













1
IIHSLMKSI
13



 


13
KASGRGRRE
13


 


2
IHSLMKSIL
9

















TABLE XXXIV







V1-HLA-A1-



10mers-158P1D7


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













669
TTERPSASLY
33



 


307
PTKAPGLIPY
25


 


430
MFLGLHNLEY
23


 


796
ELKLMETLMY
23


 


191
RGNQLQTLPY
21


 


435
HNLEYLYLEY
21


 


456
NPMPKLKVLY
21


 


649
RDNSPVHLQY
21


 


743
STEFLSFQDA
21


 


747
LSFQDASSLY
21


 


134
GLENLEFLQA
20


 


150
VIEPSAFSKL
20


 


264
ESICPTPPVY
20


 


276
HEDPSGSLHL
20


 


728
SPLTGSNMKY
20


 


781
QLQPDMEAHY
20


 


203
FLEHIGRILD
19


 


820
YFELKANLHA
19


 


377
SLMKSDLVEY
18


 


630
VLVLHRRRRY
18


 


652
SPVHLQYSMY
18


 


805
YSRPRKVLVE
18


 


128
KEDTFHGLEN
17


 


408
MNLTRLQKLY
17


 


432
LGLHNLEYLY
17


 


502
ILDDLDLLTQ
17


 


518
PWDCSCDLVG
17


 


540
VTDDILCTSP
17


 


601
LTDAVPLSVL
17


 


681
HMVSPMVHVY
17


 


759
ILEKERELQQ
17


 


811
VLVEQTKNEY
17


 


830
EPDYLEVLEQ
17


 


297
STKTTSILKL
16


 


317
ITKPSTQLPG
16


 


351
NIESLSDLRP
16


 


561
NSEILCPGLV
16


 


723
EQENHSPLTG
16


 


765
ELQQLGITEY
16


 


771
ITEYLRKNIA
16

















TABLE XXXIV







V3-HLA-A1-



10mers-158P1D7


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













4
LYEQHMGAHE
11



 


8
HMGAHEELKL
8


 


2
ASLYEQHMGA
5

















TABLE XXXIV







V4-HLA-A1-



10mers-158P1D7


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













4
HSLMKSILWS
10



 


3
IHSLMKSILW
6


 


12
WSKASGRGRR
5


 


8
KSILWSKASG
4

















TABLE XXXV







V1-HLA-A2-



10mers-158P1D7


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













369
ILAGNIIHSL
33



 


417
YLNGNHLTKL
31


 


166
ILNDNAIESL
30


 


70
SLLNNGLTML
28


 


158
KLNRLKVLIL
27


 


189
DLRGNQLQTL
27


 


465
YLNNNLLQVL
27


 


613
GLLIMFITIV
27


 


407
FMNLTRLQKL
26


 


610
LILGLLIMFI
26


 


126
ILKEDTFHGL
25


 


431
FLGLHNLEYL
25


 


600
SLTDAVPLSV
25


 


174
SLPPNIFRFV
24


 


393
HLGNNRIEVL
24


 


473
VLPPHIFSGV
24


 


551
HLDKKELKAL
24


 


94
HLGFNNIADI
23


 


118
HINHNSLEIL
23


 


425
KLSKGMFLGL
23


 


441
YLEYNAIKEI
23


 


592
NTADTILRSL
23


 


624
CAAGIVVLVL
23


 


150
VIEPSAFSKL
22


 


257
ILSRLKKESI
22


 


282
SLHLAATSSI
22


 


297
STKTTSILKL
22


 


343
LLIHCQERNI
22


 


401
VLEEGSFMNL
22


 


433
GLHNLEYLYL
22


 


746
FLSFQDASSL
22


 


802
TLMYSRPRKV
22


 


12
LLACISLHSQ
21


 


78
MLHTNDFSGL
21


 


377
SLMKSDLVEY
21


 


469
NLLQVLPPHI
21


 


531
WIQKLSKNTV
21


 


581
YLMVTTPATT
21


 


596
TILRSLTDAV
21


 


606
PLSVLILGLL
21


 


647
QMRDNSPVHL
21


 


721
LLEQENHSPL
21


 


44
MLINCEAKGI
20


 


52
GIKMVSEISV
20


 


86
GLTNAISIHL
20


 


110
GLGLLKQLHI
20


 


374
IIHSLMKSDL
20


 


409
NLTRLQKLYL
20


 


457
PMPKLKVLYL
20


 


478
IFSGVPLTKV
20


 


502
ILDDLDLLTQ
20


 


601
LTDAVPLSVL
20


 


603
DAVPLSVLIL
20


 


803
LMYSRPRKVL
20


 


206
HIGRILDLQL
19


 


220
WACNCDLLQL
19


 


232
WLENMPPQSI
19


 


305
KLPTKAPGLI
19


 


464
LYLNNNLLQV
19


 


488
NLKTNQFTHL
19


 


505
DLDLLTQIDL
19


 


526
VGLQQWIQKL
19


 


543
DILCTSPGHL
19


 


564
ILCPGLVNNP
19


 


605
VPLSVLILGL
19


 


616
IMFITIVFCA
19


 


619
ITIVFCAAGI
19


 


623
FCAAGIVVLV
19


 


668
HTTERPSASL
19


 


676
SLYEQHMVSP
19


 


720
SLLEQENHSP
19


 


754
SLYRNILEKE
19


 


827
LHAEPDYLEV
19


 


828
HAEPDYLEVL
19


 


4
WIHLFYSSLL
18


 


15
CISLHSQTPV
18


 


60
SVPPSRPFQL
18


 


102
DIEIGAFNGL
18


 


240
SIIGDVVCNS
18


 


295
RMSTKTTSIL
18


 


304
LKLPTKAPGL
18


 


337
VLSPSGLLIH
18


 


346
HCQERNIESL
18


 


382
DLVEYFTLEM
18


 


383
LVEYFTLEML
18


 


392
LHLGNNRIEV
18


 


500
SNILDDLDLL
18


 


7
LFYSSLLACI
17


 


104
EIGAFNGLGL
17


 


105
IGAFNGLGLL
17


 


141
LQADNNFITV
17


 


163
KVLILNDNAI
17


 


170
NAIESLPPNI
17


 


204
LEHIGRILDL
17


 


260
RLKKESICPT
17


 


308
TKAPGLIPYI
17


 


415
KLYLNGNHLT
17


 


462
KVLYLNNNLL
17


 


490
KTNQFTHLPV
17


 


519
WDCSCDLVGL
17


 


559
ALNSEILCPG
17


 


608
SVLILGLLIM
17


 


609
VLILGLLIMF
17


 


620
TIVFCAAGIV
17


 


621
IVFCAAGIVV
17


 


622
VFCAAGIVVL
17


 


674
SASLYEQHMV
17


 


760
LEKERELQQL
17


 


770
GITEYLRKNI
17


 


788
AHYPGAHEEL
17


 


798
KLMETLMYSR
17


 


3
LWIHLFYSSL
16


 


6
HLFYSSLLAC
16


 


50
AKGIKMVSEI
16


 


99
NIADIEIGAF
16


 


113
LLKQLHINHN
16


 


115
KQLHINHNSL
16


 


142
QADNNFITVI
16


 


192
GNQLQTLPYV
16


 


252
FFKGSILSRL
16


 


313
LIPYITKPST
16


 


336
KVLSPSGLLI
16


 


368
LILAGNIIHS
16


 


390
EMLHLGNNRI
16


 


412
RLQKLYLNGN
16


 


428
KGMFLGLHNL
16


 


445
NAIKEILPGT
16


 


454
TFNPMPKLKV
16


 


480
SGVPLTKVNL
16


 


494
FTHLPVSNIL
16


 


497
LPVSNILDDL
16


 


501
NILDDLDLLT
16


 


556
ELKALNSEIL
16


 


560
LNSEILCPGL
16


 


582
LMVTTPATTT
16


 


611
ILGLLIMFIT
16


 


615
LIMFITIVFC
16


 


712
SDAKHLQRSL
16


 


811
VLVEQTKNEY
16

















TABLE XXXV







V3-HLA-A2-



10mers-158P1D7


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













8
HMGAHEELKL
21



 


3
SLYEQHMGAH
16

















TABLE XXXV







V4-HLA-A2-



10mers-158P1D7


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













2
IIHSLMKSIL
20



 


1
NIIHSLMKSI
19


 


5
SLMKSILWSK
19


 


6
LMKSILWSKA
15


 


9
SILWSKASGR
13


 


10
ILWSKASGRG
13


 


14
KASGRGRREE
9

















TABLE XXXVI







V1-HLA-A0203-



10mers-158P1D7


Each peptide is a portion of


SEQ ID NO: 3; each start


position is specified, the length


of peptide 10 amino acids,


and the end position for each


peptide is the start position


plus nine.









Pos
1234567890
score













278
DPSGSLHLAA
19



 


617
MFITIVFCAA
19


 


279
PSGSLHLAAT
17


 


618
FITIVFCAAG
17


 


5
IHLFYSSLLA
10


 


41
DGTMLINCEA
10


 


81
TNDFSGLTNA
10


 


92
SIHLGFNNIA
10


 


98
NNIADIEIGA
10


 


134
GLENLEFLQA
10


 


146
NFITVIEPSA
10


 


162
LKVLILNDNA
10


 


212
DLQLEDNKWA
10


 


277
EDPSGSLHLA
10


 


301
TSILKLPTKA
10


 


362
PQNPRKLILA
10


 


437
LEYLYLEYNA
10


 


550
GHLDKKELKA
10


 


579
TSYLMVTTPA
10


 


585
TTPATTTNTA
10


 


595
DTILRSLTDA
10


 


616
IMFITIVFCA
10


 


666
THHTTERPSA
10


 


705
ERNEKEGSDA
10


 


743
STEFLSFQDA
10


 


771
ITEYLRKNIA
10


 


779
IAQLQPDMEA
10


 


784
PDMEAHYPGA
10


 


816
TKNEYFELKA
10


 


820
YFELKANLHA
10


 


6
HLFYSSLLAC
9


 


42
GTMLINCEAK
9


 


82
NDFSGLTNAI
9


 


93
IHLGFNNIAD
9


 


99
NIADIEIGAF
9


 


135
LENLEFLQAD
9


 


147
FITVIEPSAF
9


 


163
KVLILNDNAI
9


 


213
LQLEDNKWAC
9


 


302
SILKLPTKAP
9


 


363
QNPRKLILAG
9


 


438
EYLYLEYNAI
9


 


551
HLDKKELKAL
9


 


580
SYLMVTTPAT
9


 


586
TPATTTNTAD
9


 


596
TILRSLTDAV
9


 


667
HHTTERPSAS
9


 


706
RNEKEGSDAK
9


 


744
TEELSEQOAS
9


 


772
TEYLRKNIAQ
9


 


780
AQLQPDMEAH
9


 


785
DMEAHYPGAH
9


 


817
KNEYFELKAN
9


 


821
FELKANLHAE
9

















TABLE XXXVI







V3-HLA-A0203-



10mers-158P1D7


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













2
ASLYEQHMGA
10



 


3
SLYEQHMGAH
9


 


4
LYEQHMGAHE
8

















TABLE XXXVI







V4-HLA-A0203-



10mers-158P1D7


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
LMKSILWSKA
10



 


7
MKSILWSKAS
9


 


8
KSILWSKASG
8

















TABLE XXXVII







V1-HLA-A3-



10mers-158P1D7


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













149
TVIEPSAFSK
29



 


439
YLYLEYNAIK
28


 


290
SINDSRMSTK
27


 


477
HIFSGVPLTK
26


 


768
QLGITEYLRK
26


 


525
LVGLQQWIQK
24


 


632
VLHRRRRYKK
24


 


781
QLQPDMEAHY
24


 


178
NIFRFVPLTH
23


 


210
ILDLQLEDNK
23


 


446
AIKEILPGTF
23


 


631
LVLHRRRRYK
23


 


245
VVCNSPPFFK
22


 


597
ILRSLTDAVP
22


 


676
SLYEQHMVSP
22


 


729
PLTGSNMKYK
22


 


796
ELKLMETLMY
22


 


336
KVLSPSGLLI
21


 


367
KLILAGNIIH
21


 


377
SLMKSDLVEY
21


 


481
GVPLTKVNLK
21


 


614
LLIMFITIVF
21


 


655
HLQYSMYGHK
21


 


682
MVSPMVHVYR
21


 


123
SLEILKEDTF
20


 


194
QLQTLPYVGF
20


 


337
VLSPSGLLIH
20


 


357
DLRPPPQNPR
20


 


416
LYLNGNHLTK
20


 


502
ILDDLDLLTQ
20


 


798
KLMETLMYSR
20


 


45
LINCEAKGIK
19


 


158
KLNRLKVLIL
19


 


189
DLRGNQLQTL
19


 


398
RIEVLEEGSF
19


 


406
SFMNLTRLQK
19


 


472
QVLPPHIFSG
19


 


609
VLILGLLIMF
19


 


621
IVFCAAGIVV
19


 


11
SLLACISLHS
18


 


23
PVLSSRGSCD
18


 


60
SVPPSRPFQL
18


 


254
KGSILSRLKK
18


 


310
APGLIPYITK
18


 


371
AGNIIHSLMK
18


 


415
KLYLNGNHLT
18


 


463
VLYLNNNLLQ
18


 


581
YLMVTTPATT
18


 


600
SLTDAVPLSV
18


 


630
VLVLHRRRRY
18


 


746
FLSFQDASSL
18


 


754
SLYRNILEKE
18


 


759
ILEKERELQQ
18


 


44
MLINCEAKGI
17


 


106
GAFNGLGLLK
17


 


134
GLENLEFLQA
17


 


147
FITVIEPSAF
17


 


163
KVLILNDNAI
17


 


164
VLILNDNAIE
17


 


197
TLPYVGFLEH
17


 


206
HIGRILDLQL
17


 


257
ILSRLKKESI
17


 


265
SICPTPPVYE
17


 


282
SLHLAATSSI
17


 


303
ILKLPTKAPG
17


 


369
ILAGNIIHSL
17


 


608
SVLILGLLIM
17


 


628
IVVLVLHRRR
17


 


629
VVLVLHRRRR
17


 


688
HVYRSPSFGP
17


 


765
ELQQLGITEY
17


 


811
VLVEQTKNEY
17


 


2
KLWIHLFYSS
16


 


17
SLHSQTPVLS
16


 


70
SLLNNGLTML
16


 


71
LLNNGLTMLH
16


 


99
NIADIEIGAF
16


 


104
EIGAFNGLGL
16


 


112
GLLKQLHINH
16


 


116
QLHINHNSLE
16


 


171
AIESLPPNIF
16


 


214
QLEDNKWACN
16


 


312
GLIPYITKPS
16


 


409
NLTRLQKLYL
16


 


422
HLTKLSKGMF
16


 


425
KLSKGMFLGL
16


 


473
VLPPHIFSGV
16


 


633
LHRRRRYKKK
16


 


649
RDNSPVHLQY
16


 


686
MVHVYRSPSF
16


 


716
HLQRSLLEQE
16


 


720
SLLEQENHSP
16


 


753
SSLYRNILEK
16


 


774
YLRKNIAQLQ
16


 


822
ELKANLHAEP
16


 


90
AISIHLGFNN
15


 


161
RLKVLILNDN
15


 


166
ILNDNAIESL
15


 


182
FVPLTHLDLR
15


 


209
RILDLQLEDN
15


 


244
DVVCNSPPFF
15


 


260
RLKKESICPT
15


 


271
PVYEEHEDPS
15


 


300
TTSILKLPTK
15


 


305
KLPTKAPGLI
15


 


314
IPYITKPSTQ
15


 


393
HLGNNRIEVL
15


 


419
NGNHLTKLSK
15


 


450
ILPGTFNPMP
15


 


462
KVLYLNNNLL
15


 


465
YLNNNLLQVL
15


 


470
LLQVLPPHIF
15


 


507
DLLTQIDLED
15


 


528
LQQWIQKLSK
15


 


539
TVTDDILCTS
15


 


544
ILCTSPGHLD
15


 


562
SEILCPGLVN
15


 


564
ILCPGLVNNP
15


 


569
LVNNPSMPTQ
15


 


583
MVTTPATTTN
15


 


669
TTERPSASLY
15


 


706
RNEKEGSDAK
15


 


808
PRKVLVEQTK
15


 


810
KVLVEQTKNE
15


 


6
HLFYSSLLAC
14


 


68
QLSLLNNGLT
14


 


75
GLTMLHTNDF
14


 


110
GLGLLKQLHI
14


 


126
ILKEDTFHGL
14


 


150
VIEPSAFSKL
14


 


165
LILNONAIES
14


 


174
SLPPNIFRFV
14


 


200
YVGFLEHIGR
14


 


226
LLQLKTWLEN
14


 


228
QLKTWLENMP
14


 


232
WLENMPPQSI
14


 


240
SIIGDVVCNS
14


 


264
ESICPTPPVY
14


 


323
QLPGPYCPIP
14


 


382
DLVEYFTLEM
14


 


400
EVLEEGSFMN
14


 


412
RLQKLYLNGN
14


 


433
GLHNLEYLYL
14


 


460
KLKVLYLNNN
14


 


483
PLTKVNLKTN
14


 


486
KVNLKTNQFT
14


 


501
NILDDLDLLT
14


 


534
KLSKNTVTDD
14


 


563
EILCPGLVNN
14


 


596
TILRSLTDAV
14


 


604
AVPLSVLILG
14


 


613
GLLIMFITIV
14


 


643
QVDEQMRDNS
14


 


689
VYRSPSFGPK
14


 


812
LVEQTKNEYF
14


 


815
QTKNEYFELK
14

















TABLE XXXVII







V3-HLA-A3-



10mers-158P1D7


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













3
SLYEQHMGAH
22



 


7
QHMGAHEELK
14

















TABLE XXXVII







V4-HLA-A3-



10mers-158P1D7


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













5
SLMKSILWSK
23



 


9
SILWSKASGR
21


 


1
NIIHSLMKSI
13


 


2
IIHSLMKSIL
13


 


10
ILWSKASGRG
13


 


8
KSILWSKASG
12

















TABLE XXXVIII







V1-HLA-A26-



10mers-158P1D7


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













244
DVVCNSPPFF
30



 


603
DAVPLSVLIL
26


 


104
EIGAFNGLGL
24


 


264
ESICPTPPVY
24


 


595
DTILRSLTDA
24


 


765
ELQQLGITEY
24


 


129
EDTFHGLENL
23


 


173
ESLPPNIFRF
23


 


297
STKTTSILKL
23


 


307
PTKAPGLIPY
23


 


349
ERNIESLSDL
23


 


383
LVEYFTLEML
23


 


385
EYFTLEMLHL
23


 


400
EVLEEGSFMN
23


 


773
EYLRKNIAQL
23


 


58
EISVPPSRPF
22


 


274
EEHEDPSGSL
22


 


404
EGSFMNLTRL
22


 


592
NTADTILRSL
22


 


796
ELKLMETLMY
22


 


60
SVPPSRPFQL
21


 


189
DLRGNQLQTL
21


 


543
DILCTSPGHL
21


 


601
LTDAVPLSVL
21


 


102
DIEIGAFNGL
20


 


130
DTFHGLENLE
20


 


668
HTTERPSASL
20


 


669
TTERPSASLY
20


 


99
NIADIEIGAF
19


 


681
HMVSPMVHVY
19


 


686
MVHVYRSPSF
19


 


814
EQTKNEYFEL
19


 


149
TVIEPSAFSK
18


 


205
EHIGRILDLQ
18


 


462
KVLYLNNNLL
18


 


539
TVTDDILCTS
18


 


556
ELKALNSEIL
18


 


563
EILCPGLVNN
18


 


589
TTTNTADTIL
18


 


609
VLILGLLIMF
18


 


708
EKEGSDAKHL
18


 


801
ETLMYSRPRK
18


 


812
LVEQTKNEYF
18


 


423
LTKLSKGMFL
17


 


497
LPVSNILDDL
17


 


500
SNILDDLDLL
17


 


505
DLDLLTQIDL
17


 


538
NTVTDDILCT
17


 


652
SPVHLQYSMY
17


 


713
DAKHLQRSLL
17


 


738
KTTNQSTEFL
17


 


751
DASSLYRNIL
17


 


55
MVSEISVPPS
16


 


118
HINHNSLEIL
16


 


217
DNKWACNCDL
16


 


446
AIKEILPGTF
16


 


472
QVLPPHIFSG
16


 


494
FTHLPVSNIL
16


 


516
DNPWDCSCDL
16


 


608
SVLILGLLIM
16


 


621
IVFCAAGIVV
16


 


811
VLVEQTKNEY
16


 


819
EYFELKANLH
16


 


39
EKDGTMLINC
15


 


147
FITVIEPSAF
15


 


150
VIEPSAFSKL
15


 


277
EDPSGSLHLA
15


 


346
HCQERNIESL
15


 


377
SLMKSDLVEY
15


 


382
DLVEYFTLEM
15


 


449
EILPGTFNPM
15


 


604
AVPLSVLILG
15


 


671
ERPSASLYEQ
15


 


747
LSFQDASSLY
15


 


760
LEKERELQQL
15


 


763
ERELQQLGIT
15


 


830
EPDYLEVLEQ
15


 


3
LWIHLFYSSL
14


 


63
PSRPFQLSLL
14


 


70
SLLNNGLTML
14


 


125
EILKEDTFHG
14


 


166
ILNDNAIESL
14


 


181
RFVPLTHLDL
14


 


182
FVPLTHLDLR
14


 


195
LQTLPYVGFL
14


 


206
HIGRILDLQL
14


 


220
WACNCDLLQL
14


 


300
TTSILKLPTK
14


 


374
IIHSLMKSDL
14


 


398
RIEVLEEGSF
14


 


480
SGVPLTKVNL
14


 


481
GVPLTKVNLK
14


 


485
TKVNLKTNQF
14


 


546
CTSPGHLDKK
14


 


605
VPLSVLILGL
14


 


628
IVVLVLHRRR
14


 


630
VLVLHRRRRY
14


 


705
ERNEKEGSDA
14

















TABLE XXXVIII







V3-HLA-A26-



10mers-158P1D


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













6
EQHMGAHEEL
18



 


8
HMGAHEELKL
10

















TABLE XXXVIII







V4-HLA-A26-



10mers-158P1D


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













2
IIHSLMKSIL
14



 


1
NIIHSLMKSI
9


 


5
SLMKSILWSK
6


 


9
SILWSKASGR
6

















TABLE XXXIX







V1-HLA-B0702-



10mers-158P1D7


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













62
PPSRPFQLSL
24



 


176
PPNIFRFVPL
24


 


790
YPGAHEELKL
24


 


278
DPSGSLHLAA
23


 


475
PPHIFSGVPL
23


 


329
CPIPCNCKVL
22


 


359
RPPPQNPRKL
22


 


361
PPQNPRKLIL
22


 


605
VPLSVLILGL
22


 


548
SPGHLDKKEL
21


 


807
RPRKVLVEQT
21


 


249
SPPFFKGSIL
20


 


497
LPVSNILDDL
20


 


482
VPLTKVNLKT
18


 


566
CPGLVNNPSM
18


 


575
MPTQTSYLMV
18


 


237
PPQSIIGDVV
17


 


360
PPPQNPRKLI
17


 


425
KLSKGMFLGL
17


 


624
CAAGIVVLVL
17


 


152
EPSAFSKLNR
16


 


198
LPYVGFLEHI
16


 


236
MPPQSIIGDV
16


 


517
NPWDCSCDLV
16


 


104
EIGAFNGLGL
15


 


598
LRSLTDAVPL
15


 


830
EPDYLEVLEQ
15


 


16
ISLHSQTPVL
14


 


155
AFSKLNRLKV
14


 


158
KLNRLKVLIL
14


 


179
IFRFVPLTHL
14


 


181
RFVPLTHLDL
14


 


189
DLRGNQLQTL
14


 


276
HEDPSGSLHL
14


 


295
RMSTKTTSIL
14


 


319
KPSTQLPGPY
14


 


331
IPCNCKVLSP
14


 


339
SPSGLLIHCQ
14


 


364
NPRKLILAGN
14


 


369
ILAGNIIHSL
14


 


404
EGSFMNLTRL
14


 


456
NPMPKLKVLY
14


 


457
PMPKLKVLYL
14


 


603
DAVPLSVLIL
14


 


622
VFCAAGIVVL
14


 


647
QMRDNSPVHL
14


 


672
RPSASLYEQH
14


 


63
PSRPFQLSLL
13


 


65
RPFQLSLLNN
13


 


206
HIGRILDLQL
13


 


306
LPTKAPGLIP
13


 


310
APGLIPYITK
13


 


324
LPGPYCPIPC
13


 


385
EYFTLEMLHL
13


 


417
YLNGNHLTKL
13


 


480
SGVPLTKVNL
13


 


551
HLDKKELKAL
13


 


560
LNSEILCPGL
13


 


572
NPSMPTQTSY
13


 


573
PSMPTQTSYL
13


 


586
TPATTTNTAD
13


 


601
LTDAVPLSVL
13


 


708
EKEGSDAKHL
13


 


738
KTTNQSTEFL
13


 


751
DASSLYRNIL
13


 


788
AHYPGAHEEL
13


 


9
YSSLLACISL
12


 


25
LSSRGSCDSL
12


 


105
IGAFNGLGLL
12


 


126
ILKEDTFHGL
12


 


132
FHGLENLEFL
12


 


150
VIEPSAFSKL
12


 


175
LPPNIFRFVP
12


 


183
VPLTHLDLRG
12


 


195
LQTLPYVGFL
12


 


204
LEHIGRILDL
12


 


220
WACNCDLLQL
12


 


252
FFKGSILSRL
12


 


263
KESICPTPPV
12


 


297
STKTTSILKL
12


 


304
LKLPTKAPGL
12


 


380
KSDLVEYFTL
12


 


393
HLGNNRIEVL
12


 


409
NLTRLQKLYL
12


 


428
KGMFLGLHNL
12


 


433
GLHNLEYLYL
12


 


451
LPGTFNPMPK
12


 


478
IFSGVPLTKV
12


 


488
NLKTNQFTHL
12


 


499
VSNILDDLDL
12


 


519
WDCSCDLVGL
12


 


556
ELKALNSEIL
12


 


606
PLSVLILGLL
12


 


692
SPSFGPKHLE
12


 


712
SDAKHLQRSL
12


 


746
FLSFQDASSL
12


 


773
EYLRKNIAQL
12


 


783
QPDMEAHYPG
12


 


803
LMYSRPRKVL
12


 


814
EQTKNEYFEL
12


 


825
ANLHAEPDYL
12


 


828
HAEPDYLEVL
12


 


22
TPVLSSRGSC
11


 


36
NCEEKDGTML
11


 


60
SVPPSRPFQL
11


 


61
VPPSRPFQLS
11


 


70
SLLNNGLTML
11


 


78
MLHTNDFSGL
11


 


102
DIEIGAFNGL
11


 


108
FNGLGLLKQL
11


 


115
KQLHINHNSL
11


 


129
EDTFHGLENL
11


 


153
PSAFSKLNRL
11


 


156
FSKLNRLKVL
11


 


166
ILNDNAIESL
11


 


218
NKWACNCDLL
11


 


224
CDLLQLKTWL
11


 


267
CPTPPVYEEH
11


 


274
EEHEDPSGSL
11


 


314
IPYITKPSTQ
11


 


315
PYITKPSTQL
11


 


349
ERNIESLSDL
11


 


374
IIHSLMKSDL
11


 


401
VLEEGSFMNL
11


 


423
LTKLSKGMFL
11


 


431
FLGLHNLEYL
11


 


452
PGTFNPMPKL
11


 


455
FNPMPKLKVL
11


 


462
KVLYLNNNLL
11


 


465
YLNNNLLQVL
11


 


474
LPPHIFSGVP
11


 


505
DLDLLTQIDL
11


 


589
TTTNTADTIL
11


 


592
NTADTILRSL
11


 


623
FCAAGIVVLV
11


 


668
HTTERPSASL
11


 


684
SPMVHVYRSP
11


 


691
RSPSFGPKHL
11


 


713
DAKHLQRSLL
11


 


721
LLEQENHSPL
11


 


757
RNILEKEREL
11


 


762
KERELQQLGI
11


 


766
LQQLGITEYL
11


 


818
NEYFELKANL
11

















TABLE XXXIX







V3-HLA-B0702-



10mers-158P1D7


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













8
HMGAHEELKL
14



 


6
EQHMGAHEEL
11


 


2
ASLYEQHMGA
8


 


9
MGAHEELKLM
7

















TABLE XXXIX







V4-HLA-B0702-



10mers-158P1D7


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 starts position


plus nine.









Pos
1234567890
score













2
IIHSLMKSIL
11



 


1
NIIHSLMKSI
6


 


6
LMKSILWSKA
6


 


14
KASGRGRREE
6

















TABLE XL







V1-HLA-B08-



10mers-158P1D7









Pos
1234567890
score










NoResultsFound.

















TABLE XL







V3-HLA-B08-



10mers-158P1D7









Pos
1234567890
score










NoResultsFound.

















TABLE XL







V4-HLA-B08-



10mers-158P1D7









Pos
1234567890
score










NoResultsFound.

















TABLE XLI







V1-HLA-B1510-



10mers-158P1D7









Pos
1234567890
score










NoResultsFound.

















TABLE XLI







V3-HLA-B1510-



10mers-158P1D7









Pos
1234567890
score










NoResultsFound.

















TABLE XLI







V4-HLA-B1510-



10mers-158P1D7









Pos
1234567890
score










NoResultsFound.

















TABLE XLII







V1-HLA-B2705-



10mers-158P1D7









Pos
1234567890
score










NoResultsFound.

















TABLE XLII







V3-HLA-B2705-



10mers-158P1D7









Pos
1234567890
score










NoResultsFound.

















TABLE XLII







V4-HLA-B2705-



10mers-158P1D7









Pos
1234567890
score










NoResultsFound.

















TABLE XLIII







V4-HLA-B2709-



10mers-158P107









Pos
1234567890
score










NoResultsFound.

















TABLE XLIII







V3-HLA-B2709-



10mers-158P1D7









Pos
1234567890
score










NoResultsFound.

















TABLE XLIII







V4-HLA-B2709-



10mers-158P1D7









Pos
1234567890
score










NoResultsFound.

















TABLE XLIV







V1-HLA-B4402-



10mers-158P1D7


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













276
HEDPSGSLHL
25



 


138
LEFLQADNNF
24


 


204
LEHIGRILDL
24


 


274
EEHEDPSGSL
22


 


760
LEKERELQQL
22


 


794
HEELKLMETL
22


 


442
LEYNAIKEIL
21


 


818
NEYFELKANL
21


 


37
CEEKDGTMLI
20


 


555
KELKALNSEI
20


 


762
KERELQQLGI
20


 


173
ESLPPNIFRF
19


 


329
CPIPCNCKVL
19


 


233
LENMPPQSII
18


 


773
EYLRKNIAQL
18


 


60
SVPPSRPFQL
17


 


99
NIADIEIGAF
17


 


223
NCDLLQLKTW
17


 


264
ESICPTPPVY
17


 


297
STKTTSILKL
17


 


359
RPPPQNPRKL
17


 


456
NPMPKLKVLY
17


 


500
SNILDDLDLL
17


 


562
SEILCPGLVN
17


 


829
AEPDYLEVLE
17


 


385
EYFTLEMLHL
16


 


448
KEILPGTFNP
16


 


551
HLDKKELKAL
16


 


609
VLILGLLIMF
16


 


614
LLIMFITIVF
16


 


708
EKEGSDAKHL
16


 


788
AHYPGAHEEL
16


 


44
MLINCEAKGI
15


 


57
SEISVPPSRP
15


 


63
PSRPFQLSLL
15


 


82
NDFSGLTNAI
15


 


103
IEIGAFNGLG
15


 


108
FNGLGLLKQL
15


 


150
VIEPSAFSKL
15


 


156
FSKLNRLKVL
15


 


171
AIESLPPNIF
15


 


304
LKLPTKAPGL
15


 


315
PYITKPSTQL
15


 


369
ILAGNIIHSL
15


 


393
HLGNNRIEVL
15


 


408
MNLTRLQKLY
15


 


446
AIKEILPGTF
15


 


455
FNPMPKLKVL
15


 


480
SGVPLTKVNL
15


 


510
TQIDLEDNPW
15


 


522
SCDLVGLQQW
15


 


526
VGLQQWIQKL
15


 


573
PSMPTQTSYL
15


 


603
DAVPLSVLIL
15


 


605
VPLSVLILGL
15


 


622
VFCAAGIVVL
15


 


744
TEFLSFQDAS
15


 


757
RNILEKEREL
15


 


765
ELQQLGITEY
15


 


796
ELKLMETLMY
15


 


821
FELKANLHAE
15


 


825
ANLHAEPDYL
15


 


828
HAEPDYLEVL
15


 


38
EEKDGTMLIN
14


 


58
EISVPPSRPF
14


 


70
SLLNNGLTML
14


 


124
LEILKEDTFH
14


 


128
KEDTFHGLEN
14


 


135
LENLEFLQAD
14


 


142
QADNNFITVI
14


 


151
IEPSAFSKLN
14


 


158
KLNRLKVLIL
14


 


166
ILNDNAIESL
14


 


181
RFVPLTHLDL
14


 


186
THLDLRGNQL
14


 


201
VGFLEHIGRI
14


 


220
WACNCDLLQL
14


 


308
TKAPGLIPYI
14


 


319
KPSTQLPGPY
14


 


352
IESLSDLRPP
14


 


377
SLMKSDLVEY
14


 


380
KSDLVEYFTL
14


 


403
EEGSFMNLTR
14


 


404
EGSFMNLTRL
14


 


425
KLSKGMFLGL
14


 


428
KGMFLGLHNL
14


 


438
EYLYLEYNAI
14


 


462
KVLYLNNNLL
14


 


485
TKVNLKTNQF
14


 


588
ATTTNTADTI
14


 


592
NTADTILRSL
14


 


598
LRSLTDAVPL
14


 


624
CAAGIVVLVL
14


 


670
TERPSASLYE
14


 


691
RSPSFGPKHL
14


 


702
EEEERNEKEG
14


 


728
SPLTGSNMKY
14


 


772
TEYLRKNIAQ
14


 


795
EELKLMETLM
14


 


803
LMYSRPRKVL
14


 


3
LWIHLFYSSL
13


 


9
YSSLLACISL
13


 


16
ISLHSQTPVL
13


 


62
PPSRPFQLSL
13


 


91
ISIHLGFNNI
13


 


104
EIGAFNGLGL
13


 


115
KQLHINHNSL
13


 


117
LHINHNSLEI
13


 


129
EDTFHGLENL
13


 


147
FITVIEPSAF
13


 


157
SKLNRLKVLI
13


 


163
KVLILNDNAI
13


 


170
NAIESLPPNI
13


 


172
IESLPPNIFR
13


 


189
DLRGNQLQTL
13


 


206
HIGRILDLQL
13


 


211
LDLQLEDNKW
13


 


215
LEDNKWACNC
13


 


248
NSPPFFKGSI
13


 


263
KESICPTPPV
13


 


295
RMSTKTTSIL
13


 


305
KLPTKAPGLI
13


 


346
HCQERNIESL
13


 


349
ERNIESLSDL
13


 


360
PPPQNPRKLI
13


 


389
LEMLHLGNNR
13


 


402
LEEGSFMNLT
13


 


407
FMNLTRLQKL
13


 


409
NLTRLQKLYL
13


 


417
YLNGNHLTKL
13


 


430
MFLGLHNLEY
13


 


441
YLEYNAIKEI
13


 


457
PMPKLKVLYL
13


 


465
YLNNNLLQVL
13


 


488
NLKTNQFTHL
13


 


505
DLDLLTQIDL
13


 


548
SPGHLDKKEL
13


 


601
LTDAVPLSVL
13


 


606
PLSVLILGLL
13


 


610
LILGLLIMFI
13


 


612
LGLLIMFITI
13


 


630
VLVLHRRRRY
13


 


647
QMRDNSPVHL
13


 


669
TTERPSASLY
13


 


681
HMVSPMVHVY
13


 


701
EEEEERNEKE
13


 


703
EEERNEKEGS
13


 


704
EERNEKEGSD
13


 


709
KEGSDAKHLQ
13


 


738
KTTNQSTEFL
13


 


747
LSFQDASSLY
13


 


751
DASSLYRNIL
13


 


764
RELQQLGITE
13


 


781
QLQPDMEAHY
13


 


4
WIHLFYSSLL
12


 


25
LSSRGSCDSL
12


 


50
AKGIKMVSEI
12


 


67
FQLSLLNNGL
12


 


75
GLTMLHTNDF
12


 


78
MLHTNDFSGL
12


 


86
GLTNAISIHL
12


 


102
DIEIGAFNGL
12


 


105
IGAFNGLGLL
12


 


123
SLEILKEDTF
12


 


126
ILKEDTFHGL
12


 


131
TFHGLENLEF
12


 


132
FHGLENLEFL
12


 


139
EFLQADNNFI
12


 


153
PSAFSKLNRL
12


 


176
PPNIFRFVPL
12


 


191
RGNQLQTLPY
12


 


194
QLQTLPYVGF
12


 


195
LQTLPYVGFL
12


 


202
GFLEHIGRIL
12


 


218
NKWACNCDLL
12


 


224
CDLLQLKTWL
12


 


249
SPPFFKGSIL
12


 


252
FFKGSILSRL
12


 


307
PTKAPGLIPY
12


 


322
TQLPGPYCPI
12


 


334
NCKVLSPSGL
12


 


335
CKVLSPSGLL
12


 


336
KVLSPSGLLI
12


 


343
LLIHCQERNI
12


 


348
QERNIESLSD
12


 


361
PPQNPRKLIL
12


 


390
EMLHLGNNRI
12


 


414
QKLYLNGNHL
12


 


431
FLGLHNLEYL
12


 


432
LGLHNLEYLY
12


 


433
GLHNLEYLYL
12


 


435
HNLEYLYLEY
12


 


461
LKVLYLNNNL
12


 


470
LLQVLPPHIF
12


 


494
FTHLPVSNIL
12


 


503
LDDLDLLTQI
12


 


514
LEDNPWDCSC
12


 


519
WDCSCDLVGL
12


 


536
SKNTVTDDIL
12


 


543
DILCTSPGHL
12


 


556
ELKALNSEIL
12


 


572
NPSMPTQTSY
12


 


619
ITIVFCAAGI
12


 


649
RDNSPVHLQY
12


 


700
LEEEEERNEK
12


 


707
NEKEGSDAKH
12


 


712
SDAKHLQRSL
12


 


713
DAKHLQRSLL
12


 


740
TNQSTEFLSF
12


 


746
FLSFQDASSL
12


 


766
LQQLGITEYL
12


 


790
YPGAHEELKL
12


 


800
METLMYSRPR
12


 


814
EQTKNEYFEL
12


 


824
KANLHAEPDY
12

















TABLE XLIV







V4-HLA-B4402-



10mers-158P1D7


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













1
NIIHSLMKSI
14



3
IHSLMKSILW
14


2
IIHSLMKSIL
10

















TABLE XLV







V1-HLA-B5101-



10mers-158P1D7









Pos
1234567890
score










NoResultsFound.

















TABLE XLV







V3-HLA-B5101-



10mers-158P1D7









Pos
1234567890
score










NoResultsFound.

















TABLE XLV







V4-HLA-B5101-



10mers-158P1D7









Pos
1234567890
score










NoResultsFound.

















TABLE XLVI







V1-HLA-DRB-0101-



15mers-158P1D7


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













6
HLFYSSLLACISLHS
34



 


300
TTSILKLPTKAPGLI
33


 


73
NNGLTMLHTNDFSGL
32


 


554
KKELKALNSEILCPG
31


 


744
TEFLSFQDASSLYRN
31


 


145
NNFITVIEPSAFSKL
30


 


169
DNAIESLPPNIFRFV
30


 


468
NNLLQVLPPHIFSGV
30


 


153
PSAFSKLNRLKVLIL
29


 


444
YNAIKEILPGTFNPM
29


 


15
CISLHSQTPVLSSRG
28


 


42
GTMLINCEAKGIKMV
28


 


177
PNIFRFVPLTHLDLR
28


 


230
KTWLENMPPQSIIGD
28


 


467
NNNLLQVLPPHIFSG
28


 


572
NPSMPTQTSYLMVTT
28


 


606
PLSVLILGLLIMFIT
28


 


121
HNSLEILKEDTFHGL
27


 


129
EDTFHGLENLEFLQA
27


 


161
RLKVLILNDNAIESL
27


 


179
IFRFVPLTHLDLRGN
27


 


200
YVGFLEHIGRILDLQ
27


 


364
NPRKLILAGNIIHSL
27


 


383
LVEYFTLEMLHLGNN
27


 


420
GNHLTKLSKGMFLGL
27


 


436
NLEYLYLEYNAIKEI
27


 


491
TNQFTHLPVSNILDD
27


 


1
MKLWIHLFYSSLLAC
26


 


81
TNDFSGLTNAISIHL
26


 


102
DIEIGAFNGLGLLKQ
26


 


192
GNQLQTLPYVGFLEH
26


 


452
PGTFNPMPKLKVLYL
26


 


455
FNPMPKLKVLYLNNN
26


 


476
PHIFSGVPLTKVNLK
26


 


529
QQWIQKLSKNTVTDD
26


 


595
DTILRSLTDAVPLSV
26


 


611
ILGLLIMFITIVFCA
26


 


618
FITIVFCAAGIVVLV
26


 


817
KNEYFELKANLHAEP
26


 


19
HSQTPVLSSRGSCDS
25


 


94
HLGFNNIADIEIGAF
25


 


108
FNGLGLLKQLHINHN
25


 


132
FHGLENLEFLQADNN
25


 


135
LENLEFLQADNNFIT
25


 


156
FSKLNRLKVLILNDN
25


 


279
PSGSLHLAATSSIND
25


 


313
LIPYITKPSTQLPGP
25


 


314
IPYITKPSTQLPGPY
25


 


332
PCNCKVLSPSGLLIH
25


 


388
TLEMLHLGNNRIEVL
25


 


396
NNRIEVLEEGSFMNL
25


 


407
FMNLTRLQKLYLNGN
25


 


431
FLGLHNLEYLYLEYN
25


 


441
YLEYNAIKEILPGTF
25


 


503
LDDLDLLTQIDLEDN
25


 


551
HLDKKELKALNSEIL
25


 


559
ALNSEILCPGLVNNP
25


 


50
AKGIKMVSEISVPPS
24


 


144
DNNFITVIEPSAFSK
24


 


163
KVLILNDNAIESLPP
24


 


184
PLTHLDLRGNQLQTL
24


 


229
LKTWLENMPPQSIIG
24


 


255
GSILSRLKKESICPT
24


 


334
NCKVLSPSGLLIHCQ
24


 


349
ERNIESLSDLRPPPQ
24


 


363
QNPRKLILAGNIIHS
24


 


372
GNIIHSLMKSDLVEY
24


 


412
RLQKLYLNGNHLTKL
24


 


460
KLKVLYLNNNLLQVL
24


 


604
AVPLSVLILGLLIMF
24


 


605
VPLSVLILGLLIMFI
24


 


608
SVLILGLLIMFITIV
24


 


615
LIMFITIVFCAAGIV
24


 


619
ITIVFCAAGIVVLVL
24


 


645
DEQMRDNSPVHLQYS
24


 


686
MVHVYRSPSFGPKHL
24


 


724
QENHSPLTGSNMKYK
24


 


797
LKLMETLMYSRPRKV
24


 


800
METLMYSRPRKVLVE
24


 


2
KLWIHLFYSSLLACI
23


 


22
TPVLSSRGSCDSLCN
23


 


52
GIKMVSEISVPPSRP
23


 


56
VSEISVPPSRPFQLS
23


 


84
FSGLTNAISIHLGFN
23


 


97
FNNIADIEIGAFNGL
23


 


242
IGDVVCNSPPFFKGS
23


 


280
SGSLHLAATSSINDS
23


 


310
APGLIPYITKPSTQL
23


 


380
KSDLVEYFTLEMLHL
23


 


483
PLTKVNLKTNQFTHL
23


 


578
QTSYLMVTTPATTTN
23


 


598
LRSLTDAVPLSVLIL
23


 


612
LGLLIMFITIVFCAA
23


 


683
VSPMVHVYRSPSFGP
23


 


718
QRSLLEQENHSPLTG
23


 


732
GSNMKYKTTNQSTEF
23


 


780
AQLQPDMEAHYPGAH
23


 


794
HEELKLMETLMYSRP
23


 


9
YSSLLACISLHSQTP
22


 


12
LLACISLHSQTPVLS
22


 


49
EAKGIKMVSEISVPP
22


 


53
IKMVSEISVPPSRPF
22


 


58
EISVPPSRPFQLSLL
22


 


166
ILNDNAIESLPPNIF
22


 


204
LEHIGRILDLQLEDN
22


 


223
NCDLLQLKTWLENMP
22


 


235
NMPPQSIIGDVVCNS
22


 


239
QSIIGDVVCNSPPFF
22


 


293
DSRMSTKTTSILKLP
22


 


303
ILKLPTKAPGLIPYI
22


 


352
IESLSDLRPPPQNPR
22


 


357
DLRPPPQNPRKLILA
22


 


541
TDDILCTSPGHLDKK
22


 


577
TQTSYLMVTTPATTT
22


 


594
ADTILRSLTDAVPLS
22


 


641
KKQVDEQMRDNSPVH
22


 


674
SASLYEQHMVSPMVH
22


 


684
SPMVHVYRSPSFGPK
22


 


776
RKNIAQLQPDMEAHY
22


 


100
IADIEIGAFNGLGLL
21


 


105
IGAFNGLGLLKQLHI
21


 


260
RLKKESICPTPPVYE
21


 


373
NIIHSLMKSDLVEYF
21


 


487
VNLKTNQFTHLPVSN
21


 


651
NSPVHLQYSMYGHKT
21


 


736
KYKTTNQSTEFLSFQ
21


 


55
MVSEISVPPSRPFQL
20


 


182
FVPLTHLDLRGNQLQ
20


 


198
LPYVGFLEHIGRILD
20


 


410
LTRLQKLYLNGNHLT
20


 


423
LTKLSKGMFLGLHNL
20


 


445
NAIKEILPGTFNPMP
20


 


472
QVLPPHIFSGVPLTK
20


 


497
LPVSNILDDLDLLTQ
20


 


549
PGHLDKKELKALNSE
20


 


569
LVNNPSMPTQTSYLM
20


 


676
SLYEQHMVSPMVHVY
20


 


760
LEKERELQQLGITEY
20


 


772
TEYLRKNIAQLQPDM
20


 


88
TNAISIHLGFNNIAD
19


 


124
LEILKEDTFHGLENL
19


 


250
PPFFKGSILSRLKKE
19


 


304
LKLPTKAPGLIPYIT
19


 


397
NRIEVLEEGSFMNLT
19


 


405
GSFMNLTRLQKLYLN
19


 


415
KLYLNGNHLTKLSKG
19


 


438
EYLYLEYNAIKEILP
19


 


473
VLPPHIFSGVPLTKV
19


 


614
LLIMFITIVFCAAGI
19


 


620
TIVFCAAGIVVLVLH
19


 


753
SSLYRNILEKERELQ
19


 


793
AHEELKLMETLMYSR
19


 


818
NEYFELKANLHAEPD
19


 


5
IHLFYSSLLACISLH
18


 


13
LACISLHSQTPVLSS
18


 


39
EKDGTMLINCEAKGI
18


 


65
RPFQLSLLNNGLTML
18


 


68
QLSLLNNGLTMLHTN
18


 


76
LTMLHTNDFSGLTNA
18


 


137
NLEFLQADNNFITVI
18


 


146
NFITVIEPSAFSKLN
18


 


187
HLDLRGNQLQTLPYV
18


 


210
ILDLQLEDNKWACNC
18


 


227
LQLKTWLENMPPQSI
18


 


286
AATSSINDSRMSTKT
18


 


302
SILKLPTKAPGLIPY
18


 


404
EGSFMNLTRLQKLYL
18


 


421
NHLTKLSKGMFLGLH
18


 


426
LSKGMFLGLHNLEYL
18


 


428
KGMFLGLHNLEYLYL
18


 


462
KVLYLNNNLLQVLPP
18


 


465
YLNNNLLQVLPPHIF
18


 


471
LQVLPPHIFSGVPLT
18


 


481
GVPLTKVNLKTNQFT
18


 


486
KVNLKTNQFTHLPVS
18


 


580
SYLMVTTPATTTNTA
18


 


592
NTADTILRSLTDAVP
18


 


616
IMFITIVFCAAGIVV
18


 


617
MFITIVFCAAGIVVL
18


 


675
ASLYEQHMVSPMVHV
18


 


703
EEERNEKEGSDAKHL
18


 


743
STEFLSFQDASSLYR
18


 


763
ERELQQLGITEYLRK
18


 


768
QLGITEYLRKNIAQL
18


 


771
ITEYLRKNIAQLQPD
18


 


802
TLMYSRPRKVLVEQT
18


 


7
LFYSSLLACISLHSQ
17


 


34
LCNCEEKDGTMLINC
17


 


35
CNCEEKDGTMLINCE
17


 


44
MLINCEAKGIKMVSE
17


 


66
PFQLSLLNNGLTMLH
17


 


67
FQLSLLNNGLTMLHT
17


 


82
NDFSGLTNAISIHLG
17


 


89
NAISIHLGFNNIADI
17


 


90
AISIHLGFNNIADIE
17


 


92
SIHLGFNNIADIEIG
17


 


111
LGLLKQLHINHNSLE
17


 


116
QLHINHNSLEILKED
17


 


148
ITVIEPSAFSKLNRL
17


 


159
LNRLKVLILNDNAIE
17


 


164
VLILNDNAIESLPPN
17


 


172
IESLPPNIFRFVPLT
17


 


226
LLQLKTWLENMPPQS
17


 


247
CNSPPFFKGSILSRL
17


 


254
KGSILSRLKKESICP
17


 


257
ILSRLKKESICPTPP
17


 


261
LKKESICPTPPVYEE
17


 


278
DPSGSLHLAATSSIN
17


 


299
KTTSILKLPTKAPGL
17


 


318
TKPSTQLPGPYCPIP
17


 


341
SGLLIHCQERNIESL
17


 


376
HSLMKSDLVEYFTLE
17


 


386
YFTLEMLHLGNNRIE
17


 


419
NGNHLTKLSKGMFLG
17


 


429
GMFLGLHNLEYLYLE
17


 


439
YLYLEYNAIKEILPG
17


 


458
MPKLKVLYLNNNLLQ
17


 


463
VLYLNNNLLQVLPPH
17


 


464
LYLNNNLLQVLPPHI
17


 


478
IFSGVPLTKVNLKTN
17


 


522
SCDLVGLQQWIQKLS
17


 


525
LVGLQQWIQKLSKNT
17


 


528
LQQWIQKLSKNTVTD
17


 


537
KNTVTDDILCTSPGH
17


 


539
TVTDDILCTSPGHLD
17


 


546
CTSPGHLDKKELKAL
17


 


576
PTQTSYLMVTTPATT
17


 


586
TPATTTNTADTILRS
17


 


596
TILRSLTDAVPLSVL
17


 


601
LTDAVPLSVLILGLL
17


 


607
LSVLILGLLIMFITI
17


 


609
VLILGLLIMFITIVF
17


 


625
AAGIVVLVLHRRRRY
17


 


626
AGIVVLVLHRRRRYK
17


 


627
GIVVLVLHRRRRYKK
17


 


637
RRYKKKQVDEQMRDN
17


 


706
RNEKEGSDAKHLQRS
17


 


711
GSDAKHLQRSLLEQE
17


 


741
NQSTEFLSFQDASSL
17


 


801
ETLMYSRPRKVLVEQ
17


 


820
YFELKANLHAEPDYL
17


 


21
QTPVLSSRGSCDSLC
16


 


110
GLGLLKQLHINHNSL
16


 


123
SLEILKEDTFHGLEN
16


 


142
QADNNFITVIEPSAF
16


 


147
FITVIEPSAFSKLNR
16


 


160
NRLKVLILNDNAIES
16


 


207
IGRILDLQLEDNKWA
16


 


222
CNCDLLQLKTWLENM
16


 


233
LENMPPQSIIGDVVC
16


 


238
PQSIIGDVVCNSPPF
16


 


248
NSPPFFKGSILSRLK
16


 


269
TPPVYEEHEDPSGSL
16


 


271
PVYEEHEDPSGSLHL
16


 


319
KPSTQLPGPYCPIPC
16


 


321
STQLPGPYCPIPCNC
16


 


325
PGPYCPIPCNCKVLS
16


 


328
YCPIPCNCKVLSPSG
16


 


333
CNCKVLSPSGLLIHC
16


 


366
RKLILAGNIIHSLMK
16


 


367
KLILAGNIIHSLMKS
16


 


369
ILAGNIIHSLMKSDL
16


 


378
LMKSDLVEYFTLEML
16


 


381
SDLVEYFTLEMLHLG
16


 


391
MLHLGNNRIEVLEEG
16


 


395
GNNRIEVLEEGSFMN
16


 


399
IEVLEEGSFMNLTRL
16


 


402
LEEGSFMNLTRLQKL
16


 


434
LHNLEYLYLEYNAIK
16


 


446
AIKEILPGTFNPMPK
16


 


447
IKEILPGTFNPMPKL
16


 


448
KEILPGTFNPMPKLK
16


 


500
SNILDDLDLLTQIDL
16


 


511
QIDLEDNPWDCSCDL
16


 


519
WDCSCDLVGLQQWIQ
16


 


542
DDILCTSPGHLDKKE
16


 


558
KALNSEILCPGLVNN
16


 


562
SEILCPGLVNNPSMP
16


 


563
EILCPGLVNNPSMPT
16


 


581
YLMVTTPATTTNTAD
16


 


603
DAVPLSVLILGLLIM
16


 


658
YSMYGHKTTHHTTER
16


 


671
ERPSASLYEQHMVSP
16


 


689
VYRSPSFGPKHLEEE
16


 


719
RSLLEQENHSPLTGS
16


 


735
MKYKTTNQSTEFLSF
16


 


746
FLSFQDASSLYRNIL
16


 


749
FQDASSLYRNILEKE
16


 


769
LGITEYLRKNIAQLQ
16


 


810
KVLVEQTKNEYFELK
16


 


821
FELKANLHAEPDYLE
16

















TABLE XLVI







V3-HLA-DRB-0101-



15mers-158P1D7


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













7
ASLYEQHMGAHEELK
18



 


11
EQHMGAHEELKLMET
18


 


3
ERPSASLYEQHMGAH
16


 


8
SLYEQHMGAHEELKL
15


 


6
SASLYEQHMGAHEEL
14


 


5
PSASLYEQHMGAHEE
10


 


12
QHMGAHEELKLMETL
10


 


9
LYEQHMGAHEELKLM
9


 


14
MGAHEELKLMETLMY
8

















TABLE XLVI







V4-HLA-DRB-0101-



15mers-158P1D7


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













10
SLMKSILWSKASGRG
26



 


5
GNIIHSLMKSILWSK
24


 


9
HSLMKSILWSKASGR
24


 


14
SILWSKASGRGRREE
23


 


12
MKSILWSKASGRGRR
18


 


4
AGNIIHSLMKSILWS
17


 


2
ILAGNIIHSLMKSIL
16


 


1
LILAGNIIHSLMKSI
14


 


13
KSILWSKASGRGRRE
14


 


6
NIIHSLMKSILWSKA
13


 


3
LAGNIIHSLMKSILW
12

















TABLE XLVII







V1-HLA-DRB-0301-



15mers-158P1D7


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













779
IAQLQPDMEAHYPGA
36



 


376
HSLMKSDLVEYFTLE
31


 


124
LEILKEDTFHGLENL
30


 


460
KLKVLYLNNNLLQVL
28


 


809
RKVLVEQTKNEYFEL
27


 


138
LEFLQADNNFITVIE
26


 


407
FMNLTRLQKLYLNGN
26


 


420
GNHLTKLSKGMFLGL
26


 


628
IVVLVLHRRRRYKKK
26


 


801
ETLMYSRPRKVLVEQ
26


 


121
HNSLEILKEDTFHGL
25


 


372
GNIIHSLMKSDLVEY
25


 


396
NNRIEVLEEGSFMNL
25


 


428
KGMFLGLHNLEYLYL
25


 


499
VSNILDDLDLLTQID
25


 


503
LDDLDLLTQIDLEDN
25


 


810
KVLVEQTKNEYFELK
25


 


129
EDTFHGLENLEFLQA
24


 


163
KVLILNDNAIESLPP
22


 


238
PQSIIGDVVCNSPPF
22


 


794
HEELKLMETLMYSRP
22


 


68
QLSLLNNGLTMLHTN
21


 


73
NNGLTMLHTNDFSGL
21


 


145
NNFITVIEPSAFSKL
21


 


169
DNAIESLPPNIFRFV
21


 


399
IEVLEEGSFMNLTRL
21


 


405
GSFMNLTRLQKLYLN
21


 


444
YNAIKEILPGTFNPM
21


 


498
PVSNILDDLDLLTQI
21


 


537
KNTVTDDILCTSPGH
21


 


541
TDDILCTSPGHLDKK
21


 


607
LSVLILGLLIMFITI
21


 


645
DEQMRDNSPVHLQYS
21


 


756
YRNILEKERELQQLG
21


 


2
KLWIHLFYSSLLACI
20


 


41
DGTMLINCEAKGIKM
20


 


97
FNNIADIEIGAFNGL
20


 


148
ITVIEPSAFSKLNRL
20


 


156
FSKLNRLKVLILNDN
20


 


185
LTHLDLRGNQLQTLP
20


 


187
HLDLRGNQLQTLPYV
20


 


192
GNQLQTLPYVGFLEH
20


 


204
LEHIGRILDLQLEDN
20


 


206
HIGRILDLQLEDNKW
20


 


211
LDLQLEDNKWACNCD
20


 


242
IGDVVCNSPPFFKGS
20


 


254
KGSILSRLKKESICP
20


 


272
VYEEHEDPSGSLHLA
20


 


351
NIESLSDLRPPPQNP
20


 


355
LSDLRPPPQNPRKLI
20


 


388
TLEMLHLGNNRIEVL
20


 


431
FLGLHNLEYLYLEYN
20


 


455
FNPMPKLKVLYLNNN
20


 


463
VLYLNNNLLQVLPPH
20


 


549
PGHLDKKELKALNSE
20


 


612
LGLLIMFITIVFCAA
20


 


679
EQHMVSPMVHVYRSP
20


 


718
QRSLLEQENHSPLTG
20


 


768
QLGITEYLRKNIAQL
20


 


50
AKGIKMVSEISVPPS
19


 


56
VSEISVPPSRPFQLS
19


 


58
EISVPPSRPFQLSLL
19


 


65
RPFQLSLLNNGLTML
19


 


84
FSGLTNAISIHLGFN
19


 


100
IADIEIGAFNGLGLL
19


 


102
DIEIGAFNGLGLLKQ
19


 


108
FNGLGLLKQLHINHN
19


 


116
QLHINHNSLEILKED
19


 


162
LKVLILNDNAIESLP
19


 


179
IFRFVPLTHLDLRGN
19


 


183
VPLTHLDLRGNQLQT
19


 


200
YVGFLEHIGRILDLQ
19


 


208
GRILDLQLEDNKWAC
19


 


226
LLQLKTWLENMPPQS
19


 


301
TSILKLPTKAPGLIP
19


 


365
PRKLILAGNIIHSLM
19


 


375
IHSLMKSDLVEYFTL
19


 


413
LQKLYLNGNHLTKLS
19


 


415
KLYLNGNHLTKLSKG
19


 


423
LTKLSKGMFLGLHNL
19


 


429
GMFLGLHNLEYLYLE
19


 


459
PKLKVLYLNNNLLQV
19


 


461
LKVLYLNNNLLQVLP
19


 


468
NNLLQVLPPHIFSGV
19


 


486
KVNLKTNQFTHLPVS
19


 


547
TSPGHLDKKELKALN
19


 


554
KKELKALNSEILCPG
19


 


604
AVPLSVLILGLLIMF
19


 


697
PKHLEEEEERNEKEG
19


 


745
EFLSFQDASSLYRNI
19


 


763
ERELQQLGITEYLRK
19


 


826
NLHAEPDYLEVLEQQ
19


 


13
LACISLHSQTPVLSS
18


 


66
PFQLSLLNNGLTMLH
18


 


76
LTMLHTNDFSGLTNA
18


 


90
AISIHLGFNNIADIE
18


 


164
VLILNDNAIESLPPN
18


 


177
PNIFRFVPLTHLDLR
18


 


201
VGFLEHIGRILDLQL
18


 


222
CNCDLLQLKTWLENM
18


 


287
ATSSINDSRMSTKTT
18


 


293
DSRMSTKTTSILKLP
18


 


328
YCPIPCNCKVLSPSG
18


 


340
PSGLLIHCQERNIES
18


 


341
SGLLIHCQERNIESL
18


 


342
GLLIHCQERNIESLS
18


 


367
KLILAGNIIHSLMKS
18


 


381
SDLVEYFTLEMLHLG
18


 


391
MLHLGNNRIEVLEEG
18


 


406
SFMNLTRLQKLYLNG
18


 


430
MFLGLHNLEYLYLEY
18


 


437
LEYLYLEYNAIKEIL
18


 


452
PGTFNPMPKLKVLYL
18


 


454
TFNPMPKLKVLYLNN
18


 


478
IFSGVPLTKVNLKTN
18


 


484
LTKVNLKTNQFTHLP
18


 


507
DLLTQIDLEDNPWDC
18


 


514
LEDNPWDCSCDLVGL
18


 


529
QQWIQKLSKNTVTDD
18


 


620
TIVFCAAGIVVLVLH
18


 


627
GIVVLVLHRRRRYKK
18


 


629
VVLVLHRRRRYKKKQ
18


 


641
KKQVDEQMRDNSPVH
18


 


684
SPMVHVYRSPSFGPK
18


 


707
NEKEGSDAKHLQRSL
18


 


719
RSLLEQENHSPLTGS
18


 


726
NHSPLTGSNMKYKTT
18


 


744
TEFLSFQDASSLYRN
18


 


31
CDSLCNCEEKDGTML
17


 


96
GFNNIADIEIGAFNG
17


 


114
LKQLHINHNSLEILK
17


 


137
NLEFLQADNNFITVI
17


 


210
ILDLQLEDNKWACNC
17


 


250
PPFFKGSILSRLKKE
17


 


255
GSILSRLKKESICPT
17


 


269
TPPVYEEHEDPSGSL
17


 


389
LEMLHLGNNRIEVLE
17


 


509
LTQIDLEONPWDCSC
17


 


522
SCDLVGLQQWIQKLS
17


 


525
LVGLQQWIQKLSKNT
17


 


630
VLVLHRRRRYKKKQV
17


 


639
YKKKQVDEQMRDNSP
17


 


683
VSPMVHVYRSPSFGP
17


 


755
LYRNILEKERELQQL
17


 


757
RNILEKERELQQLGI
17


 


788
AHYPGAHEELKLMET
17


 


816
TKNEYFELKANLHAE
17

















TABLE XLVII







V3-HLA-DRB-0301-



15mers-158P1D7


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












11
EQHMGAHEELKLMET
27

















TABLE XLVII







V4-HLA-DRB-0301-



15mers-158P1D7


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












5
GNIIHSLMKSILWSK
25



 


9
HSLMKSILWSKASGR
14


 


12
MKSILWSKASGRGRR
13


 


4
AGNIIHSLMKSILWS
12

















TABLE XLVIII







V1-HLA-DR1-0401-



15mers-158P1D7


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













81
TNDFSGLTNAISIHL
28



 


137
NLEFLQADNNFITVI
28


 


153
PSAFSKLNRLKVLIL
28


 


179
IFRFVPLTHLDLRGN
28


 


404
EGSFMNLTRLQKLYL
28


 


578
QTSYLMVTTPATTTN
28


 


2
KLWIHLFYSSLLACI
26


 


66
PFQLSLLNNGLTMLH
26


 


84
FSGLTNAISIHLGFN
26


 


108
FNGLGLLKQLHINHN
26


 


138
LEFLQADNNFITVIE
26


 


210
ILDLQLEDNKWACNC
26


 


280
SGSLHLAATSSINDS
26


 


388
TLEMLHLGNNRIEVL
26


 


398
RIEVLEEGSFMNLTR
26


 


437
LEYLYLEYNAIKEIL
26


 


460
KLKVLYLNNNLLQVL
26


 


503
LDDLDLLTQIDLEDN
26


 


522
SCDLVGLQQWIQKLS
26


 


554
KKELKALNSEILCPG
26


 


683
VSPMVHVYRSPSFGP
26


 


719
RSLLEQENHSPLTGS
26


 


1
MKLWIHLFYSSLLAC
22


 


6
HLFYSSLLACISLHS
22


 


94
HLGFNNIADIEIGAF
22


 


105
IGAFNGLGLLKQLHI
22


 


129
EDTFHGLENLEFLQA
22


 


144
DNNFITVIEPSAFSK
22


 


177
PNIFRFVPLTHLDLR
22


 


325
PGPYCPIPCNCKVLS
22


 


383
LVEYFTLEMLHLGNN
22


 


414
QKLYLNGNHLTKLSK
22


 


428
KGMFLGLHNLEYLYL
22


 


436
NLEYLYLEYNAIKEI
22


 


476
PHIFSGVPLTKVNLK
22


 


491
TNQFTHLPVSNILDD
22


 


615
LIMFITIVFCAAGIV
22


 


620
TIVFCAAGIVVLVLH
22


 


655
HLQYSMYGHKTTHHT
22


 


743
STEFLSFQDASSLYR
22


 


746
FLSFQDASSLYRNIL
22


 


787
EAHYPGAHEELKLME
22


 


9
YSSLLACISLHSQTP
20


 


10
SSLLACISLHSQTPV
20


 


13
LACISLHSQTPVLSS
20


 


43
TMLINCEAKGIKMVS
20


 


50
AKGIKMVSEISVPPS
20


 


52
GIKMVSEISVPPSRP
20


 


53
IKMVSEISVPPSRPF
20


 


73
NNGLTMLHTNDFSGL
20


 


90
AISIHLGFNNIADIE
20


 


102
DIEIGAFNGLGLLKQ
20


 


111
LGLLKQLHINHNSLE
20


 


123
SLEILKEDTFHGLEN
20


 


124
LEILKEDTFHGLENL
20


 


132
FHGLENLEFLQADNN
20


 


135
LENLEFLQADNNFIT
20


 


156
FSKLNRLKVLILNDN
20


 


159
LNRLKVLILNDNAIE
20


 


161
RLKVLILNDNAIESL
20


 


163
KVLILNDNAIESLPP
20


 


182
FVPLTHLDLRGNQLQ
20


 


198
LPYVGFLEHIGRILD
20


 


201
VGFLEHIGRILDLQL
20


 


204
LEHIGRILDLQLEDN
20


 


207
IGRILDLQLEDNKWA
20


 


223
NCDLLQLKTWLENMP
20


 


238
PQSIIGDVVCNSPPF
20


 


255
GSILSRLKKESICPT
20


 


258
LSRLKKESICPTPPV
20


 


269
TPPVYEEHEDPSGSL
20


 


300
TTSILKLPTKAPGLI
20


 


310
APGLIPYITKPSTQL
20


 


311
PGLIPYITKPSTQLP
20


 


340
PSGLLIHCQERNIES
20


 


352
IESLSDLRPPPQNPR
20


 


365
PRKLILAGNIIHSLM
20


 


372
GNIIHSLMKSDLVEY
20


 


380
KSDLVEYFTLEMLHL
20


 


381
SDLVEYFTLEMLHLG
20


 


386
YFTLEMLHLGNNRIE
20


 


407
FMNLTRLQKLYLNGN
20


 


410
LTRLQKLYLNGNHLT
20


 


413
LQKLYLNGNHLTKLS
20


 


431
FLGLHNLEYLYLEYN
20


 


434
LHNLEYLYLEYNAIK
20


 


455
FNPMPKLKVLYLNNN
20


 


458
MPKLKVLYLNNNLLQ
20


 


461
LKVLYLNNNLLQVLP
20


 


467
NNNLLQVLPPHIFSG
20


 


481
GVPLTKVNLKTNQFT
20


 


499
VSNILDDLDLLTQID
20


 


500
SNILDDLDLLTQIDL
20


 


506
LDLLTQIDLEDNPWD
20


 


509
LTQIDLEDNPWDCSC
20


 


525
LVGLQQWIQKLSKNT
20


 


529
QQWIQKLSKNTVTDD
20


 


537
KNTVTDDILCTSPGH
20


 


566
CPGLVNNPSMPTQTS
20


 


572
NPSMPTQTSYLMVTT
20


 


581
YLMVTTPATTTNTAD
20


 


594
ADTILRSLTDAVPLS
20


 


598
LRSLTDAVPLSVLIL
20


 


604
AVPLSVLILGLLIMF
20


 


606
PLSVLILGLLIMFIT
20


 


608
SVLILGLLIMFITIV
20


 


609
VLILGLLIMFITIVF
20


 


612
LGLLIMFITIVFCAA
20


 


619
ITIVFCAAGIVVLVL
20


 


626
AGIVVLVLHRRRRYK
20


 


627
GIVVLVLHRRRRYKK
20


 


641
KKQVDEQMRDNSPVH
20


 


757
RNILEKERELQQLGI
20


 


768
QLGITEYLRKNIAQL
20


 


808
PRKVLVEQTKNEYFE
20


 


19
HSQTPVLSSRGSCDS
18


 


35
CNCEEKDGTMLINCE
18


 


39
EKDGTMLINCEAKGI
18


 


65
RPFQLSLLNNGLTML
18


 


77
TMLHTNDFSGLTNAI
18


 


113
LLKQLHINHNSLEIL
18


 


134
GLENLEFLQADNNFI
18


 


146
NFITVIEPSAFSKLN
18


 


149
TVIEPSAFSKLNRLK
18


 


160
NRLKVLILNDNAIES
18


 


183
VPLTHLDLRGNQLQT
18


 


215
LEDNKWACNCDLLQL
18


 


220
WACNCDLLQLKTWLE
18


 


251
PFFKGSILSRLKKES
18


 


252
FFKGSILSRLKKESI
18


 


272
VYEEHEDPSGSLHLA
18


 


281
GSLHLAATSSINDSR
18


 


287
ATSSINDSRMSTKTT
18


 


343
LLIHCQERNIESLSD
18


 


368
LILAGNIIHSLMKSD
18


 


369
ILAGNIIHSLMKSDL
18


 


488
NLKTNQFTHLPVSNI
18


 


514
LEDNPWDCSCDLVGL
18


 


553
DKKELKALNSEILCP
18


 


563
EILCPGLVNNPSMPT
18


 


564
ILCPGLVNNPSMPTQ
18


 


582
LMVTTPATTTNTADT
18


 


591
TNTADTILRSLTDAV
18


 


673
PSASLYEQHMVSPMV
18


 


698
KHLEEEEERNEKEGS
18


 


704
EERNEKEGSDAKHLQ
18


 


711
GSDAKHLQRSLLEQE
18


 


749
FQDASSLYRNILEKE
18


 


760
LEKERELQQLGITEY
18


 


807
RPRKVLVEQTKNEYF
18


 


313
LIPYITKPSTQLPGP
17


 


528
LQQWIQKLSKNTVTD
17


 


658
YSMYGHKTTHHTTER
17


 


818
NEYFELKANLHAEPD
17


 


5
IHLFYSSLLACISLH
16


 


197
TLPYVGFLEHIGRIL
16


 


200
YVGFLEHIGRILDLQ
16


 


217
DNKWACNCDLLQLKT
16


 


229
LKTWLENMPPQSIIG
16


 


250
PPFFKGSILSRLKKE
16


 


384
VEYFTLEMLHLGNNR
16


 


441
YLEYNAIKEILPGTF
16


 


452
PGTFNPMPKLKVLYL
16


 


462
KVLYLNNNLLQVLPP
16


 


675
ASLYEQHMVSPMVHV
16


 


687
VHVYRSPSFGPKHLE
16


 


734
NMKYKTTNQSTEFLS
16


 


753
SSLYRNILEKERELQ
16


 


802
TLMYSRPRKVLVEQT
16


 


817
KNEYFELKANLHAEP
16


 


22
TPVLSSRGSCDSLCN
15


 


185
LTHLDLRGNQLQTLP
15


 


293
DSRMSTKTTSILKLP
15


 


484
LTKVNLKTNQFTHLP
15


 


629
VVLVLHRRRRYKKKQ
15


 


630
VLVLHRRRRYKKKQV
15


 


732
GSNMKYKTTNQSTEF
15


 


756
YRNILEKERELQQLG
15


 


801
ETLMYSRPRKVLVEQ
15


 


15
CISLHSQTPVLSSRG
14


 


42
GTMLINCEAKGIKMV
14


 


56
VSEISVPPSRPFQLS
14


 


58
EISVPPSRPFQLSLL
14


 


68
QLSLLNNGLTMLHTN
14


 


69
LSLLNNGLTMLHTND
14


 


76
LTMLHTNDFSGLTNA
14


 


88
TNAISIHLGFNNIAD
14


 


92
SIHLGFNNIADIEIG
14


 


97
FNNIADIEIGAFNGL
14


 


100
IADIEIGAFNGLGLL
14


 


110
GLGLLKQLHINHNSL
14


 


114
LKQLHINHNSLEILK
14


 


116
QLHINHNSLEILKED
14


 


121
HNSLEILKEDTFHGL
14


 


145
NNFITVIEPSAFSKL
14


 


147
FITVIEPSAFSKLNR
14


 


148
ITVIEPSAFSKLNRL
14


 


162
LKVLILNDNAIESLP
14


 


164
VLILNDNAIESLPPN
14


 


169
DNAIESLPPNIFRFV
14


 


172
IESLPPNIFRFVPLT
14


 


176
PPNIFRFVPLTHLDL
14


 


187
HLDLRGNQLQTLPYV
14


 


192
GNQLQTLPYVGFLEH
14


 


195
LQTLPYVGFLEHIGR
14


 


208
GRILDLQLEDNKWAC
14


 


212
DLQLEDNKWACNCDL
14


 


230
KTWLENMPPQSIIGD
14


 


239
QSIIGDVVCNSPPFF
14


 


243
GDVVCNSPPFFKGSI
14


 


282
SLHLAATSSINDSRM
14


 


288
TSSINDSRMSTKTTS
14


 


314
IPYITKPSTQLPGPY
14


 


328
YCPIPCNCKVLSPSG
14


 


334
NCKVLSPSGLLIHCQ
14


 


341
SGLLIHCQERNIESL
14


 


342
GLLIHCQERNIESLS
14


 


349
ERNIESLSDLRPPPQ
14


 


355
LSDLRPPPQNPRKLI
14


 


366
RKLILAGNIIHSLMK
14


 


367
KLILAGNIIHSLMKS
14


 


376
HSLMKSDLVEYFTLE
14


 


389
LEMLHLGNNRIEVLE
14


 


391
MLHLGNNRIEVLEEG
14


 


396
NNRIEVLEEGSFMNL
14


 


399
IEVLEEGSFMNLTRL
14


 


405
GSFMNLTRLQKLYLN
14


 


415
KLYLNGNHLTKLSKG
14


 


420
GNHLTKLSKGMFLGL
14


 


423
LTKLSKGMFLGLHNL
14


 


427
SKGMFLGLHNLEYLY
14


 


429
GMFLGLHNLEYLYLE
14


 


439
YLYLEYNAIKEILPG
14


 


444
YNAIKEILPGTFNPM
14


 


447
IKEILPGTFNPMPKL
14


 


448
KEILPGTFNPMPKLK
14


 


463
VLYLNNNLLQVLPPH
14


 


468
NNLLQVLPPHIFSGV
14


 


471
LQVLPPHIFSGVPLT
14


 


475
PPHIFSGVPLTKVNL
14


 


479
FSGVPLTKVNLKTNQ
14


 


486
KVNLKTNQFTHLPVS
14


 


496
HLPVSNILDDLDLLT
14


 


511
QIDLEDNPWDCSCDL
14


 


523
CDLVGLQQWIQKLSK
14


 


541
TDDILCTSPGHLDKK
14


 


557
LKALNSEILCPGLVN
14


 


561
NSEILCPGLVNNPSM
14


 


567
PGLVNNPSMPTQTSY
14


 


579
TSYLMVTTPATTTNT
14


 


580
SYLMVTTPATTTNTA
14


 


595
DTILRSLTDAVPLSV
14


 


611
ILGLLIMFITIVFCA
14


 


613
GLLIMFITIVFCAAG
14


 


614
LLIMFITIVFCAAGI
14


 


616
IMFITIVFCAAGIVV
14


 


618
FITIVFCAAGIVVLV
14


 


625
AAGIVVLVLHRRRRY
14


 


628
IVVLVLHRRRRYKKK
14


 


645
DEQMRDNSPVHLQYS
14


 


651
NSPVHLQYSMYGHKT
14


 


653
PVHLQYSMYGHKTTH
14


 


657
QYSMYGHKTTHHTTE
14


 


680
QHMVSPMVHVYRSPS
14


 


684
SPMVHVYRSPSFGPK
14


 


686
MVHVYRSPSFGPKHL
14


 


697
PKHLEEEEERNEKEG
14


 


718
QRSLLEQENHSPLTG
14


 


727
HSPLTGSNMKYKTTN
14


 


744
TEFLSFQDASSLYRN
14


 


763
ERELQQLGITEYLRK
14


 


766
LQQLGITEYLRKNIA
14


 


772
TEYLRKNIAQLQPDM
14


 


776
RKNIAQLQPDMEAHY
14


 


779
IAQLQPDMEAHYPGA
14


 


794
HEELKLMETLMYSRP
14


 


797
LKLMETLMYSRPRKV
14


 


800
METLMYSRPRKVLVE
14


 


810
KVLVEQTKNEYFELK
14


 


820
YFELKANLHAEPDYL
14


 


824
KANLHAEPDYLEVLE
14

















TABLE XLVIII







V3-HLA-DR1-0401-



15mers-158P10D


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













5
PSASLYEQHMGAHEE
18



 


11
EQHMGAHEELKLMET
14


 


1
TTERPSASLYEQHMG
12


 


3
ERPSASLYEQHMGAH
12


 


9
LYEQHMGAHEELKLM
12


 


10
YEQHMGAHEELKLME
12


 


12
QHMGAHEELKLMETL
12


 


14
MGAHEELKLMETLMY
12


 


7
ASLYEQHMGAHEELK
10


 


6
SASLYEQHMGAHEEL
8

















TABLE XLVIII







V4-HLA-DR1-0401-



15mers-158P1D7


Each peptide is a portion of SEQ ID


NO: 9; each start position is


specified, the length of pepfide is 15


amino acids, and the end position


for each peptide is the start position


plus fourteen.









Pos
123456789012345
score













5
GNIIHSLMKSILWSK
20



 


9
HSLMKSILWSKASGR
20


 


1
LILAGNIIHSLMKSI
18


 


2
ILAGNIIHSLMKSIL
18


 


10
SLMKSILWSKASGRG
18


 


14
SILWSKASGRGRREE
16


 


4
AGNIIHSLMKSILWS
14


 


8
IHSLMKSILWSKASG
14


 


13
KSILWSKASGRGRRE
9

















TABLE XLIX







V1-HLA-DRB1-1101-



15mers-158P1D7


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













683
VSPMVHVYRSPSFGP
26



 


153
PSAFSKLNRLKVLIL
25


 


452
PGTFNPMPKLKVLYL
25


 


179
IFRFVPLTHLDLRGN
24


 


404
EFSFMNLTRLQKLYL
24


 


615
LIMFITIVFCAAGIV
24


 


627
GIVVLVLHRRRRYKK
24


 


6
HLFYSSLLACISLHS
23


 


81
TNDFSGLTNAISIHL
23


 


441
YLEYNAIKEILPGTF
23


 


626
AGIVVLVLHRRRRYK
23


 


144
DNNRITVIEPSAFSK
22


 


407
FMNLTRLQKLYLNGN
22


 


420
GNHLTKLSKGMFLGL
22


 


680
QHMVSPMVHVYRSPS
22


 


173
ESLPPNIFRFVPLTH
21


 


201
VGFLEHIGRILDLQL
21


 


328
YCPIPCNCKVLSPSG
21


 


769
LGITEYLRKNIAQLQ
21


 


198
LPYVGFLEHIGRILD
20


 


239
QSIIGDVVCNSPPFF
20


 


254
KGSILSRLKKESICP
20


 


255
GSILSRLKKESICPT
20


 


301
TSILKLPTKAPGLIP
20


 


311
PGLIPYITKPSTQLP
20


 


349
ERNIESLSDLRPPPQ
20


 


372
GNIIHSLMKSDLVEY
20


 


529
QQWIQKLSKNTVTDD
20


 


616
IMFITIVFCAAGIVV
20


 


641
KKQVDEQMRDNSPVH
20


 


797
LKLMETLMYSRPRKV
20


 


820
YFELKANLHAEPDYL
20


 


384
VEYFTLEMLHLGNNR
19


 


595
DTILRSLTDAVPLSV
19


 


802
TLMYSRPRKVLVEQT
19


 


53
IKMVSEISVPPSRPF
18


 


132
FHGLENLEFLQADNN
18


 


300
TTSILKLPTKAPGLI
18


 


414
QKLYLNGNHLTKLSK
18


 


491
TNQFTHLPVSNILDD
18


 


655
HLQYSMYGHKTTHHT
18


 


817
KNEYFELKANLHAEP
18


 


105
IGAGNGLGLLKQLHI
17


 


197
TLPYVGFLEHIGRIL
17


 


383
LVEYFTLEMLHLGNN
17


 


476
PHIFSGVPLTKVNLK
17


 


516
DNPWDCSCDLVGLQQ
17


 


625
AAGIVVLVLHRRRRY
17


 


628
IVVLVLHRRRRYKKK
17


 


1
MKLWIHLFYSSLLAC
16


 


18
LHSQTPVLSSRGSCD
16


 


64
SRPTQLSLLNNGLTM
16


 


94
HLGFNNIADIEIGAF
16


 


129
EDTFHGLENLEFLQA
16


 


177
PNIFRFVPLTHLDLR
16


 


229
LKTWLENMPPQSIIG
16


 


270
PPVYEEHEDPSGSLH
16


 


297
STKTTSILKLPTKAP
16


 


325
PGPYCPIPCNCKVLS
16


 


427
SKGMFLGLHNLEYLY
16


 


428
KGMFLGLHNLEYLYL
16


 


436
NLEYLYLEYNAIKEI
16


 


578
QTSYLMVTTPATTTN
16


 


743
STEFLSFQDASSLYR
16


 


753
SSLYRNILEKERELQ
16


 


754
SLYRNILEKERELQQ
16


 


768
QLGITEYLRKNIAQL
16


 


818
NEYFELKANLHAEPD
16


 


43
TMLINCEAKGIKMVS
15


 


46
INCEAKGIKMVSEIS
15


 


49
EAKGIKMVSEISVPP
15


 


107
AFNGLGLLKQLHINH
15


 


145
NNFITVIEPSAFSKL
15


 


182
FVPLTHLDLRGNQLQ
15


 


252
FFKGSILSRLKKESI
15


 


314
IPYITKPSTQLPGPY
15


 


342
GLLIHCQERNIESLS
15


 


368
LILAGNIIHSLMKSD
15


 


591
TNTADTILRSLTDAV
15


 


602
TDAVPLSVLILGLLI
15


 


629
VVLVLHRRRRYKKKQ
15


 


630
VLVLHRRRRYKKKQV
15


 


673
PSASLYEQHMVSPMV
15


 


711
GSDAKHLQRSLLEQE
15


 


749
FQDASSLYRNILEKE
15


 


756
YRNILEKERELQQLG
15


 


801
ETLMYSRPRKVLVEQ
15


 


19
HSQTPVLSSRGSCDS
14


 


39
EKDGTMLINCEAKGI
14


 


55
MVSEISVPPSRPFQL
14


 


72
LNNGLTMLHTNDFSG
14


 


73
NNGLTMLHTNDFSGL
14


 


110
GLGLLKQLHINHNSL
14


 


113
LLKQLHINHNSLEIL
14


 


120
NHNSLEILKEDTFHG
14


 


227
LQLKTWLENMPPQSI
14


 


238
PQSIIGDVVCNSPPF
14


 


268
PTPPVYEEHEDPSGS
14


 


276
HEDPSGSLHLAATSS
14


 


291
INDSRMSTKTTSILK
14


 


338
LSPSGLLIHCQERNI
14


 


351
NIESLSDLRPPPQNP
14


 


385
EYFTLEMLHLGNNRI
14


 


388
TLEMLHLGNNRIEVL
14


 


417
YLNGNHLTKLSKGMF
14


 


468
NNLLQVLPPHIFSGV
14


 


469
NLLQVLPPHIFSGVP
14


 


478
IFSGVPLTKVNLKTN
14


 


481
GVPLTKVNLKTNQFT
14


 


506
LDLLTQIDLEDNPWD
14


 


526
VGLQQWIQKLSKNTV
14


 


537
KNTVTDDILCTSPGH
14


 


546
CTSPGHLDKKELKAL
14


 


563
EILCPGLVNNPSMPT
14


 


577
TQTSYLMVTTPATTT
14


 


604
AVPLSVLILGLLIMF
14


 


664
KTTHHTTERPSASLY
14


 


681
HMVSPMVHVYRSPSF
14


 


701
EEEEERNEKEGSDAK
14


 


719
RSLLEQENHSPLTGS
14


 


781
QLQPDMEAHYPGAHE
14


 


809
RKVLVEQTKNEYFEL
14


 


15
CISLHSQTPVLSSRG
13


 


41
DGTMLINCEAKGIKM
13


 


66
PFQLSLLNNGLTMLH
13


 


85
SGLTNAISIHLGFNN
13


 


90
AISIHLGFNNIADIE
13


 


156
FSKLNRLKVLILNDN
13


 


159
LNRLKVLILNDNAIE
13


 


169
DNAIESLPPNIFRFV
13


 


223
NCDLLQLKTWLENMP
13


 


240
SIIGDVVCNSPPFFK
13


 


321
STQLPGPYCPIPCNC
13


 


396
NNRIEVLEEGSFMNL
13


 


458
MPKLKVLYLNNNLLQ
13


 


460
KLKVLYLNNNLLQVL
13


 


464
LYLNNNLLQVLPPHI
13


 


472
QVLPPHIFSGVPLTK
13


 


496
HLPVSNILDDLDLLT
13


 


522
SCDLVGLQQWIQKLS
13


 


525
LVGLQQWIQKLSKNT
13


 


554
KKELKALNSEILCPG
13


 


606
PLSVLILGLLIMFIT
13


 


609
VLILGLLIMFITIVF
13


 


611
ILGLLIMFITIVFCA
13


 


614
LLIMFITIVFCAAGI
13


 


9
YSSLLACISLHSQTP
12


 


10
SSLLACISLHSQTPV
12


 


12
LLACISLHSQTPVLS
12


 


22
TPVLSSRGSCDSLCN
12


 


31
CDSLCNCEEKDGTML
12


 


50
AKGIKMVSEISVPPS
12


 


52
GIKMVSEISVPPSRP
12


 


75
GLTMLHTNDFSGLTN
12


 


97
FNNIADIEIGAFNGL
12


 


99
NIADIEIGAFNGLGL
12


 


108
FNGLGLLKQLHINHN
12


 


111
LGLLKQLHINHNSLE
12


 


121
HNSLEILKEDTFHGL
12


 


123
SLEILKEDTFHGLEN
12


 


135
LENLEFLQADNNFIT
12


 


142
QADNNFITVIEPSAF
12


 


160
NRLKVLILNDNAIES
12


 


161
RLKVLILNDNAIESL
12


 


163
KVLILNDNAIESLPP
12


 


166
ILNDNAIESLPPNIF
12


 


192
GNQLQTLPYVGFLEH
12


 


195
LQTLPYVGFLEHIGR
12


 


200
YVGFLEHIGRILDLQ
12


 


204
LEHIGRILDLQLEDN
12


 


207
IGRILDLQLEDNKWA
12


 


210
ILDLQLEDNKWACNC
12


 


226
LLQLKTWLENMPPQS
12


 


230
KTWLENMPPQSIIGD
12


 


250
PPFFKGSILSRLKKE
12


 


260
RLKKESICPTPPVYE
12


 


269
TPPVYEEHEDPSGSL
12


 


279
PSGSLHLAATSSIND
12


 


310
APGLIPYITKPSTQL
12


 


331
IPCNCKVLSPSGLLI
12


 


352
IESLSDLRPPPQNPR
12


 


366
RKLILAGNIIHSLMK
12


 


386
YFTLEMLHLGNNRIE
12


 


395
GNNRIEVLEEGSFMN
12


 


410
LTRLQKLYLNGNHLT
12


 


431
FLGLHNLEYLYLEYN
12


 


434
LHNLEYLYLEYNAIK
12


 


444
YNAIKEILPGTFNPM
12


 


448
KEILPGTFNPMPKLK
12


 


455
FNPMPKLKVLYLNNN
12


 


465
YLNNNLLQVLPPHIF
12


 


467
NNNLLQVLPPHIFSG
12


 


470
LLQVLPPHIFSGVPL
12


 


500
SNILDDLDLLTQIDL
12


 


503
LDDLDLLTQIDLEDN
12


 


511
QIDLEDNPWDCSCDL
12


 


538
NTVTDDILCTSPGHL
12


 


539
TVTDDILCTSPGHLD
12


 


551
HLDKKELKALNSEIL
12


 


557
LKALNSEILCPGLVN
12


 


562
SEILCPGLVNNPSMP
12


 


569
LVNNPSMPTQTSYLM
12


 


576
PTQTSYLMVTTPATT
12


 


608
SVLILGLLIMFITIV
12


 


613
GLLIMFITIVFCAAG
12


 


642
KQVDEQMRDNSPVHL
12


 


648
MRDNSPVHLQYSMYG
12


 


651
NSPVHLQYSMYGHKT
12


 


674
SASLYEQHMVSPMVH
12


 


686
MVHVYRSPSFGPKHL
12


 


718
QRSLLEQENHSPLTG
12


 


732
GSNMKYKTTNQSTEF
12


 


741
NQSTEFLSFQDASSL
12


 


763
ERELQQLGITEYLRK
12


 


773
EYLRKNIAQLQPDME
12


 


776
RKNIAQLQPDMEAHY
12


 


780
AQLQPDMEAHYPGAH
12


 


794
HEELKLMETLMYSRP
12

















TABLE XLIX







V3-HLA-DRB-1101-



15mers-158P1D7


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













5
PSASLYEQHMGAHEE
14



 


7
ASLYEQHMGAHEELK
10


 


9
LYEQHMGAHEELKLM
8


 


13
HMGAHEELKLMETLM
8


 


11
EQHMGAHEELKLMET
7


 


3
ERPSASLYEQHMGAH
6


 


4
RPSASLYEQHMGAHE
6


 


6
SASLYEQHMGAHEEL
6


 


8
SLYEQHMGAHEELKL
6


 


14
MGAHEELKLMETLMY
6

















TABLE XLIX







V4-HLA-DRB-1101-



15mers-158P1D7


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













5
GNIIHSLMKSILWSK
21



 


9
HSLMKSILWSKASGR
19


 


1
LILAGNIIHSLMKSI
15


 


11
LMKSILWSKASGRGR
14


 


13
KSILWSKASGRGRRE
14


 


10
SLMKSILWSKASGRG
12


 


14
SILWSKASGRGRREE
11

















TABLE XXII







158P1D7



v.6 - HLA-A1-9-mers


Each peptide is a portion


of SEQ ID NO: 13; each


start position is specified,


the length of peptide is 9


amino acids, and the end


position for each peptide


is the start position plus


eight.









Pos
123456789
score













11
SFGPKHLEE
12



 


10
PSFGPKHLE
8


 


1
GNIIHSLMN
7


 


7
LMNPSFGPK
7

















TABLE XXIII







158P1D7



v.6 - HLA-A0201-9-mers


Each peptide is a portion


of SEQ ID NO: 13; each


start position is specified,


the length of peptide is 9


amino acids, and the end


position for each peptide


is the start position plus


eight.









Pos
123456789
score













6
SLMNPSFGP
15



 


2
NIIHSLMNP
14


 


7
LMNPSFGPK
13


 


3
IIHSLMNPS
12


 


9
NPSFGPKHL
10


 


11
SFGPKHLEE
8

















TABLE XXIV







158P1D7 v.6-



HLA-A0203-9-mers









Pos
123456789
score






No results found

















TABLE XXV







158P1D7



v.6 - HLA-A3-9-mers


Each peptide is a portion


of SEQ ID NO: 13; each


start position is specified,


the length of peptide is 9


amino acids, and the end


position for each peptide


is the start position plus


eight.









Pos
123456789
score













7
LMNPSFGPK
14



 


2
NIIHSLMNP
12


 


6
SLMNPSFGP
12


 


3
IIHSLMNPS
10


 


15
KHLEEEEER
10


 


4
IHSLMNPSF
9


 


1
GNIIHSLMN
8


 


11
SFGPKHLEE
8


 


8
MNPSFGPKH
7

















TABLE XXVI







158P1D7



v.6 - HLA-A26-9-mers


Each peptide is a portion


of SEQ ID NO: 13; each


start position is specified,


the length of peptide is 9


amino acids, and the end


position for each peptide


is the start position plus


eight.









Pos
123456789
score













2
NIIHSLMNP
12



 


4
IHSLMNPSF
9


 


9
NPSFGPKHL
8


 


1
GNIIHSLMN
6


 


3
IIHSLMNPS
6

















TABLE XXVII







158P1D7



v.6 - HLA-B0702-9-mers


Each peptide is a portion


of SEQ ID NO: 13; each


start position is specified,


the length of peptide is 9


amino acids, and the end


position for each peptide


is the start position plus


eight.









Pos
123456789
score













9
NPSFGPKHL
22



 


4
IHSLMNPSF
10


 


13
GPKHLEEEE
10

















TABLE XXVIII







158P1D7



v.6 - HLA-B08-9-mers


Each pepdde is a portion


of SEQ ID NO: 13; each


start position is specified,


the length of peptide is 9


amino acids, and the end


position for each peptide


is the start position plus


eight.









Pos
123456789
score













13
GPKHLEEEE
18



 


9
NPSFGPKHL
17


 


11
SFGPKHLEE
13


 


4
IHSLMNPSF
9


 


6
SLMNPSFGP
8

















TABLE XXIX







158P1D7



v.6 - HLA-B1510-9-mers


Each peptide is a portion


of SEQ ID NO: 13; each


start position is specified,


the length of peptide is 9


amino acids, and the end


position for each peptide


is the start position plus


eight.









Pos
123456789
score













4
IHSLMNPSF
20



 


9
NPSFGPKHL
13


 


15
KHLEEEEER
12

















TABLE XXX







158P1D7



v.6 - HLA-82705-9-mers


Each peptide is a portion


of SEQ ID NO: 13; each


start position is specified,


the length of peptide is 9


amino acids, and the end


position for each peptide


is the start position plus


eight.









Pos
123456789
score













15
KHLEEEEER
17



 


4
IHSLMNPSF
15


 


7
LMNPSFGPK
12


 


9
NPSFGPKHL
12


 


8
MNPSFGPKH
11

















TABLE XXXI







158P1D7



v.6 - HLA-B2709-9-mers


Each peptide is a portion


of SEQ ID NO: 13; each


start position is specified,


the length of peptide is 9


amino acids, and the end


position for each peptide


is the start position plus


eight.









Pos
123456789
score













4
IHSLMNPSF
10



 


9
NPSFGPKHL
10


 


1
GNIIHSLMN
5

















TABLE XXXII







158P1D7



v.6 - HLA-V4402-9-mers


Each peptide is a portion


of SEQ ID NO: 13; each


start position is specified,


the length of peptide is 9


amino acids, and the end


position for each peptide


is the start posifion plus


eight.









Pos
123456789
score













9
NPSFGPKHL
15



 


4
IHSLMNPSF
12

















TABLE XXXIII







158P1D7



v.6 - HLA-B5101-9-mers


Each peptide is a portion


of SEQ ID NO: 13; each


start position is specified,


the length of peptide is 9


amino acids, and the end


position for each peptide


is the start position plus


eight.









Pos
123456789
score













9
NPSFGPKHL
20



 


12
FGPKHLEEE
10


 


13
GPKHLEEEE
10

















TABLE XXXIV







158P1D7



v.6 - HLA-A1-10-mers


Each peptide is a portion of


SEQ ID NO: 13; each start


position is specified, the


length of peptide is 9


amino acids, and the end


position for each peptide is


the start position plus


eight.









Pos
1234567890
score













11
PSFGPKHLEE
11



 


1
AGNIIHSLMN
8


 


8
LMNPSFGPKH
7


 


7
SLMNPSFGPK
6


 


12
SFGPKHLEEE
6

















TABLE XXXV







158P1D7



v.6 - HLA-A0201-10-mers


Each peptide is a portion of


SEQ ID NO: 13; each start


position is specified, the


length of peptide is 10


amino acids, and the end


position for each peptide is


the start position plus nine.









Pos
1234567890
score













8
LMNPSFGPKH
16



 


4
IIHSLMNPSF
13


 


7
SLMNPSFGPK
13


 


3
NIIHSLMNPS
11


 


12
SFGPKHLEEE
10


 


9
MNPSFGPKHL
9

















TABLE XXXVI







158P1D7 v.6-



HLA-A0203-10-mers









Pos
1234567890
score






No Results found

















TABLE XXXVII







158P1D7



v.6 - HLA-A3-10-mers


Each peptide is a portion of


SEQ ID NO: 13; each start


position is specified, the


length of peptide is 10


amino acids, and the end


position for each peptide is


the start position plus nine.









Pos
1234567890
score













7
SLMNPSFGPK
23



 


4
IIHSLMNPSF
16


 


3
NIIHSLMNPS
11


 


8
LMNPSFGPKH
10

















TABLE XXVIII







158P1D7



v.6 - HLA-A26-10-mers


Each peptide is a portion of


SEQ ID NO: 13; each start


position is specified, the


length of peptide is 10


amino acids, and the end


position for each peptide is


the start position plus nine.









Pos
1234567890
score













4
IIHSLMNPSF
14



 


9
MNPSFGPKHL
9


 


2
GNIIHSLMNP
8


 


3
NIIHSLMNPS
8


 


12
SFGPKHLEEE
6

















TABLE XXXIX







158P1D7



v.6 - HLA-A0702-10-mers


Each peptide is a portion of


SEQ ID NO: 13; each start


position is specified, the


length of peptide is 10


amino acids, and the end


position for each pepdde is


the start position plus nine.









Pos
1234567890
score













10
NPSFGPKHLE
12



 


9
MNPSFGPKHL
10


 


14
GPKHLEEEEE
10


 


4
IIHSLMNPSF
8

















TABLE XL







158P1D7 v.6 -



HLA-B08-10-mers









Pos
1234567890
score






No Results Found

















TABLE XLI







158P1D7 v.6 



HLA-B1510-10-mers









Pos
1234567890
score






No Results Found

















TABLE XLII







158P1D7 v.6 -



HLA-B2705-10-mers









Pos
1234567890
score






No Results Found
















TABLE XLIII







158P1D7 v.6 -


HLA-B2709-10-mers









Pos
1234567890
score






No Results Found
















TABLE XLIV







158P1D7 v.6 -


HLA-B4402-10-mers


Each peptide is a portion of


SEQ ID NO: 13; each start


position is specified, the


length of peptide is 10


amino acids, and the end


position for each peptide is


the start position plus nine.









Pos
1234567890
score













9
MNPSFGPKHL
14



 


4
IIHSLMNPSF
10

















TABLE XLV







158P1D7 v.6-HLA-



B5101-10-mers









Pos
1234567890
score










No Results Found.

















TABLE XLVI







158P1D7 v.6-HLA-



DRB 0101-15-mers


Each peptide is a portion of SEQ


ID NO: 13; each start position is


specified, the length of peptide is


15 amino acids, and the end


position for each peptide is the


start position plus fourteen.









Pos
123456789012345
score













7
GNIIHSLMNPSFGPK
24



 


11
HSLMNPSFGPKHLEE
18


 


9
IIHSLMNPSFGPKHL
17


 


1
RKLILAGNIIHSLMN
16


 


2
KLILAGNIIHSLMNP
16


 


4
ILAGNIIHSLMNPSF
16


 


6
AGNIIHSLMNPSFGP
16


 


12
SLMNPSFGPKHLEEE
16


 


3
LILAGNIIHSLMNPS
14


 


8
NIIHSLMNPSFGPKH
13

















TABLE XLVII







158P1D7 v.6-HLA-



DRB-0301-15-mers


Each peptide is a portion of SEQ


ID NO: 13; each start position is


specified, the length of peptide is


15 amino acids, and the end


position for each peptide is the


start position plus fourteen.









Pos
123456789012345
score













7
GNIIHSLMNPSFGPK
25



 


2
KLILAGNIIHSLMNP
18


 


6
AGNIIHSLMNPSFGP
13


 


1
RKLILAGNIIHSLMN
12


 


11
HSLMNPSFGPKHLEE
12

















TABLE XLVIII







158P1D7 v.6-HLA-



DRB 0410-15-mers


Each peptide is a portion of SEQ


ID NO: 13; each start position is


specified, the length of peptide is


15 amino acids, and the end


position for each peptide is the


start position plus fourteen.









Pos
123456789012345
score













7
GNIIHSLMNPSFGPK
20



 


3
LILAGNIIHSLMNPS
18


 


4
ILAGNIIHSLMNPSF
18


 


1
RKLILAGNIIHSLMN
14


 


2
KLILAGNIIHSLMNP
14


 


6
AGNIIHSLMNPSFGP
14


 


10
IHSLMNPSFGPKHLE
14


 


12
SLMNPSFGPKHLEEE
12

















TABLE XLIX







158P1D7 v.6-HLA-



DRB 1101-15-mers


Each peptide is a portion of SEQ


ID NO: 13; each start position is


specified, the length of peptide is


15 amino acids, and the end


position for each peptide is the


start position plus fourteen.









Pos
123456789012345
score













3
LILAGNIIHSLMNPS
15



 


1
RKLILAGNIIHSLMN
12


 


6
AGNIIHSLMNPSFGP
12


 


7
GNIIHSLMNPSFGPK
12


 


8
NIIHSLMNPSFGPKH
12


 


15
NPSFGPKHLEEEEER
10


 


13
LMNPSFGPKHLEEEE
9


 


14
MNPSFGPKHLEEEEE
9


 


11
HSLMNPSFGPKHLEE
7
















TABLE L







Exon boundaries of transcript 158P1D7v.1












Exon
Start
End
Length







1
1
2555
2555

















TABLE LI(a)





Nucleotide sequence of transcript variant 158P1D7 v.3 (SEQ ID NO: 70)

















tcggatttca tcacatgaca acatgaagct gtggattcat ctcttttatt catctctcct
60



 


tgcctgtata tctttacact cccaaactcc agtgctctca tccagaggct cttgtgattc
120


 


tctttgcaat tgtgaggaaa aagatggcac aatgctaata aattgtgaag caaaaggtat
180


 


caagatggta tctgaaataa gtgtgccacc atcacgacct ttccaactaa gcttattaaa
240


 


taacggcttg acgatgcttc acacaaatga cttttctggg cttaccaatg ctatttcaat
300


 


acaccttgga tttaacaata ttgcagatat tgagataggt gcatttaatg gccttggcct
360


 


cctgaaacaa cttcatatca atcacaattc tttagaaatt cttaaagagg atactttcca
420


 


tggactggaa aacctggaat tcctgcaagc agataacaat tttatcacag tgattgaacc
480


 


aagtgccttt agcaagctca acagactcaa agtgttaatt ttaaatgaca atgctattga
540


 


gagtcttcct ccaaacatct tccgatttgt tcctttaacc catctagatc ttcgtggaaa
600


 


tcaattacaa acattgcctt atgttggttt tctcgaacac attggccgaa tattggatct
660


 


tcagttggag gacaacaaat gggcctgcaa ttgtgactta ttgcagttaa aaacttggtt
720


 


ggagaacatg cctccacagt ctataattgg tgatgttgtc tgcaacagcc ctccattttt
780


 


taaaggaagt atactcagta gactaaagaa ggaatctatt tgccctactc caccagtgta
840


 


tgaagaacat gaggatcctt caggatcatt acatctggca gcaacatctt caataaatga
900


 


tagtcgcatg tcaactaaga ccacgtccat tctaaaacta cccaccaaag caccaggttt
960


 


gataccttat attacaaagc catccactca acttccagga ccttactgcc ctattccttg
1020


 


taactgcaaa gtcctatccc catcaggact tctaatacat tgtcaggagc gcaacattga
1080


 


aagcttatca gatctgagac ctcctccgca aaatcctaga aagctcattc tagcgggaaa
1140


 


tattattcac agtttaatga agtctgatct agtggaatat ttcactttgg aaatgcttca
1200


 


cttgggaaac aatcgtattg aagttcttga agaaggatcg tttatgaacc taacgagatt
1260


 


acaaaaactc tatctaaatg gtaaccacct gaccaaatta agtaaaggca tgttccttgg
1320


 


tctccataat cttgaatact tatatcttga atacaatgcc attaaggaaa tactgccagg
1380


 


aacctttaat ccaatgccta aacttaaagt cctgtattta aataacaacc tcctccaagt
1440


 


tttaccacca catatttttt caggggttcc tctaactaag gtaaatctta aaacaaacca
1500


 


gtttacccat ctacctgtaa gtaatatttt ggatgatctt gatttactaa cccagattga
1560


 


ccttgaggat aacccctggg actgctcctg tgacctggtt ggactgcagc aatggataca
1620


 


aaagttaagc aagaacacag tgacagatga catcctctgc acttcccccg ggcatctcga
1680


 


caaaaaggaa ttgaaagccc taaatagtga aattctctgt ccaggtttag taaataaccc
1740


 


atccatgcca acacagacta gttaccttat ggtcaccact cctgcaacaa caacaaatac
1800


 


ggctgatact attttacgat ctcttacgga cgctgtgcca ctgtctgttc taatattggg
1860


 


acttctgatt atgttcatca ctattgtttt ctgtgctgca gggatagtgg ttcttgttct
1920


 


tcaccgcagg agaagataca aaaagaaaca agtagatgag caaatgagag acaacagtcc
1980


 


tgtgcatctt cagtacagca tgtatggcca taaaaccact catcacacta ctgaaagacc
2040


 


ctctgcctca ctctatgaac agcacatggg agcccacgaa gagctgaagt taatggaaac
2100


 


attaatgtac tcacgtccaa ggaaggtatt agtggaacag acaaaaaatg agtattttga
2160


 


acttaaagct aatttacatg ctgaacctga ctatttagaa gtcctggagc agcaaacata
2220


 


gatggaga
2228
















TABLE LII(a)





Nucleotide sequence alignment of 158P1D7 v.1 (SEQ ID NO: 71) and


158P1D7 v.3 (SEQ ID NO: 72)





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 























TABLE LIII(a)





Peptide sequences of protein coded



by 158P1D7 v.3 (SEQ ID NO: 73)

















MKLWIHLFYS SLLACISLHS QTPVLSSRGS CDSLCNCEEK DGTMLINCEA KGIKMVSEIS
60



 


VPPSRPFQLS LLNNGLTMLH TNDFSGLTNA ISIHLGFNNI ADIEIGAFNG LGLLKQLHIN
120


 


HNSLEILKED TFHGLENLEF LQADNNFITV IEPSAFSKLN RLKVLILNDN AIESLPPNIF
180


 


RFVPLTHLDL RGNQLQTLPY VGFLEHIGRI LDLQLEDNKW ACNCDLLQLK TWLENMPPQS
240


 


IIGDVVCNSP PFFKGSILSR LKKESICPTP PVYEEHEDPS GSLHLAATSS INDSRMSTKT
300


 


TSILKLPTKA PGLIPYITKP STQLPGPYCP IPCNCKVLSP SGLLIHCQER NIESLSDLRP
360


 


PPQNPRKLIL AGNIIHSLMK SDLVEYFTLE MLHLGNNRIE VLEEGSFMNL TRLQKLYLNG
420


 


NHLTKLSKGM FLGLHNLEYL YLEYNAIKEI LPGTFNPMPK LKVLYLNNNL LQVLPPHIFS
480


 


GVPLTKVNLK TNQFTHLPVS NILDDLDLLT QIDLEDNPWD CSCDLVGLQQ WIQKLSKNTV
540


 


TDDILCTSPG HLDKKELKAL NSEILCPGLV NNPSMPTQTS YLMVTTPATT TNTADTILRS
600


 


LTDAVPLSVL ILGLLIMFIT IVFCAAGIVV LVLHRRRRYK KKQVDEQMRD NSPVHLQYSM
660


 


YCHKTTHHTT ERPSASLYEQ HMGAHEELKL METLMYSRPR KVLVEQTKNE YFELKANLHA
720


 


EPDYLEVLEO OT
732
















TABLE LIV(a)





Amino acid sequence alignment of 158P1D7 v.1 (SEQ ID NO: 74) and


158P1D7 v.3 (SEQ ID NO: 75)





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 























TABLE LI(b)





Nucleotide sequence of transcript variant



158P1D7 v.4 (SEQ ID NO: 76)

















tcggatttca tcacatgaca acatgaagct gtggattcat ctcttttatt catctctcct
60



 


tgcctgtata tctttacact cccaaactcc agtgctctca tccagaggct cttgtgattc
120


 


tctttgcaat tgtgaggaaa aagatggcac aatgctaata aattgtgaag caaaaggtat
180


 


caagatggta tctgaaataa gtgtgccacc atcacgacct ttccaactaa gcttattaaa
240


 


taacggcttg acgatgcttc acacaaatga cttttctggg cttaccaatg ctatttcaat
300


 


acaccttgga tttaacaata ttgcagatat tgagataggt gcatttaatg gccttggcct
360


 


cctgaaacaa cttcatatca atcacaattc tttagaaatt cttaaagagg atactttcca
420


 


tggactggaa aacctggaat tcctgcaagc agataacaat tttatcacag tgattgaacc
480


 


aagtgccttt agcaagctca acagactcaa agtgttaatt ttaaatgaca atgctattga
540


 


gagtcttcct ccaaacatct tccgatttgt tcctttaacc catctagatc ttcgtggaaa
600


 


tcaattacaa acattgcctt atgttggttt tctcgaacac attggccgaa tattggatct
660


 


tcagttggag gacaacaaat gggcctgcaa ttgtgactta ttgcagttaa aaacttggtt
720


 


ggagaacatg cctccacagt ctataattgg tgatgttgtc tgcaacagcc ctccattttt
780


 


taaaggaagt atactcagta gactaaagaa ggaatctatt tgccctactc caccagtgta
840


 


tgaagaacat gaggatcctt caggatcatt acatctggca gcaacatctt caataaatga
900


 


tagtcgcatg tcaactaaga ccacgtccat tctaaaacta cccaccaaag caccaggttt
960


 


gataccttat attacaaagc catccactca acttccagga ccttactgcc ctattccttg
1020


 


taactgcaaa gtcctatccc catcaggact tctaatacat tgtcaggagc gcaacattga
1080


 


aagcttatca gatctgagac ctcctccgca aaatcctaga aagctcattc tagcgggaaa
1140


 


tattattcac agtttaatga agtccatcct ttggtccaaa gcatctggaa gaggaagaag
1200


 


agaggaatga gaaagaagga agtgatgcaa aacatctcca aagaagtctt ttggaacagg
1260


 


aaaatcattc accactcaca gggtcaaata tgaaatacaa aaccacgaac caatcaacag
1320


 


aatttttatc cttccaagat gccagctcat tgtacagaaa cattttagaa aaagaaaggg
1380


 


aacttcagca actgggaatc acagaatacc taaggaaaaa cattgctcag ctccagcctg
1440


 


atatggaggc acattatcct ggagcccacg aagagctgaa gttaatggaa acattaatgt
1500


 


actcacgtcc aaggaaggta ttagtggaac agacaaaaaa tgagtatttt gaacttaaag
1560


 


ctaatttaca tgctgaacct gactattiag aagtcctgga gcagcaaaca tagatggaga
1620
















TABLE LII(b)





Nucleotide sequence alignment of 158P1D7 v.1 (SEQ ID NO: 77) and


158P1D7 v.4 (SEQ ID NO: 78)





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE LII(b)





Peptide sequences of protein coded by 158P1D7 v.4 (SEQ ID NO:79)






















MKLWIHLFYS
SLLACISLHS
QTPVLSSRGS
CDSLCNCEEK
DGTMLINCEA
KGIKMVSEIS
 60



 


VPPSRPFQLS
LLNNGLTMLH
TNDFSGLTNA
ISIHLGFNNI
ADIEIGAFNG
LGLLKQLHIN
120


 


HNSLEILKED
TFHGLENLEF
LQADNNFITV
IEPSAFSKLN
RLKVLILNDN
AIESLPPNIF
180


 


RFVPLTHLDL
RGNQLQTLPY
VGFLEHIGRI
LDLQLEDNKW
ACNCDLLQLK
TWLENNPPQS
240


 


IIGDVVCNSP
PFFKGSILSR
LKKESICPTP
PVYEEHEDPS
GSLHLAATSS
INDSRMSTKT
300


 


TSILKLPTKA
PGLIPYITKP
STQLPGPYCP
IPCNCKVLSP
SGLLIHCQER
NIESLSDLRP
360


 


PPQNPRKLIL
AGNIIHSLMK
SILWSKASGR
GRREE


395
















TABLE LIV(b)





Amino acid sequence alignment of 158P1D7 v.1 (SEQ ID NO: 80) and


158P1D7 v.4 (SEQ ID NO: 81)





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 























TABLE LI(c)





Nucleotide sequence of transcript variant 158P1D7 v.5



(SEQ ID NO:82)






















gcgtcgacaa
caagaaatac
tagaaaagga
ggaaggagaa
cattgctgca
gcttggatct
60



 


acaacctaag
aaagcaagag
tgatcaatct
cagctctgtt
aaacatcttg
tttacttact
120


 


gcattcagca
gcttgcaaat
ggttaactat
atgcaaaaaa
gtcagcatag
ctgtgaagta
180


 


tgccgtgaat
tttaattgag
ggaaaaagga
caattgcttc
aggatgctct
agtatgcact
240


 


ctgcttgaaa
tattttcaat
gaaatgctca
gtattctatc
tttgaccaga
ggttttaact
300


 


ttatgaagct
atgggacttg
acaaaaagtg
atatttgaga
agaaagtacg
cagtggttgg
360


 


tgttttcttt
tttttaataa
aggaattgaa
ttactttgaa
cacctcttcc
agctgtgcat
420


 


tacagataac
gtcaggaaga
gtctctgctt
tacagaatcg
gatttcatca
catgacaaca
480


 


tgaagctgtg
gattcatctc
ttttattcat
ctctccttgc
ctgtatatct
ttacactccc
540


 


aaactccagt
gctctcatcc
agaggctctt
gtgattctct
ttgcaattgt
gaggaaaaag
600


 


atggcacaat
gctaataaat
tgtgaagcaa
aaggtatcaa
gatggtatct
gaaataagtg
660


 


tgccaccatc
acgacctttc
caactaagct
tattaaataa
cggcttgacg
atgcttcaca
720


 


caaatgactt
ttctgggctt
accaatgcta
tttcaataca
ccttggattt
aacaatattg
780


 


cagatattga
gataggtgca
tttaatggcc
ttggcctcct
gaaacaactt
catatcaata
840


 


acaattcttt
agaaattctt
aaagaggata
ctttccatgg
actggaaaac
ctggaattcc
900


 


tgcaagcaga
taacaatttt
atcacagtga
ttgaaccaag
tgcctttagc
aagctcaaca
960


 


gactcaaagt
gttaatttta
aatgacaatg
ctattgagag
tcttcctcca
aacatcttcc
1020


 


gatttgttcc
tttaacccat
ctagatcttc
gtggaaatca
attacaaaca
ttgccttatg
1080


 


ttggttttct
cgaacacatt
ggccgaatat
tggatcttca
gttggaggac
aacaaatggg
1140


 


cctgcaattg
tgacttattg
cagttaaaaa
cttggttgga
gaacatgcct
ccacagtcta
1200


 


taattggtga
tgttgtctgc
aacagccctc
cattttttaa
aggaagtata
ctcagtagac
1260


 


taaagaagga
atctatttgc
cctactccac
cagtgtatga
agaacatgag
gatccttcag
1320


 


gatcattaca
tctggcagca
acatcttcaa
taaatgatag
tcgcatgtca
actaagacca
1380


 


cgtccattct
aaaactaccc
accaaagcac
caggtttgat
accttatatt
acaaagccat
1440


 


ccactcaact
tccaggacct
tactgcccta
ttccttgtaa
ctgcaaagtc
ctatccccat
1500


 


caggacttct
aatacattgt
caggagcgca
acattgaaag
cttatcagat
ctgagacctc
1560


 


ctccgcaaaa
tcctagaaag
ctcattctag
cgggaaatat
tattcacagt
ttaatgaagt
1620


 


ctgatctagt
ggaatatttc
actttggaaa
tgcttcactt
gggaaacaat
cgtattgaag
1680


 


ttcttgaaga
aggatcgttt
atgaacctaa
cgagattaca
aaaactctat
ctaaatggta
1740


 


accacctgac
caaattaagt
aaaggcatgt
tccttggtct
ccataatctt
gaatacttat
1800


 


atcttgaata
caatgccatt
aaggaaatac
tgccaggaac
ctttaatcca
atgcctaaac
1860


 


ttaaagtcct
gtatttaaat
aacaacctcc
tccaagtttt
accaccacat
attttttcag
1920


 


gggttcctct
aactaaggta
aatcttaaaa
caaaccagtt
tacccatcta
cctgtaagta
1980


 


atattttgga
tgatcttgat
ttactaaccc
agattgacct
tgaggataac
ccctgggact
2040


 


gctcctgtga
cctggttgga
ctgcagcaat
ggatacaaaa
gttaagcaag
aacacagtga
2100


 


cagatgacat
cctctgcact
tcccccgggc
atctcgacaa
aaaggaattg
aaagccctaa
2160


 


atagtgaaat
tctctgtcca
ggtttagtaa
ataacccatc
catgccaaca
cagactagtt
2220


 


accttatggt
caccactcct
gcaacaacaa
caaatacggc
tgatactatt
ttacgatctc
2280


 


ttacggacgc
tgtgccactg
tctgttctaa
tattgggact
tctgattatg
ttcatcacta
2340


 


ttgttttctg
tgctgcaggg
atagtggttc
ttgttcttca
ccgcaggaga
agatacaaaa
2400


 


agaaacaagt
agatgagcaa
atgagagaca
acagtcctgt
gcatcttcag
tacagcatgt
2460


 


atggccataa
aaccactcat
cacactactg
aaagaccctc
tgcctcactc
tatgaacagc
2520


 


acatggtgag
ccccatggtt
catgtctata
gaagtccatc
ctttggtcca
aagcatctgg
2580


 


aagaggaaga
agagaggaat
gagaaagaag
gaagtgatgc
aaaacatctc
caaagaagtc
2640


 


ttttggaaca
ggaaaatcat
tcaccactca
cagggtcaaa
tatgaaatac
aaaaccacga
2700


 


accaatcaac
agaattttta
tccttccaag
atgccagctc
attgtacaga
aacattttag
2760


 


aaaaagaaag
ggaacttcag
caactgggaa
tcacagaata
cctaaggaaa
aacattgctc
2820


 


agctccagcc
tgatatggag
gcacattatc
ctggagccca
cgaagagctg
aagttaatgg
2880


 


aaacattaat
gtactcacgt
ccaaggaagg
tattagtgga
acagacaaaa
aatgagtatt
2940


 


ttgaacttaa
agctaattta
catgctgaac
ctgactattt
agaagtcctg
gagcagcaaa
3000


 


catagatgga
gagttgaggg
ctttcgccag
aaatgctgtg
attctgttat
taagtccata
3060


 


ccttgtaaat
aagtgcctta
cgtgagtgtg
tcatcaatca
gaacctaagc
acagagtaaa
3120


 


ctatggggaa
aaaaaaagaa
gacgaaacag
aaactcaggg
atcactggga
gaagccatgg
3180


 


cataatcttc
aggcaattta
gtctgtccca
aataaacata
catccttggc
atgtaaatca
3240


 


tcaagggtaa
tagtaatatt
catatacctg
aaacgtgtct
cataggagtc
ctctctgcac
3300
















TABLE LII(c)





Nucleotide sequence alignment of 158P1D7 v.1 (SEQ ID NO: 83) and


158P1D7 v.5 (SEQ ID NO: 84)





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE LIII(c)





Peptide sequences of protein coded by 158P1D7 v.5 (SEQ ID NO:85)






















MKLWIHLFYS
SLLACISLHS
QTPVLSSRGS
CDSLCNCEEK
DGTMLINCEA
KGIKMVSEIS
60



 


VPPSRPFQLS
LLNNGLTMLH
TNDFSGLTNA
ISIHLGFNNI
ADIEIGAFNG
LGLLKQLHIN
120


 


HNSLEILKED
TFHGLENLEF
LQADNNFITV
IEPSAFSKLN
RLKVLILNDN
AIESLPPNIF
180


 


RFVPLTHLDL
RGNQLQTLPY
VGFLEHIGRI
LDLQLEDNKW
ACNCDLLQLK
TWLENMPPQS
240


 


IIGDVVCNSP
PFFKGSILSR
LKKESICPTP
PVYEEHEDPS
GSLHLAATSS
INDSRMSTKT
300


 


TSILKLPTKA
PGLIPYITKP
STQLPGPYCP
IPCNCKVLSP
SGLLIHCQER
NIESLSDLRP
360


 


PPQNPRKLIL
AGNIIHSLMK
SDLVEYFTLE
MLHLGNNRIE
VLEEGSFMNL
TRLQKLYLNG
420


 


NHLTKLSKGM
FLGLHNLEYL
YLEYNAIKEI
LPGTFNPMPK
LKVLYLNNNL
LQVLPPHIFS
480


 


GVPLTKVNLK
TNQFTHLPVS
NILDDLDLLT
QIDLEDNPWD
CSCDLVGLQQ
WIQKLSKNTV
540


 


TDDILCTSPG
HLDKKELKAL
NSEILCPGLV
NNPSMPTQTS
YLMVTTPATT
TNTADTILRS
600


 


LTDAVPLSVL
ILGLLIMFIT
IVFCAAGIVV
LVLHRRRRYK
KKQVDEQMRD
NSPVHLQYSM
660


 


YGHKTTHHTT
ERPSASLYEQ
HMVSPMVHVY
RSPSFGPKHL
EEEEERNEKE
GSDAKHLQRS
720


 


LLEQENHSPL
TGSNMKYKTT
NQSTEFLSFQ
DASSLYRNIL
EKERELQQLG
ITEYLRKNIA
780


 


QLQPDMEAHY
PGAHEELKLM
ETLMYSRPRK
VLVEQTKNEY
FELKANLHAE
PDYLEVLEQQ
840


 


T





841
















TABLE LIV(c)





Amino acid sequence alignment of 158P1D7 v.1 (SEQ ID NO: 86) and


158P1D7 v.5 (SEQ ID NO: 87)





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE LI(d)





Nucleotide sequence of transcript variant 158P1D7 v.6 (SEQ ID NO:88)






















tcggatttca
tcacatgaca
acatgaagct
gtggattcat
ctcttttatt
catctctcct
60



 


tgcctgtata
tctttacact
cccaaactcc
agtgctctca
tccagaggct
cttgtgattc
120


 


tctttgcaat
tgtgaggaaa
aagatggcac
aatgctaata
aattgtgaag
caaaaggtat
180


 


caagatggta
tctgaaataa
gtgtgccacc
atcacgacct
ttccaactaa
gcttattaaa
240


 


taacggcttg
acgatgcttc
acacaaatga
cttttctggg
cttaccaatg
ctatttcaat
300


 


acaccttgga
tttaacaata
ttgcagatat
tgagataggt
gcatttaatg
gccttggcct
360


 


cctgaaacaa
cttcatatca
atcacaattc
tttagaaatt
cttaaagagg
atactttcca
420


 


tggactggaa
aacctggaat
tcctgcaagc
agataacaat
tttatcacag
tgattgaacc
480


 


aagtgccttt
agcaagctca
acagactcaa
agtgttaatt
ttaaatgaca
atgctattga
540


 


gagtcttcct
ccaaacatct
tccgatttgt
tcctttaacc
catctagatc
ttcgtggaaa
600


 


tcaattacaa
acattgcctt
atgttggttt
tctcgaacac
attggccgaa
tattggatct
660


 


tcagttggag
gacaacaaat
gggcctgcaa
ttgtgactta
ttgcagttaa
aaacttggtt
720


 


ggagaacatg
cctccacagt
ctataattgg
tgatgttgtc
tgcaacagcc
ctccattttt
780


 


taaaggaagt
atactcagta
gactaaagaa
ggaatctatt
tgccctactc
caccagtgta
840


 


tgaagaacat
gaggatcctt
caggatcatt
acatctggca
gcaacatctt
caataaatga
900


 


tagtcgcatg
tcaactaaga
ccacgtccat
tctaaaacta
cccaccaaag
caccaggttt
960


 


gataccttat
attacaaagc
catccactca
acttccagga
ccttactgcc
ctattccttg
1020


 


taactgcaaa
gtcctatccc
catcaggact
tctaatacat
tgtcaggagc
gcaacattga
1080


 


aagcttatca
gatctgagac
ctcctccgca
aaatcctaga
aagctcattc
tagcgggaaa
1140


 


tattattcac
agtttaatga
atccatcctt
tggtccaaag
catctggaag
aggaagaaga
1200


 


gaggaatgag
aaagaaggaa
gtgatgcaaa
acatctccaa
agaagtcttt
tggaacagga
1260


 


aaatcattca
ccactcacag
ggtcaaatat
gaaatacaaa
accacgaacc
aatcaacaga
1320


 


atttttatcc
ttccaagatg
ccagctcatt
gtacagaaac
attttagaaa
aagaaaggga
1380


 


acttcagcaa
ctgggaatca
cagaatacct
aaggaaaaac
attgctcagc
tccagcctga
1440


 


tatggaggca
cattatcctg
gagcccacga
agagctgaag
ttaatggaaa
cattaatgta
1500


 


ctcacgtcca
aggaaggtat
tagtggaaca
gacaaaaaat
gagtattttg
aacttaaagc
1560


 


taatttacat
gctgaacctg
actatttaga
agtcctggag
cagcaaacat
agatggaga
1619
















TABLE LII(d)





Nucleotide sequence alignment of 158P1D7 v.1 (SEQ ID NO: 89) and


158P1D7 v.6 (SEQ ID NO: 90)





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE LIII(d)





Peptide sequences of protein coded by 158P1D7 v.6 (SEQ ID NO:91)






















MKLWIHLFYS
SLLACISLHS
QTPVLSSRGS
CDSLCNCEEK
DGTMLINCEA
KGIKMVSEIS
60



 


VPPSRPFQLS
LLNNGLTMLH
TNDFSGLTNA
ISIHLGFNNI
ADIEIGAFNG
LGLLKQLHIN
120


 


HNSLEILKED
TFHGLENLEF
LQADNNFITV
IEPSAFSKLN
RLKVLILNDN
AIESLPPNIF
180


 


RFVPLTHLDL
RGNQLQTLPY
VGFLEHIGRI
LDLQLEDNKW
ACNCDLLQLK
TWLENMPPQS
240


 


IIGDVVCNSP
PFFKGSILSR
LKKESICPTP
PVYEEHEDPS
GSLHLAATSS
INDSRMSTKT
300


 


TSILKLPTKA
PGLIPYITKP
STQLPGPYCP
IPCNCKVLSP
SGLLIHCQER
NIESLSDLRP
360


 


PPQNPRKLIL
AGNIIHSLMN
PSFGPKHLEE
EEERNEKEGS
DAKHLQRSLL
EQENHSPLTG
420


 


SNMKYKTTNQ
STEFLSFQDA
SSLYRNILEK
ERELQQLGIT
EYLRKNIAQL
QPDMEAHYPG
480


 


AHEELKLMET
LMYSRPRKVL
VEQTKNEYFE
LKANLHAEPD
YLEVLEQQT

529
















TABLE LIV(d)





Amino acid sequence alignment of 158P1D7 v.1 (SEQ ID NO: 92) and


158P1D7 v.6 (SEQ ID NO: 93)





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE LV





Search peptides















158P1D7,variant 1: 9-mers 10-mers and 15-mers (SEQ ID NO: 94)


MKLWIHLFYS SLLACISLHS QTPVLSSRGS CDSLCNCEEK DGTMLINCEA KGIKMVSEIS


 


VPPSRPFQLS LLNNGLTMLH TNDFSGLTNA ISIHLGFNNI ADIEIGAFNG LGLLKQLHIN


 


HNSLEILKED TFHGLENLEF LQADNNFITV IEPSAFSKLN RLKVLILNDN AIESLPPNIF


 


RFVPLTHLDL RGNQLQTLPY VGFLEHIGRI LDLQLEDNKW ACNCDLLQLK TWLENMPPQS


 


IIGDVVCNSP PFFKGSILSR LKKESICPTP PVYEEHEDPS GSLHLAATSS INDSRMSTKT


 


TSILKLPTKA PGLIPYITKP STQLPGPYCP IPCNCKVLSP SGLLIHCQER NIESLSDLRP


 


PPQNPRKLIL AGNIIHSLMK SDLVEYFTLE MLHLGNNRIE VLEEGSFMNL TRLQKLYLNG


 


NHLTKLSKGM FLGLHNLEYL YLEYNAIKEI LPGTFNPMPK LKVLYLNNNL LQVLPPHIFS


 


GVPLTKVNLK TNQFTHLPVS NILDDLDLLT QIDLEDNPWD CSCDLVGLQQ WIQKLSKNTV


 


TDDILCTSPG HLDKKELKAL NSEILCPGLV NNPSMPTQTS YLMVTTPATT TNTADTILRS


 


LTDAVPLSVL ILGLLIMFIT IVFCAAGIVV LVLHRRRRYK KKQVDEQMRD NSPVHLQYSM


 


YGHKTTHHTT ERPSASLYEQ HMVSPMVHVY RSPSFGPKHL EEEEERNEKE GSDAKHLQRS


 


LLEQENHSPL TGSNMKYKTT NQSTEFLSFQ DASSLYRNIL EKERELQQLG ITEYLRKNIA


 


QLQPDMEAHY PGAHEELKLM ETLMYSRPRK VLVEQTKNEY FELKANLHAE PDYLEVLEQQ


 


T


 


158P1D7 Variant 3:


9-mers


ASLYEQHMGAHEELKL (SEQ ID NO: 95) start position 675


10-mers


SASLYEQHMGAHEELKLM (SEQ ID NO: 96) start position 674


15-mers


TTERPSASLYEQHMGAHEELKLMETLMY (SEQ ID NO: 97) start position 669


 


158P1D7 Variant 4:


9-mers


IIHSLMKSILWSKASGRGRREE (SEQ ID NO: 98) start position 674


10-mers


NIIHSLMKSILWSKASGRGRREE (SEQ ID NO: 99) start position 673


15-mers


LILAGNIIHSLMKSILWSKASGRGRREE (SEQ ID NO: 100) start position 668


 


158P1D7 Variant 6:


9-mers


GNIIHSLMNPSFGPKHLEEEEER (SEQ ID NO: 101) start position 372


10-mers


AGNIIHSLMNPSFGPKHLEEEEER (SEQ ID NO: 102) start position 371


15-mers


RKLILAGNIIHSLMNPSFGPKHLEEEEER (SEQ ID NO: 103) start position 366
















TABLE LVI







Protein Characteristics of 158P1D7











Bioinformatic





Program
URL
Outcome





ORF
ORF finder

2555 bp


Protein length


841 aa


Transmembrane region
TM Pred
http://www.ch.embnet.org/
One TM, aa609-aa633



HMMTop
http://www.enzim.hu/hmmtop/
One TM, aa609-aa633



Sosui
http://www.genome.ad.jp/SOSui/
One TM, aa608-aa630



TMHMM
http://www.cbs.dtu.dk/services/TMHMM
One TM, aa611-aa633


Signal Peptide
Signal P
http://www.cbs.dtu.dk/services/SignalP/
Signal peptide, aa3-aa25


pl
pl/MW tool
http://www.expasy.ch/tools/
pl 6.07


Molecular weight
pl/MW tool
http://www.expasy.ch/tools/
95.1 kD


Localization
PSORT
http://psort.nibb.ac.jp/
Plasma membrane



PSORT II
http://psort.nibb.ac.jp/
65% nuclear, 8% cytoplasmic,





4% plasma membrane


Motifs
Pfam
http://www.sanger.ac.uk/Pfam/
Leucine-rich repeat;





mannosyl transferase



Prints
http://bioinf.man.ac.uk/cgi-bin/dbbrowser
Leucine-rich repeats; Relaxin





receptor



Blocks
http://www.blocks.fhcrc.org/
Leucine rich repeats;





cysteine-rich flanking region
















TABLE LVII







Characteristics of 158P1D7 specific antibodies














Affinity





mAb
Isotype
(nM)
FACS
Internalization
Western















X68(2)22.1.1
IgG2b/k
3.8
+
+
+


X68(2)31.1.1
IgG2a/k
14
+
+
+


X68(2)18.1.1
IgG2a/k
19
+
+
+


X68(2)120.1.1
IgG2a/k
19
+
+
+
















TABLE LVIII







Detection of 158P1D7 protein by immunohistochemistry


in various cancer patient specimens.










TISSUE
Number Positive
Number tested
% No. Positive













Bladder TCC
35
71
49.3


Lung Carcinoma
26
6
23.1


Breast Carcinoma
11
10
90.9

## Claims

1. An isolated polynucleotide that encodes a 158P1D7 protein, wherein the polynucleotide is selected from the group consisting of:
(a) a polynucleotide comprising the sequence of SEQ ID NO:6, from nucleotide residue numbers 23 through 2221;
(b) a polynucleotide comprising the sequence of SEQ ID NO:8, from nucleotide residue numbers 23 through 1210; and
(c) a polynucleotide comprising the sequence of SEQ ID NO:10, from nucleotide residue numbers 23 through 1612.

2. An isolated polynucleotide that is fully complementary to a polynucleotide of claim 1.

3. An isolated polynucleotide of claim 1 that encodes the polypeptide sequence shown in SEQ ID NO: 7, 9, or 11.

4. The isolated polynucleotide of claim 1 wherein the polynucleotide is contained within a recombinant expression vector.