FIELD OF THE INVENTION The invention described herein relates to a novel gene and its encoded protein, termed 83P2H3, and to diagnostic and therapeutic methods and compositions useful in the management of various cancers that express 83P2H3.

BACKGROUND OF THE INVENTION Cancer is the second leading cause of human death next to coronary disease. Worldwide, millions of people die from cancer every year. In the United States alone, as reported by the American Cancer Society, cancer causes the death of well over a half-million people annually, with over 1.2 million new cases diagnosed per year. While deaths from heart disease have been declining significantly, those resulting from cancer generally are on the rise. In the early part of the next century, cancer is predicted to become the leading cause of death.

Worldwide, several cancers stand out as the leading killers. In particular, carcinomas of the lung, prostate, breast, colon, pancreas, and ovary represent the primary causes of cancer death. These and virtually all other carcinomas share a common lethal feature. With very few exceptions, metastatic disease from a carcinoma is fatal. Moreover, even for those cancer patients who initially survive their primary cancers, common experience has shown that their lives are dramatically altered. Many cancer patients experience strong anxieties driven by the awareness of the potential for recurrence or treatment failure. Many cancer patients experience physical debilitations following treatment. Furthermore, many cancer patients experience a recurrence.

Worldwide, prostate cancer is the fourth most prevalent cancer in men. In North America and Northern Europe, it is by far the most common cancer in males and is the second leading cause of cancer death in men. In the United States alone, well over 30,000 men die annually of this disease- second only to lung cancer. Despite the magnitude of these figures, there is still no effective treatment for metastatic prostate cancer. Surgical prostatectomy, radiation therapy, hormone ablation therapy, surgical castration and chemotherapy continue to be the main treatment modalities. Unfortunately, these treatments are ineffective for many and are often associated with undesirable consequences.

On the diagnostic front, the lack of a prostate tumor marker that can accurately detect early- stage, localized tumors remains a significant limitation in the diagnosis and management of this disease. Although the serum prostate specific antigen (PSA) assay has been a very useful tool, however its specificity and general utility is widely regarded as lacking in several important respects.

Progress in identifying additional specific markers for prostate cancer has been improved by the generation of prostate cancer xenografts that can recapitulate different stages of the disease in mice.

The LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer xenografts that have survived passage in severe combined immune deficient (SCID) mice and have exhibited the capacity to mimic the transition from androgen dependence to androgen independence (Klein et al., 1997, Nat. Med.

3 : 402). More recently identified prostate cancer markers include PCTA-1 (Su et al., 1996, Proc. Natl.

Acad. Sci. USA 93: 7252), prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res 1996 Sep 2 (9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad Sci U S A. 1999 Dec 7; 96 (25): 14523-8) and prostate stem cell antigen (PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).

While previously identified markers such as PSA, PSM, PCTA and PSCA have facilitated efforts to diagnose and treat prostate cancer, there is need for the identification of additional markers and therapeutic targets for prostate and related cancers in order to further improve diagnosis and therapy.

Renal cell carcinoma (RCC) accounts for approximately 3 percent of adult malignancies.

Once adenomas reach a diameter of 2 to 3 cm, malignant potential exists. In the adult, the two principal malignant renal tumors are renal cell adenocarcinoma and transitional cell carcinoma of the renal pelvis or ureter. The incidence of renal cell adenocarcinoma is estimated at more than 29,000 cases in the United States, and more than 11,600 patients died of this disease in 1998. Transitional cell carcinoma is less frequent, with an incidence of approximately 500 cases per year in the United States.

Surgery has been the primary therapy for renal cell adenocarcinoma for many decades. Until recently, metastatic disease has been refractory to any systemic therapy. With recent developments in systemic therapies, particularly immunotherapies, metastatic renal cell carcinoma may be approached aggressively in appropriate patients with a possibility of durable responses. Nevertheless, there is a remaining need for effective therapies for these patients.

Of all new cases of cancer in the United States, bladder cancer represents approximately 5 percent in men (fifth most common neoplasm) and 3 percent in women (eighth most common neoplasm). The incidence is increasing slowly, concurrent with an increasing older population. In 1998, there was an estimated 54,500 cases, including 39,500 in men and 15,000 in women. The age- adjusted incidence in the United States is 32 per 100,000 for men and 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.

Most bladder cancers recur in the bladder. Bladder cancer is managed with a combination of transurethral resection of the bladder (TUR) and intravesical chemotherapy or immunotherapy. The multifocal and recurrent nature of bladder cancer points out the limitations of TUR. Most muscle-

invasive cancers are not cured by TUR alone. Radical cystectomy and urinary diversion is the most effective means to eliminate the cancer but carry an undeniable impact on urinary and sexual function.

There continues to be a significant need for treatment modalities that are beneficial for bladder cancer patients.

An estimated 130,200 cases of colorectal cancer occurred in 2000 in the United States, including 93,800 cases of colon cancer and 36,400 of rectal cancer. Colorectal cancers are the third most common cancers in men and women. Incidence rates declined significantly during (- 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 occur among women in the United States during 2000. Additionally, about 1,400 new cases of breast cancer were expected to be diagnosed in men in 2000. After increasing about 4% per year in the 1980s, breast cancer incidence rates in women have leveled off in the 1990s to about 110.6 cases per 100,000.

In the U. S. alone, there were an estimated 41,200 deaths (40,800 women, 400 men) in 2000 due to breast cancer. Breast cancer ranks second among cancer deaths in women. According to the most recent data, mortality rates declined significantly during 1992-1996 with the largest decreases in younger women, both white and black. These decreases were probably the result of earlier detection and improved treatment.

Taking into account the medical circumstances and the patient's preferences, treatment of breast cancer may involve lumpectomy (local removal of the tumor) and removal of the lymph nodes under the arm; mastectomy (surgical removal of the breast) and removal of the lymph nodes under the arm; radiation therapy; chemotherapy; or hormone therapy. Often, two or more methods are used in combination. Numerous studies have shown that, for early stage disease, long-term survival rates after lumpectomy plus radiotherapy are similar to survival rates after modified radical mastectomy.

Significant advances in reconstruction techniques provide several options for breast reconstruction after mastectomy. Recently, such reconstruction has been done at the same time as the mastectomy.

Local excision of ductal carcinoma in situ (DCIS) with adequate amounts of surrounding normal breast tissue may prevent the local recurrence of the DCIS. Radiation to the breast and/or tamoxifen may reduce the chance of DCIS occurring in the remaining breast tissue. This is important because DCIS, if left untreated, may develop into invasive breast cancer. Nevertheless, there are serious side effects or sequelae to these treatments. There is, therefore, a need for efficacious breast cancer treatments.

There were an estimated 23,100 new cases of ovarian cancer in the United States in 2000. It accounts for 4% of all cancers among women and ranks second among gynecologic cancers. During 1992-1996, ovarian cancer incidence rates were significantly declining. Consequent to ovarian cancer, there were an estimated 14,000 deaths in 2000. Ovarian cancer causes more deaths than any other cancer of the female reproductive system.

Surgery, radiation therapy, and chemotherapy are treatment options for ovarian cancer.

Surgery usually includes the removal of one or both ovaries, the fallopian tubes (salpingo- oophorectomy), and the uterus (hysterectomy). In some very early tumors, only the involved ovary will be removed, especially in young women who wish to have children. In advanced disease, an attempt is made to remove all intra-abdominal disease to enhance the effect of chemotherapy. There continues to be an important need for effective treatment options for ovarian cancer.

There were an estimated 28,300 new cases of pancreatic cancer in the United States in 2000.

Over the past 20 years, rates of pancreatic cancer have declined in men. Rates among women have remained approximately constant but may be beginning to decline. Pancreatic cancer caused an

estimated 28, 200 deaths in 2000 in the United States. Over the past 20 years, there has been a slight but significant decrease in mortality rates among men (about-0.9% per year) while rates have increased slightly among women.

Surgery, radiation therapy, and chemotherapy are treatment options for pancreatic cancer.

These treatment options can extend survival and/or relieve symptoms in many patients but are not likely to produce a cure for most. There is a significant need for additional therapeutic and diagnostic options for pancreatic cancer.

SUMMARY OF THE INVENTION The present invention relates to a novel gene, designated 83P2H3, that is over-expressed in multiple cancers listed in Table I. Northern blot expression analysis of 83P2H3 gene expression in normal tissues shows a restricted expression pattern in adult tissues. The nucleotide (Figure 2) and amino acid (Figure 2, and Figure 3) sequences of 83P2H3 are provided. The tissue-related profile of 83P2H3 in normal adult tissues, combined with the over-expression observed in prostate and other tumors, shows that 83P2H3 is aberrantly over-expressed in at least some cancers, and thus serves as a useful diagnostic and/or therapeutic target for cancers of tissues such as prostate.

The invention provides polynucleotides corresponding or complementary to all or part of the 83P2H3 genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding 83P2H3-related proteins and fragments of 4,5,6,7,8,9,10,11,12,13,14, 15,16,17,18,19,20,21,22,23,24,25, or more than 25 contiguous amino acids; at least 30,35,40, 45,50,55,60,65,70,80,85,90,95,100 or more than 100 contiguous amino acids of a 83P2H3- related protein, as well as the peptides/proteins themselves; DNA, RNA, DNA/RNA hybrids, and related molecules, polynucleotides or oligonucleotides complementary or having at least a 90% homology to the 83P2H3 genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the 83P2H3 genes, mRNAs, or to 83P2H3-encoding polynucleotides.

Also provided are means for isolating cDNAs and the genes encoding 83P2H3. Recombinant DNA molecules containing 83P2H3 polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of 83P2H3 gene products are also provided. The invention further provides antibodies that bind to 83P2H3 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 further provides methods for detecting the presence and status of 83P2H3 polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express 83P2H3. A typical embodiment of this invention provides methods for monitoring 83P2H3 gene products in a tissue or hematology sample having or suspected of having some form of growth dysregulation such as cancer.

The invention further provides various immunogenic or therapeutic compositions and strategies for treating cancers that express 83P2H3 such as prostate cancers, including therapies aimed at inhibiting the transcription, translation, processing or function of 83P2H3 as well as cancer vaccines.

BRIEF DESCRIPTION OF THE FIGURES Figure 1A. 83P2H3 SSH sequence. The 83P2H3 SSH sequence contains 405 bp (SEQ ID NO: 701).

Figure 1B. CaTrF2E11 nucleic acid sequence (SEQ ID NO: 704) and amino acid sequence (SEQ ID NO: 705).

Figure 2A-B. The cDNA (SEQ ID NO : 702) and amino acid sequence (SEQ ID NO : 703) of 83P2H3.

Figure 2C-D. The cDNA (SEQ ID NO : 706) and amino acid sequence (SEQ ID NO : 707) of CaTrF2E11.

Figure 3A. Amino acid sequence of 83P2H3 (SEQ ID NO : 703). The 83P2H3 protein has 725 amino acids with calculated molecular weight of 83. 2 kDa, and pI of 7.56.83P2H3 is predicted to be a cell surface protein that functions as an ion transporter.

Figure 3B. Amino acid sequence of CaTrF2Ell (SEQ ID NO : 707).

Figure 4A-E. 83P2H3 BLAST alignment with Homo sapiens gene for CaT-like B protein, Genbank accession HSA243501. The sequences are 99% identical.

Figure 5A-B. Northern blot analysis of 83P2H3 expression in various normal human tissues. Two multiple tissue northern blots (Clontech) were probed with the 83P2H3 SSH fragment.

Size standards in kilobases (kb) are indicated on the side. Each lane contains 2 g of mRNA. The results show the expression of 83P2H3 in prostate, and, to a lesser extent, in placenta and pancreas.

Lanes in Fig. 5A represent the following tissues: 1) heart; 2) brain; 3) placenta; 4) lung; 5) liver; 6) skeletal muscle; 7) kidney; 8) pancreas. Lanes in Figure 5B represent the following tissues: 1) spleen; 2) thymus; 3) prostate; 4) testis; 5) ovary; 6) small intestine; 7) colon; 8) leukocytes.

Figure 6. Northern blot analysis of 83P2H3 expression in prostate cancer cell lines and xenografts. RNA was extracted from the LAPC xenografts and prostate cancer cell lines. Northern blots with 10 jig of total RNA per lane were probed with the 83P2H3 SSH fragment. Size standards in kilobases (kb) are indicated on the side. Lanes represent: 1) PrEC ; 2) LAPC-4 AD; 3) LAPC-4 AI; 4) LAPC-9 AD; 5) LAPC-9 AI; 6) LNCaP ; 7) PC-3; 8) DU145; 9) TsuPrl ; 10) LAPC-4 CL.

Figure 7. Expression of 83P2H3 in prostate cancer patient samples. RNA was extracted from prostate tumors and normal adjacent tissue derived from prostate cancer patients. Northern blots with 10 jig of total RNA per lane were probed with the 83P2H3 SSH fragment. Size standards in kilobases (kb) are indicated on the side. Lanes represent: 1) Patient 1, normal adjacent tissue; 2) Patient 1, Gleason 9 tumor; 3) Patient 2, normal adjacent tissue; 4) Patient 2, Gleason 7 tumor.

Figure 8A-C. RT-PCR Expression of CaTrF2E11 in Normal Tissues and in Bladder and Kidney Cancer. First strand cDNA was prepared from normal tissues, and from bladder cancer pool and kidney cancer pool. Normalization was performed by PCR using primers to actin and GAPDH.

Semi-quantitative PCR, using primers to CaTrF2El 1, was performed at 30 cycles of amplification.

Expression of CaTrF2El 1 is observed in normal kidney and prostate, and in bladder cancer pool and kidney cancer pool. Lanes represent: 1) Colon; 2) Ovaries; 3) Leukocytes; 4) Prostate; 5) Small Intestine; 6) Spleen; 7) Testis; 8) Thymus; 9) Brain; 10) Heart; 11) Kidney; 12) Liver; 13) Lung; 14) Pancreas ; 15) Placenta; 16) Skeletal muscle; 17) Prostate; 18) Bladder Cancer Pool; 19) Kidney Cancer Pool.

Figure 9. Expression of CaTrF2E11 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), bladder cancer pool, kidney cancer pool, and lung cancer pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to CaTrF2El 1, was performed at 30 cycles of amplification. Expression of CaTrF2Ell is observed in bladder cancer pool, kidney cancer pool, lung cancer pool and VP1.. Lower level expression is also detected in ovarian cancer pool and VP2. Lane 1, Vital Pool 1; Lane 2, Vital Pool 2; Lane 3, Bladder Cancer Pool; Lane 4, Kidney Cancer Pool; Lane 5, Lung Cancer Pool; Lane 6, Ovarian Cancer Pool.

Figure 10A-B. Expression of CaTrF2E11 in normal human tissues. Two multiple tissue northern blots, with 2 ug ofmRNA/lane, were probed with the CaTrF2El 1 fragment. Size standards in kilobases (kb) are indicated on the side. The results show expression of CaTrF2El 1 in kidney and to lower levels in placenta and prostate. Lanes in Figure 10A represent: 1) Heart; 2) Brain; 3) Placenta; 4) Lung; 5) Liver; 6) Skeletal Muscle; 7) Kidney; 8) Pancreas. Lanes in Figure 10B represent: 1) Spleen; 2) Thymus; 3) Prostate; 4) Testis; 5) Ovary; 6) Small Intestine; 7) Colon; 8) Leukocytes.

Figure 11. Expression of CaTrF2E11 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 blots with 10 ug of total RNA/lane were probed with the CaTrF2El 1 fragment. Size standards in kilobases (kb) are indicated on the side. The results show expression of CaTrF2E 11 in 1 of 3 lung cancer cell lines and in 2 lung tumors. The expression detected in one NAT (isolated from diseased tissues) but not in normal tissue, isolated from healthy donors, may indicate that this tissue is not fully normal and that CaTrF2El 1 may be expressed in early stage tumors. Pt. l, Squamous carcinoma, stage IB; Pt. 2, Squamous carcinoma, stage IIB. Cell lines listed in order: CALU-1, A427, NCI-H82.

Figure 12. Expression of 83P2H3 in human tumors by RT-PCR. First strand cDNA was prepared from a vital pool 1 (VP1 : liver, lung and kidney), a vital pool 2 (VP2: pancreas, colon and stomach), a LAPC xenograft pool (LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC-9AI), a prostate ; cancer pool, and a metastatic cancer pool. The metastatic cancer pool consisted of metastatic tissues

from cancers of the following organs : breast, ovarian, pancreas, colon, prostate and bladder.

Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 83p2H3, was performed at 30 cycles of amplification. Results show expression of 83P2H3 in VP2, xenograft pool, prostate cancer pool and metastatic cancer pool. Lane 1 is VP1 ; lane 2 is VP2; lane 3 is xenograft pool; lane 4 is prostate cancer pool; lane 5 is metastasis pool; lane 6 is water.

Figure 13A-B. Two Projected Models for 83P2H3 PCaT. 83P2H3 may be expressed at the cell surface in either of two configurations, namely containing five or six transmembrane domains. Both configurations show the amino terminal end to be intracellular. The six transmembrane model predicts the C-terminus to be intracellular, while the five transmembrane model predicts the C-terminus to be extracellular. The models exhibit an ion channel signature, predicted pore structure and ankyrin repeats (ANK).

Figure 14A. Hydrophilicity amino acid profile of 83P2H3 determined by computer algorithm sequence analysis using the method of Hopp and Woods (Hopp T. P., Woods K. R., 1981. Proc. Natl.

Acad. Sci. U. S. A. 78: 3824-3828) accessed on the Protscale website (www. expasy. ch/cgi- bin/protscale. pl) through the ExPasy molecular biology server.

Figure 14B. Hydrophilicity amino acid profile ofCaTrF2El 1 determined by computer algorithm sequence analysis using the method of Hopp and Woods (Hopp T. P., Woods K. R., 1981.

Proc. Natl. Acad. Sci. U. S. A. 78: 3824-3828) accessed on the Protscale website (www. expasy. ch/cgi- bin/protscale. pl) through the ExPasy molecular biology server.

Figure 15A. Hydropathicity amino acid profile of 83P2H3 determined by computer algorithm sequence analysis using the method of Kyte and Doolittle (Kyte J., Doolittle R. F., 1982. J.

Mol. Biol. 157: 105-132) accessed on the ProtScale website (www. expasy. ch/cgi-bin/protscale. pl) through the ExPasy molecular biology server.

Figure 15B. Hydropathicity amino acid profile of CaTrF2E 11 determined by computer algorithm sequence analysis using the method of Kyte and Doolittle (Kyte J., Doolittle R. F., 1982. J.

Mol. Biol. 157: 105-132) accessed on the ProtScale website (www. expasy. ch/cgi-bin/protscale. pl) through the ExPasy molecular biology server.

Figure 16A. Percent accessible residues amino acid profile of 83P2H3 determined by computer algorithm sequence analysis using the method of Janin (Janin J., 1979 Nature 277: 491-492) accessed on the ProtScale website (www. expasy. ch/cgi-bin/protscale. pl) through the ExPasy molecular biology server.

Figure 16B. Percent accessible residues amino acid profile ofCaTrF2El 1 determined by computer algorithm sequence analysis using the method of Janin (Janin J., 1979 Nature 277: 491-492) accessed on the ProtScale website (www. expasy. ch/cgi-bin/protscale. pl) through the ExPasy molecular biology server.

Figure 17A. Average flexibility amino acid profile of 83P2H3 determined by computer algorithm sequence analysis using the method of Bhaskaran and Ponnuswamy (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32: 242-255) accessed on the ProtScale website (www. expasy. ch/cgi-bin/protscale. pl) through the ExPasy molecular biology server.

Figure 17B. Average flexibility amino acid profile of CaTrF2El 1 determined by computer algorithm sequence analysis using the method of Bhaskaran and Ponnuswamy (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res. 32: 242-255) accessed on the ProtScale website (www. expasy. ch/cgi-bin/protscale. pl) through the ExPasy molecular biology server.

Figure 18A. Beta-turn amino acid profile of 83P2H3 determined by computer algorithm sequence analysis using the method of Deleage and Roux (Deleage, G., Roux B. 1987 Protein Engineering 1: 289-294) accessed on the ProtScale website (www. expasy. ch/cgi-bin/protscale. pl) through the ExPasy molecular biology server.

Figure 18B. Beta-turn amino acid profile of CaTrF2El 1 determined by computer algorithm sequence analysis using the method of Deleage and Roux (Deleage, G., Roux B. 1987 Protein Engineering 1: 289-294) accessed on the ProtScale website (www. expasy. ch/cgi-bin/protscale. pl) through the ExPasy molecular biology server.

Figure 19A-F. Plasma membrane staining of 83P2H3 by C-terminal-directed antibodies.

Panels A-C: Rabbit and mouse polyclonal antibodies specific for C-terminal amino acids 615-725 of 83P2H3 protein and an anti-HIS tag polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) were used to stain 293T cells transfected with either empty vector, or with a pCDNA 3.1 83P2H3 expression vector that contains a terminal HIS tag. Staining was detected by incubation with species specific FITC-conjugated secondary antibodies and analysis on a Coulter Epics XL flow cytometer. The respective fluorescent profiles of the two populations are indicated with arrows.

Panels D-E: 293T-83P2H3 HIS tagged cells were stained with anti-HIS polyclonal antibody and FITC-conjugated secondary antibody and examined by bright field and fluorescent microscopy. A representative stained cell is shown. Panel F: Immunohistochemical analysis of 83P2H3 expression in 293T cells. Parrain embedded 293T-83P2H3 cells were sectioned, mounted and stained with anti- 83P2H3 rabbit polyclonal antibody (5 llg/ml). Staining was visualized by incubation with biotinylated anti-rabbit IgG secondary antibody, followed by avidin-conjugated HRP then developed with diaminobenzidine substrate. Arrows mark areas indicative of plasma membrane staining.

Figure 20A-F. Recognition of 83P2H3 in 293T cells by anti-83P2H3 mouse polyclonal antibodies and hybridoma supernatants. Panels A-C: 293T cells transfected with either empty vector or with a pCDNA 3.1 83P2H3 expression vector that contains a carboxyl-terminal HIS tag. Cells were stained with a mouse polyclonal antibody from mice immunized with a GST-83P2H3 cleavage product that encodes amino acids 615-725 (20A) and with supernatants of two hybridomas (#4 (20B) and #8A (20C)) that were generated by fusion of myeloma cells with spleen cells of similarly immunized mice.

Staining was detected by incubation with anti-mouse FITC-conjugated secondary antibody and analysis on a Coulter Epics XL flow cytometer. The respective fluorescent profiles of the two populations are indicated with arrows. Panels D-F: The mouse polyclonal antibody (20D) and anti-83P2H3 hybridoma supernatants (20E-F) were also analyzed by Western blotting on 83P2H3 and vector transfected 293T cells. Cell lysates were separated by SDS-PAGE, transferred to nitrocellulose, blocked, and incubated with a 1: 200 dilution of the mouse polyclonal antibody and hybridoma supernatants. Anti-83P2H3 immunoreactive bands were detected by incubation with anti-mouse IgG HRP-conjugated secondary antibody and visualized by enhanced chemiluminescence and exposure to autoradiography film. Indicated with an arrow is a band representing full length 83P2H3 and with brackets aggregates and degradation products 83P2H3.

Figure 21A-B. Expression of hCaT in prostate cancer cells and fibroblasts induces the phosphorylation of ERK MAPK in these cell lines. Several mitogenic stimuli associated with cell growth and proliferation, have been shown to induce ERK activation (Price DT et al. J Urol. 1999, 162: 1537-42.). Control and 83P2H3/hCaT-expressing PC3 (Figure 21A) and NIH 3T3 (Figure 21B) cell lines were compared for their ability to induce ERK phosphorylation. Cells were grown in low (0.1-0.5%) concentrations of FBS and either left untreated or stimulated with 10% FBS for 5 min.

Whole cell lysates were separated by SDS-PAGE and analyzed by Western blotting using an anti- phospho-ERK monoclonal antibody (New England Nuclear, Bedford, Ma). Anti-ERK overlays were used to evaluate protein loading. The data showed that expression of hCaT alone is sufficient to induce ERK phosphorylation in PC3 and NIH 3T3 cells. ERK phosphorylation was further enhanced by FBS.

These results indicate that hCaT mediates ERK activation and mitogenic signaling in PC3 and NIH 3T3 cells.

Figure 22. Mediation of ERK phosphorylation by hCaT via a variety of ion channel activators. Control and 83P2H3/hCaT-expressing PC3 cell were compared for their ability to induce ERK phosphorylation in response to ion channel activators known to regulate intracellular calcium levels in several cell types. PC3 cells, stably transduced with pSR alpha neo or 83P2H3/hCaT were grown in 0.1% FBS and treated with 10% FBS, cAMP, forskolin, PMA, ionomycin or LPA for 5 min.

Whole cell lysates were separated by SDS-PAGE and analyzed by Western blotting using an anti- phospho-ERK monoclonal antibody (New England Nuclear, Bedford, MA). Anti-ERK overlays were used to evaluate protein loading. Treatment with each of 10% FBS, cAMP, ionomycin, PMA and LPA induced ERK phosphorylation in hCaT-expressing PC3 cells. In contrast, only PMA induced ERK phosphorylation in PC3-neo cells.

Figure 23. Alteration of tyrosine phosphorylation by hCaT in NIH 3T3 cells. Control and 83P2H3/hCaT-expressing NIH 3T3 cell lines were compared for their ability to alter the phosphorylation state of tyrosine-phosphorylated proteins. Cells were grown in 0.1% FBS and either left untreated or stimulated with 10% FBS for 5 min. Whole cell lysates were separated by SDS-PAGE and analyzed by Western blotting using an anti-phosphotyrosine monoclonal antibody. The data

showed that expression of hCaT alone is sufficient to induce phosphorylation of pl 80 and pl32 in NIH 3T3 cells. In addition, expression of hCaT induced the loss of tyrosine phosphorylation of p75-p82 in the same cell type. These results indicate that hCaT regulates the tyrosine phosphorylation state of several proteins in NIH 3T3 cells, and thereby controls downstream signaling pathways that may be critical for tumor growth and survival.

Figure 24. Expression of hCaT induces the proliferation of NIH 3T3 cells. Due to the importance of calcium transporters in cell growth, we investigated the effect of 83P2H3/hCaT on proliferation. Control and 83P2H3/hCaT-expressing NIH 3T3 cell lines were grown in 0.1% FBS and either left untreated or stimulated with 10% FBS for 24 hours. Proliferation was measured in triplicate.

NIH 3T3 cells expressing constitutively active Ras were used as a positive control. The data show that expression of hCaT induced a 3-fold increase in the proliferation of NIH 3T3 grown in the presence of FBS. This increase in cell growth was comparable to the effect of the strong oncogene Ras.

Figure 25A-C. Induction of calcium flux in prostate cancer cells by hCaT. The prostate cancer cell line PC3 was transduced with pSRalpha retrovirus carrying either the neo cassette alone or 83P2H3/hCaT. Stable PC3-neo and PC3-hCaT cells were examined for their ability to respond to extracellular stimuli by inducing calcium flux. PC3 cells were loaded with two calcium indicators, namely fura red and fluo4 (Molecular Probes, Eugene, Or) and analyzed by flow cytometry in the absence and presence of exogenous calcium. The data indicated that, while PC3-neo showed little responsiveness to calcium, exogenous calcium induced a calcium flux in PC3-CaT cells. Similar results were obtained in two separate experiments. These data indicates that 83P2H3/hCaT functions as a calcium transporter in PC3 cells.

Figure 26. Expression of hCaT induces the phosphorylation of calmodulin kinase. The transport of ions across membranes is regulated by calmodulin and calmodulin kinases (CaMK). Since the phosphorylation of CamK reflects its activation, the effect of hCaT on the phosphorylation of CaMK was investigated. Control and 83P2H3-expressing PC3 cell lines were compared for their ability to alter the phosphorylation state of CaMKII. Cells were grown in 0.1% FBS and either left untreated or stimulated with 10% FBS, ionomycin or calcium. Whole cell lysates were separated by SDS-PAGE and analyzed by Western blotting using an anti-phospho-CaMKII antibody. The results indicate that expression of hCaT was sufficient to enhance the phosphorylation and activation of CaMKII in PC3 cells.

Figure 27A-F. Cell surface expression of hCaT by C-terminal-specific antibodies. 293T cells were transfected with an expression vector encoding 83P2H3 HIS-tagged (PCDNA 3.1 MYC/HIS, Invitrogen), and the cell surface localization was determined by immunofluorescence.

Figure 27A shows detection of 293T cells carrying empty vector or hCaT using a GST-fusion polyclonal antibody. Figure 27B shows detection of 293T cells carrying empty vector or hCaT using an antibody directed against His to identify the C-terminus. Figure 27C-D show a PC3-CaT cell detected by immunofluorescence using a GST-fusion polyclonal antibody, or phase contrast

microscopy, respectively. Figure 27E-F show a 293T cell detected by phase contrast microscopy, or immunofluorescence using an antibody directed against His to identify the C-terminus, respectively.

Figure 28. Expression of CaTrF2E11 in human patient cancer specimens. RNA was extracted from a pool of 3 bladder cancer tumors, kidney cancer tumors and lung cancer tumors derived from cancer patients, and from normal prostate (NP), bladder (NB), kidney (NK) and colon (NC).

Northern blots with 10 jig of total RNA/lane were probed with the CaTrF2E11 fragment. Size standards in kilobases (kb) are indicated on the side. The results show expression of CaTrF2E11 in bladder cancer pool, kidney cancer pool, lung cancer pool, but not in the normal tissues. Bladder Cancer Pool = grade 2,3; Kidney Cancer Pool = grade 2,2,3; Lung Cancer Pool = Sq. IA, Sq. IIIA, LCC; NP = Normal Prostate; NB = Normal Bladder; NK = Normal Kidney; NC = Normal Colon.

Figure 29. Expression of CaTrF2E11 in bladder cancer patient specimens. RNA was extracted from the bladder cancer cell line SCaBER (CL), normal bladder (Nb), bladder tumors (T) and their matched normal adjacent tissue (N) isolated from bladder cancer patients. Northern blots with 10 pg of total RNA/lane were probed with the CaTrF2E11 fragment. Size standards in kilobases (kb) are indicated on the side. The results show expression of CaTrF2E11 in the bladder cancer cell line, and in the bladder cancer tissues. The expression detected in normal adjacent tissue (isolated from diseased tissues) but not in normal tissue, isolated from healthy donors, may indicate that this tissue is not fully normal and that CaTrF2E11 may be expressed in early stage tumors. P1-Transitional, grade 2; P2- Transitional, grade 2; P3-Transitional, grade 2; P4-Transitional; CL = Bladder cancer cell line SCABER; P = Patient; Nb = Normal Bladder; N = Normal adjacent tissue; T = Tumor.

Figure 30. Expression of CaTrF2E11 in kidney cancer patient specimens. RNA was extracted from kidney cancer cell lines (CL), kidney tumors (T) and their matched normal adjacent tissue (N) isolated from kidney cancer patients. Northern blots with 10 llg of total RNA/lane were probed with the CaTrF2E11 fragment. Size standards in kilobases (kb) are indicated on the side. The results show expression of CaTrF2E11 in 2 of 3 kidney cancer cell lines, and in both normal and kidney tumor tissues. CL = cell lines listed in order: 769-P, A498, Caki-1; NAT = Normal adjacent tissue; T = Tumor Pt. 1, Papillary carcinoma, grade 2; Pt. 2, Clear cell type, grade 2; Pt. 3, Clear cell type, grade 2; Pt. 4, Clear cell type, grade 2; Pt. 5, Clear cell type, grade 3; Pt. 6, Clear cell type, grade 3.

Figure 31A-C. Overexpression of 83P2H3 in an engineered cell line. PC3 human prostate cancer cells were engineered to overexpress 83P2H3 by retroviral transduction of the 83P2H3 cDNA.

Panel A: Northern blot analysis of 83P2H3 expression in PC3 or PC3-83P2H3 stably transduced cells.

Arrow indicates the retroviral transcript encoding the 83P2H3 cDNA. Panel B: Immunofluorescent analysis of 83P2H3 expression in PC3-83P2H3 cells using a rabbit polyclonal antibody directed to amino acids 615-725. Anti-83P2H3 staining of cells was detected following incubation with an FITC- conjugated anti-rabbit IgG secondary antibody. A representative stained cell is shown. Panel C: Phase contrast image of the cell depicted in Panel B.

DETAILED DESCRIPTION OF THE INVENTION Outline of Sections I.) Definitions II.) 83P2H3 Polynucleotides II. A.) Uses of 83P2H3 Polynucleotides II. A. 1.) Monitoring of Genetic Abnormalities ILA. 2.) Antisense Embodiments II. A. 3.) Primers and Primer Pairs II. A. 4.) Isolation of 83P2H3-Encoding Nucleic Acid Molecules lI. A. 5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems III.) 83P2H3-related Proteins III. A.) Motif-bearing Protein Embodiments III. B.) Expression of 83P2H3-related p III. C.) Modifications of 83P2H3-related Proteins III. D.) Uses of 83P2H3-related Proteins IV.) 83P2H3 Antibodies V.) 83P2H3 Cellular Immune Responses VI.) 83P2H3 Transgenic Animals VII.) Methods for the Detection of 83P2H3 VHI.) Methods for Monitoring the Status of 83P2H3-related Genes and Their Products IX.) Identification of Molecules That Interact With 83P2H3 X.) Therapeutic Methods and Compositions X. A.) Anti-Cancer Vaccines X. B.) 83P2H3 as a Target for Antibody-Based Therapy X. C.) 83P2H3 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 83P2H3.

XII.) Inhibition of 83P2H3 Protein Function XII. A.) Inhibition of 83P2H3 With Intracellular Antibodies XII. B.) Inhibition of 83P2H3 with Recombinant Proteins XII. C.) Inhibition of 83P2H3 Transcription or Translation XII. D.) General Considerations for Therapeutic Strategies XE.) KITS T.) 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.

As used herein"83P2H3"and"PCaT"are synonyms. Moreover any reference to"83P2H3"or "PCaT also refer to the family member CaTrF2E11, unless the context clearly indicates otherwise to one of ordinary skill in the art.

The terms"advanced prostate cancer","locally advanced prostate cancer","advanced disease" and"locally advanced disease"mean prostate cancers that have extended through the prostate capsule, and are meant to include stage C disease under the American Urological Association (AUA) system, stage C1-C2 disease under the Whitmore-Jewett system, and stage T3-T4 and N+ disease under the TNM (tumor, node, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes compared to patients having clinically localized (organ-confined) prostate cancer. Locally advanced disease is clinically identified by palpable evidence of induration beyond the lateral border of the prostate, or

asymmetry or induration above the prostate base. Locally advanced prostate cancer is presently diagnosed pathologically following radical prostatectomy if the tumor invades or penetrates the prostatic capsule, extends into the surgical margin, or invades the seminal vesicles.

"Altering the native glycosylation pattern"is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence 83P2H3 (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 83P2H3. 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 83P2H3-related protein). For example an. analog of the 83P2H3 protein can be specifically bound by an antibody or T cell that specifically binds to 83P2H3.

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-83P2H3 antibodies comprise monoclonal and polyclonal antibodies as well as fragments containing the antigen-binding domain and/or one or more complementarity determining regions of these antibodies.

An"antibody fragment"is defined as at least a portion of the variable region of the immunoglobulin molecule that binds to its target, i. e., the antigen-binding region. In one embodiment it specifically covers single anti-83P2H3 antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-83P2H3 antibody compositions with polyepitopic specificity.

The term"codon optimized sequences"refers to nucleotide sequences that have been optimized for a particular host species by replacing any codons having a usage frequency of less than about 20%. Nucleotide sequences that have been optimized for expression in a given host species by elimination of spurious polyadenylation sequences, elimination of exon/intron splicing signals, elimination of transposon-like repeats and/or optimization of GC content in addition to codon optimization are referred to herein as an"expression enhanced sequences." The term"cytotoxic agent"refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Examples of cytotoxic agents include, but are not limited to maytansinoids, yttrium, bismuth, ricin, ricin A-chain, doxorubicin, daunorubicin, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, sapaonaria officinalis inhibitor, and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes such as At", I"', 1', Y°,

Rel86, Rel$$, Sml53, 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, 8T"ED., Lange Publishing, Los Altos, CA (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/6XSSC/0.1% SDS/100 ug/ml ssDNA, in which temperatures for hybridization are above 37 degrees C and temperatures for washing in 0. 1XSSC/0. 1% SDS are above 55 degrees C.

The phrases"isolated"or"biologically pure"refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides in accordance with the invention preferably do not contain materials normally associated with the peptides in their in situ environment. For example, a polynucleotide is said to be"isolated"when it is substantially separated from contaminant polynucleotides that correspond or are complementary to genes other than the 83P2H3 gene or that encode polypeptides other than 83P2H3 gene product or fragments thereof. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated 83P2H3 polynucleotide. A protein is said to be"isolated,"for example, when physical, mechanical or chemical methods are employed to remove the 83P2H3 protein from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated 83P2H3 protein. Alternatively, an isolated protein can be prepared by chemical means.

The term"mammal"refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human.

The terms"metastatic prostate cancer"and"metastatic disease"mean prostate cancers that have spread to regional lymph nodes or to distant sites, and are meant to include stage D disease under the AUA system and stage TxNxM+ under the TNM system. As is the case with locally advanced prostate cancer, surgery is generally not indicated for patients with metastatic disease, and hormonal (androgen ablation) therapy is a preferred treatment modality. Patients with metastatic prostate cancer eventually develop an androgen-refractory state within 12 to 18 months of treatment initiation.

Approximately half of these androgen-refractory patients die within 6 months after developing that status.. The most common site for prostate cancer metastasis is bone. Prostate cancer bone metastases are often osteoblastic rather than osteolytic (i. e., resulting in net bone formation). Bone metastases are

found most frequently in the spine, followed by the femur, pelvis, rib cage, skull and humerus. Other common sites for metastasis include lymph nodes, lung, liver and brain. Metastatic prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole body radionuclide scans, skeletal radiography, and/or bone lesion biopsy.

The term"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 83P2H3-related protein, refers to any pattern of amino acids forming part of the primary sequence of a protein, that is associated with a particular function (e. g. protein-protein interaction, protein-DNA interaction, etc) or modification (e. g. that is phosphorylated, glycosylated or amidated), or localization (e. g. secretory sequence, nuclear localization sequence, etc.) or a sequence that is correlated with being immunogenic, either humorally or cellularly.

A motif can be either contiguous or capable of being aligned to certain positions that are generally correlated with a certain function or property. In the context of HLA motifs,"motif'refers to the pattern of residues in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino acids for a class II HLA motif, which is recognized by a particular HLA molecule. Peptide motifs for HLA binding are typically different for each protein encoded by each human HLA allele and differ in the pattern of the primary and secondary anchor residues.

A"pharmaceutical excipient"comprises a material such as an adjuvant, a carrier, pH- adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservative, and the like.

"Pharmaceutically acceptable"refers to a non-toxic, inert, and/or composition that is physiologically compatible with humans or other mammals.

The term"polynucleotide"means a polymeric form of nucleotides of at least 10 bases or base pairs in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide, and is meant to include single and double stranded forms of DNA and/or RNA. In the art, this term if often used interchangeably with"oligonucleotide". A polynucleotide can comprise a nucleotide sequence disclosed herein wherein thymidine (T) (as shown for example in SEQ ID NO: 702) 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".

The term"prevent"or"protect against"a condition or disease means to hinder, reduce or delay the onset or progression of the condition or disease.

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 x SSC (0.75 M NaC1, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1 % sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 llg/rnl), 0.1% SDS, and 10% dextran sulfate at 42 °C, with washes at 42°C in 0.2 x SSC (sodium chloride/sodium. citrate)

and 50% formamide at 55 °C, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 55 °C."Moderately stringent conditions"are described by, but not limited to, those in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e. g., temperature, ionic strength and % SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCI, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.

An HLA"supermotif'is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles.

A"transgenic animal" (e. g., a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e. g., an embryonic stage. A"transgene"is a DNA that is integrated into the genome of a cell from which a transgenic animal develops.

As used herein, an HLA or cellular immune response"vaccine"is a composition that contains or encodes one or more peptides of the invention. There are numerous embodiments of such vaccines, such as a cocktail of one or more individual peptides ; one or more peptides of the invention comprised by a polyepitopic peptide; or nucleic acids that encode such individual peptides or polypeptides, e. g., a minigene that encodes a polyepitopic peptide. The"one or more peptides"can include any whole unit integer from 1-150 or more, e. g., at least 2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20, 21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40, 41,42,43,44,45,46,47, 48,49,50,55,60,65,70,75,80,85,90,95,100,105,110,115,120,125, 130,135,140,145, or 150 or more peptides of the invention. The peptides or polypeptides can optionally be modified, such as by lipidation, addition of targeting or other sequences. HLA class I peptides of the invention can be admixed with, or linked to, HLA class II peptides, to facilitate activation of both cytotoxic T lymphocytes and helper T lymphocytes. HLA vaccines can also comprise peptide-pulsed antigen presenting cells, e. g., dendritic cells.

The term"variant"refers to a molecule that exhibits a variation from a described type or norm, such as a protein that has one or more different amino acid residues in the corresponding position (s) of a specifically described protein (e. g. the 83P2H3 protein shown in Figure 2 or Figure 3). An analog is an example of a variant protein.

The 83P2H3-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. Unless the context clearly indicates otherwise,"83P2H3"also refers to family members, such as the CaTrF2E11 identified herein, and any of the alternative splice variants disclosed herein. Fusion proteins that combine parts of different 83P2H3 proteins or fragments thereof, as well as fusion proteins of a 83P2H3 protein and a heterologous polypeptide are also included. Such 83P2H3 proteins are collectively referred to as the 83P2H3-related proteins, the proteins of the invention, or 83P2H3. The term"83P2H3-related protein"refers to a polypeptide fragment or an 83P2H3 protein sequence of 4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25, or more than 25 amino acids; or, at least 30,35,40,45,50,55,60,65,70,80,85,90,95,100 or more than 100 amino acids.

II.) 83P2H3 Polvnucleotides One aspect of the invention provides polynucleotides corresponding or complementary to all or part of an 83P2H3 gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding an 83P2H3-related protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to an 83P2H3 gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides that hybridize to an 83P2H3 gene, mRNA, or to an 83P2H3 encoding polynucleotide (collectively,"83P2H3 polynucleotides"). In all instances when referred to in this section, T can also be U in Figure 2.

Embodiments of a 83P2H3 polynucleotide include: a 83P2H3 polynucleotide having the sequence shown in Figure 2, the nucleotide sequence of 83P2H3 as shown in Figure 2, wherein T is U; at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in Figure 2; or, at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in Figure 2 where T is U. For example, embodiments of 83P2H3 nucleotides comprise, without limitation: (a) a polynucleotide comprising or consisting of the sequence as shown in Figure 2 (SEQ ID NO.: 702), wherein T can also be U; (b) a polynucleotide comprising or consisting of the sequence as shown in Figure 2 (SEQ ID NO.: 702), from nucleotide residue number 201 through nucleotide residue number 2378, wherein T can also be U; (c) a polynucleotide that encodes a 83P2H3-related protein whose sequence is encoded by the cDNAs contained in the plasmid designated p83P2H3-C deposited with American Type Culture Collection as Accession No. PTA-1893 ; (d) a polynucleotide that encodes an 83P2H3-related protein that is at least 90% homologous to the entire amino acid sequence shown in SEQ ID NO.: 702;

(e) a polynucleotide that encodes an 83P2H3-related protein that is at least 90% identical to the entire amino acid sequence shown in SEQ ID NO: 702; (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 Figure 3 in any whole number increment up to 725 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of Figure 14; (h) a polynucleotide that encodes a peptide region of at least 5 amino acids of Figure 3 in any whole number increment up to 725 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of Figure 15; (i) a polynucleotide that encodes a peptide region of at least 5 amino acids of Figure 3 in any whole number increment up to 725 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 16; (j) a polynucleotide that encodes a peptide region of at least 5 amino acids of Figure 3 in any whole number increment up to 725 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile on Figure 17; (k) a polynucleotide that encodes a peptide region of at least 5 amino acids of Figure 3 in any whole number increment up to 725 that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of Figure 18; (1) a polynucleotide that is fully complementary to a polynucleotide of any one of (a)- (m) a polynucleotide that selectively hybridizes under stringent conditions to a polynucleotide of (a)- (l) ; and (n) a peptide that is encoded by any of (a)- (k).

(o) a polynucleotide of any of (a)- (m) or peptide of (o) 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 83P2H3 polynucleotides that encode specific portions of the 83P2H3 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 or 725 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 83P2H3 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the 83P2H3 protein shown in Figure 2, or Figure 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the 83P2H3 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 30 to about amino acid 40 of the 83P2H3 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 40 to about amino acid 50 of the 83P2H3 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 50 to about amino acid 60 of the 83P2H3 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 60 to about amino acid 70 of the 83P2H3 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 70 to about amino acid 80 of the 83P2H3 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 80 to about amino acid 90 of the 83P2H3 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 90 to about amino acid 100 of the 83P2H3 protein shown in Figure 2 or Figure 3, in increments of about 10 amino acids, ending at the carboxyl terminal amino acid set forth in Figure 2 or Figure 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 83P2H3 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 83P2H3 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 83P2H3 protein shown in Figure 2 or Figure 3 can be generated by a variety of techniques well known in the art. These polynucleotide fragments can include any portion of the 83P2H3 sequence as shown in Figure 2 or Figure 3.

Additional illustrative embodiments of the invention disclosed herein include 83P2H3 polynucleotide fragments encoding one or more of the biological motifs contained within the 83P2H3 protein sequence, including one or more of the motif-bearing subsequences of the 83P2H3 protein set forth in Tables V-XVIII. In another embodiment, typical polynucleotide fragments of the invention encode one or more of the regions of 83P2H3 that exhibit homology to a known molecule. In another embodiment of the invention, typical polynucleotide fragments can encode one or more of the 83P2H3 N-glycosylation sites, cAMP and cGMP-dependent protein kinase phosphorylation sites, casein kinase II phosphorylation sites or N-myristoylation site and amidation sites.

With respect to 83P2H3 family members, such as CaTrF2E11 described in Figure 1, Figure 2 and Figure 3, polynucleotides encoding all or a portion of the protein are within the scope of the invention. In some embodiments, the fragment or variant of the CaTrF2E11 protein having the amino

acid sequence set forth in Figure 3 comprises the portion of CaTrF2E11 described in Figure 1B or one or more of the motifs or domains of CaTrF2E11 described in Table XIX (B) or Table XX.

II. A.) Uses of 83P2H3 Polvnucleotides II. A. 1.) Monitoring of Genetic Abnormalities The polynucleotides of the preceding paragraphs have a number of different specific uses.

The human 83P2H3 gene maps to the chromosomal location set forth in Example 3. For example, because the 83P2H3 gene maps to this chromosome, polynucleotides that encode different regions of the 83P2H3 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 (3-4): 81-83 (1998); Johansson et al., Blood 86 (10): 3905-3914 (1995) and Finger et al., P. N. A. S. 85 (23): 9158- 9162 (1988)). Thus, polynucleotides encoding specific regions of the 83P2H3 protein provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the chromosomal region that encodes 83P2H3 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 83P2H3 was shown to be highly expressed in prostate and other cancers, 83P2H3 polynucleotides are used in methods assessing the status of 83P2H3 gene products in normal versus cancerous tissues. Typically, polynucleotides that encode specific regions of the 83P2H3 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 83P2H3 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 83P2H3. 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 83P2H3 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., 83P2H3. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1: 1-5 (1988). The 83P2H3 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 83P2H3 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 83P2H3 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 83P2H3 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 83P2H3 mRNA and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, 83P2H3 antisense oligonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to 83P2H3 mRNA. Optionally, 83P2H3 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 83P2H3. Alternatively, the antisense molecules are modified to employ ribozymes in the inhibition of 83P2H3 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. Probes can be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme. Such probes and primers are used to detect the presence of a 83P2H3 polynucleotide in a sample and as a means for detecting a cell expressing a 83P2H3 protein.

Examples of such probes include polypeptides comprising all or part of the human 83P2H3 cDNA sequence shown in Figure 2. Examples of primer pairs capable of specifically amplifying 83P2H3 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 83P2H3 mRNA.

The 83P2H3 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 83P2H3 gene (s), mRNA (s), or fragments thereof; as reagents for the diagnosis and/or prognosis of prostate cancer and other cancers; as coding sequences capable of directing the expression of 83P2H3 polypeptides ; as tools for modulating or inhibiting the expression of the 83P2H3 gene (s) and/or translation of the 83P2H3 transcript (s); and as therapeutic agents.

II. A. 4.) Isolation of 83P2H3-Encoding Nucleic Acid Molecules The 83P2H3 cDNA sequences described herein enable the isolation of other polynucleotides encoding 83P2H3 gene product (s), as well as the isolation of polynucleotides encoding 83P2H3 gene product homologs, alternatively spliced isoforms, allelic variants, and mutant forms of the 83P2H3 gene product as well as polynucleotides that encode analogs of 83P2H3-related proteins. Various molecular cloning methods that can be employed to isolate full length cDNAs encoding an 83P2H3 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 83P2H3 gene cDNAs can be identified by probing with a labeled 83P2H3 cDNA or a fragment thereof. For example, in one embodiment, the 83P2H3 cDNA (Figure 2) or a portion thereof can be synthesized and used as a probe to retrieve overlapping and full-length cDNAs corresponding to a 83P2H3 gene. The 83P2H3 gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with 83P2H3 DNA probes or primers.

II. A. 5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems The invention also provides recombinant DNA or RNA molecules containing an 83P2H3 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 83P2H3 polynucleotide, fragment, analog or homologue thereof within a suitable prokaryotic or eukaryotic host cell. Examples of suitable eukaryotic host cells include a yeast cell, a plant cell, or an animal cell, such as a mammalian cell or an insect cell (e. g., a baculovirus-infectible cell such as an Sf9 or HighFive cell). Examples of suitable mammalian cells include various prostate cancer cell lines such as DU145 and TsuPrl, other transfectable or transducible prostate cancer cell lines, primary cells (PrEC), as well as a number of mammalian cells routinely used for the expression

of recombinant proteins (e. g., COS, CHO, 293,293T cells). More particularly, a polynucleotide comprising the coding sequence of 83P2H3 or a fragment, analog or homolog thereof can be used to generate 83P2H3 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 83P2H3 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 pSRatkneo (Muller et al., 1991, MCB 11: 1785). Using these expression vectors, 83P2H3 can be expressed in several prostate cancer and non-prostate cell lines, including for example 293,293T, rat-1, NIH 3T3 and TsuPrl. The host-vector systems of the invention are useful for the production of a 83P2H3 protein or fragment thereof. Such host-vector systems can be employed to study the functional properties of 83P2H3 and 83P2H3 mutations or analogs.

Recombinant human 83P2H3 protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a 83P2H3-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding 83P2H3 or fragment, analog or homolog thereof, the 83P2H3 or related protein is expressed in the 293T cells, and the recombinant 83P2H3 protein is isolated using standard purification methods (e. g., affinity purification using anti-83P2H3 antibodies). In another embodiment, a 83P2H3 coding sequence is subcloned into the retroviral vector pSRaMSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPrl, 293 and rat-1 in order to establish 83P2H3 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 83P2H3 coding sequence can be used for the generation of a secreted form of recombinant 83P2H3 protein.

As discussed herein, redundancy in the genetic code permits variation in 83P2H3 gene sequences. In particular, it is known in the art that specific host species often have specific codon preferences, and thus one can adapt the disclosed sequence as preferred for a desired host. For example, preferred analog codon sequences typically have rare codons (i. e., codons having a usage frequency of less than about 20% in known sequences of the desired host) replaced with higher frequency codons. Codon preferences for a specific species are calculated, for example, by utilizing codon usage tables available on the INTERNET such as at URL www. dna. affrc. go. jp/~nakamuralcodon ! htnll.

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) andKozakNAR 15 (20): 8125-8148 (1987)).

III. 83P2H3-related Proteins Another aspect of the present invention provides 83P2H3-related proteins. Specific embodiments of 83P2H3 proteins comprise a polypeptide having all or part of the amino acid sequence of human 83P2H3 as shown in Figure 2 or Figure 3. Alternatively, embodiments of 83P2H3 proteins comprise variant, homolog or analog polypeptides that have alterations in the amino acid sequence of 83P2H3 shown in Figure 2 or Figure 3.

In general, naturally occurring allelic variants of human 83P2H3 share a high degree of structural identity and homology (e. g., 90% or more homology). Typically, allelic variants of the 83P2H3 protein contain conservative amino acid substitutions within the 83P2H3 sequences described herein or contain a substitution of an amino acid from a corresponding position in a homologue of 83P2H3. One class of 83P2H3 allelic variants are proteins that share a high degree of homology with at least a small region of a particular 83P2H3 amino acid sequence, but further contain a radical departure from the sequence, such as a non-conservative substitution, truncation, insertion or frame shift. In comparisons of protein sequences, the terms, similarity, identity, and homology each have a distinct meaning as appreciated in the field of genetics. Moreover, orthology and paralogy can be important concepts describing the relationship of members of a given protein family in one organism to the members of the same family in other organisms.

Amino acid abbreviations are provided in Table II. Conservative amino acid substitutions can frequently be made in a protein without altering either the conformation or the function of the protein.

Proteins of the invention can comprise 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15 conservative substitutions. Such changes include substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa. Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered"conservative"in

particular environments (see, e. g. Table III herein; pages 13-15"Biochemistry"2nd ED. Lubert Stryer ed (Stanford University); Henikoff et al., PNAS 1992 Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19; 270 (20): 11882-6).

Embodiments of the invention disclosed herein include a wide variety of art-accepted variants or analogs of 83P2H3 proteins such as polypeptides having amino acid insertions, deletions and substitutions. 83P2H3 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 83P2H3 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, 83P2H3 variants, analogs or homologs, have the distinguishing attribute of having at least one epitope that is"cross reactive"with a 83P2H3 protein having the amino acid sequence of SEQ ID NO: 703. As used in this sentence,"cross reactive"means that an antibody or T cell that specifically binds to an 83P2H3 variant also specifically binds to the 83P2H3 protein having the amino acid sequence of SEQ ID NO: 703. A polypeptide ceases to be a variant of the protein shown in SEQ ID NO: 703 when it no longer contains any epitope capable of being recognized by an antibody or T cell that specifically binds to the 83P2H3 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 83P2H3-related protein variants share 70%, 75%, 80%, 85% or 90% or more similarity with the amino acid sequence of SEQ ID NO: 703 or a fragment thereof. Another specific class of 83P2H3 protein variants or analogs comprise one or more of the 83P2H3 biological motifs described herein or presently known in the art. Thus, encompassed by the present invention are analogs of 83P2H3 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 Figure 2 or Figure 3.

As discussed herein, embodiments of the claimed invention include polypeptides containing less than the full amino acid sequence of the 83P2H3 protein shown in Figure 2 or Figure 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 83P2H3 protein shown in Figure 2 or Figure 3.

Moreover, representative embodiments of the invention disclosed herein include polypeptides consisting of about amino acid 1 to about amino acid 10 of the 83P2H3 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 10 to about amino acid 20 of the 83P2H3 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 20 to about amino acid 30 of the 83P2H3 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 30 to about amino acid 40 of the 83P2H3 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 40 to about amino acid 50 of the 83P2H3 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 50 to about amino acid 60 of the 83P2H3 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 60 to about amino acid 70 of the 83P2H3 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 70 to about amino acid 80 of the 83P2H3 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 80 to about amino acid 90 of the 83P2H3 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 90 to about amino acid 100 of the 83P2H3 protein shown in Figure 2 or Figure 3, etc. throughout the entirety of the 83P2H3 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 83P2H3 protein shown in Figure 2 or Figure 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.

83P2H3-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 83P2H3-related protein. In one embodiment, nucleic acid molecules provide a means to generate defined fragments of the 83P2H3 protein (or variants, homologs or analogsthereof). ni. A.) Motif-bearing Protein Embodiments Additional illustrative embodiments of the invention disclosed herein include 83P2H3 polypeptides comprising the amino acid residues of one or more of the biological motifs contained within the 83P2H3 polypeptide sequence set forth in Figure 2 or Figure 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/strtic- » redict, html nsort. ims. u-tokyo. ac. in/ ; www. cbs. dh. dk/ ;

www. ebi. ac. uk/interpro/scan. html ; www. expasy. ch/tools/scnpsitl. htrnl ; EpimatrixT and EpimerT"', Brown University, www. brown. edu/Research/TB-HIV Lab/epimatrix/epimatrix. and BEVIAS, bimas. dcrt. nih. gov/.).

Motif bearing subsequences of the 83P2H3 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 83P2H3 motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the 83P2H3 motifs discussed above are associated with growth dysregulation and because 83P2H3 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 83P2H3 protein that are capable of optimally binding to specified HLA alleles (e. g., Table IV ; Epimatrix and Eimer, Brown University, URL www. 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 (3-4): 201-212; Sette et al., J. Immunol. 2001 166 (2): 1389-1397; Sidney et al., Hum Immunol. 1997 58 (1) : 12-20; Kondo et al., Immunogenetics 1997 45 (4): 249-258; Sidney et al., J. Immunol. 1996 157 (8): 3480-90; and Falk et al., Nature 351: 290-6 (1991); Hunt et al., Science 255: 1261-3 (1992); Parker et al., J. Immunol. 149: 3580-7 (1992); Parker et al., J. Immunol. 152: 163-75 (1994)); Kast et al., 1994 152 (8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61 (3): 266-278; Alexander et al., J. Immunol. 2000 164 (3); 164 (3): 1625-1633; Alexander et al., PMID: 7895164, UI : 95202582; O'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.

83P2H3-related proteins are embodied in many forms, preferably in isolated form. A purified 83P2H3 protein molecule will be substantially free of other proteins or molecules that impair the binding of 83P2H3 to antibody, T cell or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a 83P2H3-related proteins include purified 83P2H3-related proteins and functional, soluble 83P2H3-related proteins. In one embodiment, a functional, soluble 83P2H3 protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.

The invention also provides 83P2H3 proteins comprising biologically active fragments of the 83P2H3 amino acid sequence shown in Figure 2 or Figure 3. Such proteins exhibit properties of the 83P2H3 protein, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the 83P2H3 protein; to be bound by such antibodies; to elicit the activation of HTL or CTL; and/or, to be recognized by HTL or CTL.

83P2H3-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-83P2H3 antibodies, or T cells or in identifying cellular factors that bind to 83P2H3.

CTL epitopes can be determined using specific algorithms to identify peptides within an 83P2H3 protein that are capable of optimally binding to specified HLA alleles (e. g., by using the SYFPEITHI site at World Wide Web URL syfpeithi. bmi-heidelberg. com/; the listings in Table IV (A)- (E) ; Epimatrix and Eimer, Brown University, URL (www. brown. edu/Research/TB-HIV Lab/epimatrix/epimatrix. html) ; and BIMAS, URL bimas. dcrt. nih. gov/). Illustrating this, peptide epitopes from 83P2H3 that are presented in the context of human MHC class I molecules HLA-A1, A2, A3, Al 1, A24, B7 and B35 were predicted (Tables V-XVIII). Specifically, the complete amino acid sequence of the 83P2H3 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 83P2H3 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, Epimefrm and EpirnatixTm 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 83P2H3 protein. As used in this context "applied"means that the 83P2H3 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 83P2H3 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 83P2H3-related Proteins In an embodiment described in the examples that follow, 83P2H3 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 83P2H3 with a C-terminal 6XHis and MYC tag (pcDNA3. 1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, Nashville TN). The Tag5 vector provides an IgGK secretion signal that can be used to facilitate the production of a secreted 83P2H3 protein in transfected cells. The secreted HIS-tagged 83P2H3 in the culture media can be purified, e. g., using a nickel column using standard techniques.

III. C.) Modifications of 83P2H3-related Proteins Modifications of 83P2H3-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 83P2H3 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N-or C-terminal residues of the 83P2H3. Another type of covalent modification of the 83P2H3 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 83P2H3 comprises linking the 83P2H3 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. Patent Nos. 4,640,835; 4,496,689; 4,301, 144; 4,670,417; 4,791,192 or 4,179,337.

The 83P2H3-related proteins of the present invention can also be modified to form a chimeric molecule comprising 83P2H3 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 83P2H3 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 Figure 2 or Figure 3. Such a chimeric molecule can comprise multiples of the same subsequence of 83P2H3. A chimeric molecule can comprise a fusion of a 83P2H3-related protein with a polyhistidine epitope tag, which provides 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 83P2H3. In an alternative embodiment, the chimeric molecule can comprise a fusion of a 83P2H3-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 83P2H3 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, CHI, CH2 and CH3 regions of an IgGI molecule. For the production of immunoglobulin fusions see, e. g., U. S. Patent No. 5,428,130 issued June 27,1995.

III. D.) Uses of 83P2H3-related Proteins The proteins of the invention have a number of different specific uses. As 83P2H3 is highly expressed in prostate and other cancers, 83P2H3-related proteins are used in methods that assess the status of 83P2H3 gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of the 83P2H3 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 83P2H3-related proteins comprising the amino acid residues of one or more of the biological motifs contained within the 83P2H3 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, 83P2H3-related proteins that contain the amino acid residues of one or more of the biological motifs in the 83P2H3 protein are used to screen for factors that interact with that region of 83P2H3.

83P2H3 protein fragments/subsequences are particularly useful in generating and characterizing domain-specific antibodies (e. g., antibodies recognizing an extracellular or intracellular epitope of an 83P2H3 protein), for identifying agents or cellular factors that bind to 83P2H3 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 83P2H3 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 83P2H3 gene product. Antibodies raised against an 83P2H3 protein or fragment thereof are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of 83P2H3 protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers. 83P2H3-related nucleic acids or proteins are also used in generating HTL or CTL responses.

Various immunological assays useful for the detection of 83P2H3 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 83P2H3- expressing cells (e. g., in radioscintigraphic imaging methods). 83P2H3 proteins are also particularly useful in generating cancer vaccines, as further described herein.

IV.) 83P2H3 Antibodies

Another aspect of the invention provides antibodies that bind to 83P2H3-related proteins.

Preferred antibodies specifically bind to a 83P2H3-related protein and do not bind (or bind weakly) to peptides or proteins that are not 83P2H3-related proteins. For example, antibodies bind 83P2H3 can bind 83P2H3-related proteins such as the homologs or analogs thereof.

83P2H3 antibodies of the invention are particularly useful in prostate 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 83P2H3 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 83P2H3 is involved, such as advanced or metastatic prostate cancers.

The invention also provides various immunological assays useful for the detection and quantification of 83P2H3 and mutant 83P2H3-related proteins. Such assays can comprise one or more 83P2H3 antibodies capable of recognizing and binding a 83P2H3-related protein, as appropriate. These assays are performed within various immunological assay formats well known in the art, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme- linked immunofluorescent assays (ELIFA), and the like.

Immunological non-antibody assays of the invention also comprise T cell immunogenicity assays (inhibitory or stimulatory) as well as major histocompatibility complex (MHC) binding assays.

In addition, immunological imaging methods capable of detecting prostate cancer and other cancers expressing 83P2H3 are also provided by the invention, including but not limited to radioscintigraphic imaging methods using labeled 83P2H3 antibodies. Such assays are clinically useful in the detection, monitoring, and prognosis of 83P2H3 expressing cancers such as prostate cancer.

83P2H3 antibodies are also used in methods for purifying a 83P2H3-related protein and for isolating 83P2H3 homologues and related molecules. For example, a method of purifying a 83P2H3- related protein comprises incubating an 83P2H3 antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a 83P2H3-related protein under conditions that permit the 83P2H3 antibody to bind to the 83P2H3-related protein; washing the solid matrix to eliminate impurities ; and eluting the 83P2H3-related protein from the coupled antibody. Other uses of the 83P2H3 antibodies of the invention include generating anti-idiotypic antibodies that mimic the 83P2H3 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 83P2H3-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 83P2H3 can also be used, such as a 83P2H3 GST-fusion protein. In a particular embodiment, a GST fusion protein comprising all or most of the amino acid sequence of Figure 2 or Figure 3 is produced, then used as an immunogen to generate appropriate antibodies. In another embodiment, a 83P2H3-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 83P2H3-related protein or 83P2H3 expressing cells) to generate an immune response to the encoded immunogen (for review, see Donnelly et al., 1997, Ann. Rev. Immunol. 15: 617-648).

The amino acid sequence of 83P2H3 as shown in Figure 2 or Figure 3 can be analyzed to select specific regions of the 83P2H3 protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of the 83P2H3 amino acid sequence are used to identify hydrophilic regions in the 83P2H3 structure. Regions of the 83P2H3 protein that show immunogenic structure, as well as other regions and domains, can readily be identified using various other methods known in the art, such as Chou- Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis. Thus, each region identified by any of these programs or methods is within the scope of the present invention.

Methods for the generation of 83P2H3 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, IL, are effective. Administration of a 83P2H3 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.

83P2H3 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 83P2H3-related protein. When the appropriate immortalized cell culture is identified, the cells can be expanded and antibodies produced either from in vitro cultures or from ascites fluid.

The antibodies or fragments of the invention can also be produced, by recombinant means.

Regions that bind specifically to the desired regions of the 83P2H3 protein can also be produced in the context of chimeric or complementarity determining region (CDR) grafted antibodies of multiple species origin. Humanized or human 83P2H3 antibodies can also be produced, and are preferred for use in therapeutic contexts. Methods for humanizing murine and other non-human antibodies, by substituting one or more of the non-human antibody CDRs for corresponding human antibody sequences, are well known (see for example, Jones et al., 1986, Nature 321: 522-525; Riechmann et al., 1988, Nature 332: 323-327; Verhoeyen et al., 1988, Science 239: 1534-1536). See also, Carter et al., 1993, Proc. Natl. Acad. Sci. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151: 2296.

Methods for producing fully human monoclonal antibodies include phage display and transgenic methods (for review, see Vaughan et al., 1998, Nature Biotechnology 16: 535-539). Fully human 83P2H3 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 83P2H3 monoclonal antibodies can also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application W098/24893, Kucherlapati and Jakobovits et al., published December 3,1997 (see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7 (4): 607-614; U. S. patents 6,162,963 issued 19 December 2000; 6,150,584 issued 12 November 2000; and, 6,114598 issued 5 September 2000). This method avoids the in vitro manipulation required with phage display technology and efficiently produces high affinity authentic human antibodies.

Reactivity of 83P2H3 antibodies with an 83P2H3-related protein can be established by a number of well known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses using, as appropriate, 83P2H3-related proteins, 83P2H3-expressing cells or extracts thereof.

A 83P2H3 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 83P2H3 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., Wolf et al., Cancer Res. 53: 2560-2565).

V.) 83P2H3 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 Nov ; 50 (3-4) : 201-12, Review).

Furthermore, x-ray crystallographic analyses of HLA-peptide complexes have revealed pockets within the peptide binding cleft/groove of HLA molecules which accommodate, in an allele- specific mode, residues borne by peptide ligands; these residues in turn determine the HLA binding capacity of the peptides in which they are present. (See, e. g., Madden, D. R. Annu. Rev. Immunol.

13: 587,1995; Smith, et al., Immunity 4: 203,1996; Fremont et 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. etal., 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.) 83P2H3 Transeenic Animals Nucleic acids that encode a 83P2H3-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 83P2H3 can be used to clone genomic DNA that encodes 83P2H3. The cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode 83P2H3. 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. Patent Nos. 4,736,866 issued 12 April 1988, and 4,870,009 issued 26 September 1989. Typically, particular cells would be targeted for 83P2H3 transgene incorporation with tissue-specific enhancers.

Transgenic animals that include a copy of a transgene encoding 83P2H3 can be used to examine the effect of increased expression of DNA that encodes 83P2H3. 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 83P2H3 can be used to construct a 83P2H3"knock out"animal that has a defective or altered gene encoding 83P2H3 as a result of homologous recombination between the endogenous gene encoding 83P2H3 and altered genomic DNA encoding 83P2H3 introduced into an embryonic cell of the animal. For example, cDNA that encodes 83P2H3 can be used to clone genomic DNA encoding 83P2H3 in accordance with established techniques. A portion of the genomic DNA encoding 83P2H3 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 83P2H3 polypeptide.

VII.) Methods for the Detection of 83P2H3 Another aspect of the present invention relates to methods for detecting 83P2H3 polynucleotides and 83P2H3-related proteins, as well as methods for identifying a cell that expresses 83P2H3. The expression profile of 83P2H3 makes it a diagnostic marker for metastasized disease. Accordingly, the status of 83P2H3 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 83P2H3 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 83P2H3 polynucleotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, semen, cell preparations, and the like. Detectable 83P2H3 polynucleotides include, for example, a 83P2H3 gene or fragment thereof, 83P2H3 mRNA, alternative splice variant 83P2H3 mRNAs, and recombinant DNA or RNA molecules that contain a 83P2H3 polynucleotide. A number of methods for amplifying and/or detecting the presence of 83P2H3 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 83P2H3 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 83P2H3 polynucleotides as sense and antisense primers to amplify 83P2H3 cDNAs therein ; and detecting the presence of the amplified 83P2H3 cDNA. Optionally, the sequence of the amplified 83P2H3 cDNA can be determined.

In another embodiment, a method of detecting a 83P2H3 gene in a biological sample comprises first isolating genomic DNA from the sample ; amplifying the isolated genomic DNA using

83P2H3 polynucleotides as sense and antisense primers; and detecting the presence of the amplified 83P2H3 gene. Any number of appropriate sense and antisense probe combinations can be designed from the nucleotide sequence provided for the 83P2H3 (Figure 2) and used for this purpose.

The invention also provides assays for detecting the presence of an 83P2H3 protein in a tissue or other biological sample such as serum, semen, bone, prostate, urine, cell preparations, and the like.

Methods for detecting a 83P2H3-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 83P2H3-related protein in a biological sample comprises first contacting the sample with a 83P2H3 antibody, a 83P2H3-reactive fragment thereof, or a recombinant protein containing an antigen binding region of a 83P2H3 antibody; and then detecting the binding of 83P2H3-related protein in the sample.

Methods for identifying a cell that expresses 83P2H3 are also within the scope of the invention.

In one embodiment, an assay for identifying a cell that expresses a 83P2H3 gene comprises detecting the presence of 83P2H3 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 83P2H3 riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for 83P2H3, 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 83P2H3 gene comprises detecting the presence of 83P2H3-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 83P2H3-related proteins and cells that express 83P2H3-related proteins.

83P2H3 expression analysis is also useful as a tool for identifying and evaluating agents that modulate 83P2H3 gene expression. For example, 83P2H3 expression is significantly upregulated in prostate cancer, and is expressed in cancers of the tissues listed in Table I. Identification of a molecule or biological agent that inhibits 83P2H3 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 83P2H3 expression by RT-PCR, nucleic acid hybridization or antibody binding.

VIII.) Methods for Monitoring the Status of 83P2H3-related Genes and Their Products Oncogenesis is known to be a multistep process where cellular growth becomes progressively dysregulated and cells progress from a normal physiological state to precancerous and then cancerous states (see, e. g., Alers et al., Lab Invest. 77 (5): 437-438 (1997) and Isaacs et al., Cancer Surv. 23: 19-32 (1995)). In this context, examining a biological sample for evidence of dysregulated cell growth (such as aberrant 83P2H3 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 83P2H3 in a biological

sample of interest can be compared, for example, to the status of 83P2H3 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 83P2H3 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. Patent No. 5,837,501) to compare 83P2H3 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 83P2H3 expressing cells) as well as the level, and biological activity of expressed gene products (such as 83P2H3 mRNA, polynucleotides and polypeptides). Typically, an alteration in the status of 83P2H3 comprises a change in the location of 83P2H3 and/or 83P2H3 expressing cells and/or an increase in 83P2H3 mRNA and/or protein expression.

83P2H3 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 83P2H3 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 83P2H3 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 83P2H3 gene), Northern analysis and/or PCR analysis of 83P2H3 mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of 83P2H3 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 83P2H3 proteins and/or associations of 83P2H3 proteins with polypeptide binding partners). Detectable 83P2H3 polynucleotides include, for example, a 83P2H3 gene or fragment thereof, 83P2H3 mRNA, alternative splice variants, 83P2H3 mRNAs, and recombinant DNA or RNA molecules containing a 83P2H3 polynucleotide.

The expression profile of 83P2H3 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 83P2H3 provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The invention provides methods and assays for determining 83P2H3 status and diagnosing cancers that express 83P2H3, such as cancers of the tissues listed in Table 1. For example, because 83P2H3 mRNA is so highly expressed in prostate and other

cancers relative to normal prostate tissue, assays that evaluate the levels of 83P2H3 mRNA transcripts or proteins in a biological sample can be used to diagnose a disease associated with 83P2H3 dysregulation, and can provide prognostic information useful in defining appropriate therapeutic options.

The expression status of 83P2H3 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 83P2H3 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 83P2H3 in a biological sample can be examined by a number of well-known procedures in the art. For example, the status of 83P2H3 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 83P2H3 expressing cells (e. g. those that express 83P2H3 mRNAs or proteins). This examination can provide evidence of dysregulated cellular growth, for example, when 83P2H3- 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 83P2H3 in a biological sample are often associated with dysregulated cellular growth. Specifically, one indicator of dysregulated cellular growth is the metastases of cancer cells from an organ of origin (such as the prostate) to a different area of the body (such as a lymph node). In this context, evidence of dysregulated cellular growth is important for example because occult lymph node metastases can be detected in a substantial proportion of patients with prostate cancer, and such metastases are associated with known predictors of disease progression (see, e. g., Murphy et al., Prostate 42 (4): 315-317 (2000); Su et al., Semin. Surg.

Oncol. 18 (1) : 17-28 (2000) and Freeman et al., J Urol 1995 Aug 154 (2 Pt 1): 474-8).

In one aspect, the invention provides methods for monitoring 83P2H3 gene products by determining the status of 83P2H3 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 83P2H3 gene products in a corresponding normal sample. The presence of aberrant 83P2H3 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 83P2H3 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 83P2H3 mRNA can, for example, be evaluated in tissue samples including but not limited to those listed in Table I. The presence of significant 83P2H3 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 83P2H3 mRNA or express it at lower levels.

In a related embodiment, 83P2H3 status is determined at the protein level rather than at the nucleic acid level. For example, such a method comprises determining the level of 83P2H3 protein expressed by cells in a test tissue sample and comparing the level so determined to the level of 83P2H3 expressed in a corresponding normal sample. In one embodiment, the presence of 83P2H3 protein is evaluated, for example, using immunohistochemical methods. 83P2H3 antibodies or binding partners capable of detecting 83P2H3 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 83P2H3 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 83P2H3 may be indicative of the presence or promotion of a tumor. Such assays therefore have diagnostic and predictive value where a mutation in 83P2H3 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 83P2H3 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. Patent Nos. 5,382,510 issued 7 September 1999, and 5,952,170 issued 17 January 1995).

Additionally, one can examine the methylation status of the 83P2H3 gene in a biological sample.

Aberrant demethylation and/or hypermethylation of CpG islands in gene 5'regulatory regions frequently occurs in immortalized and transformed cells, and can result in altered expression of various genes. For example, promoter hypermethylation of the pi-class glutathione S-transferase (a protein expressed in normal prostate but not expressed in >90% of prostate carcinomas) appears to permanently silence transcription of this gene and is the most frequently detected genomic alteration in prostate carcinomas (De Marzo et al., Am. J. Pathol. 155 (6): 1985-1992 (1999)). In addition, this alteration is present in at least 70% of cases of high-grade prostatic intraepithelial neoplasia (PIN) (Brooks et al, Cancer Epidemiol. Biomarkers Prev., 1998,7: 531-536). In another example, expression of the LAGE-I tumor specific gene (which is not expressed in normal prostate but is expressed in 25-50% of prostate cancers) is induced by deoxy-azacytidine in lymphoblastoid cells, suggesting that tumoral expression is due to demethylation (Lethe et al., Int. J. Cancer 76 (6): 903-908 (1998)). A variety of assays for examining methylation status of a gene are well known in the art. For example, one can utilize, in Southern hybridization approaches, methylation-sensitive restriction enzymes 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 83P2H3. 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.

Biopsie 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 83P2H3 expression. The presence of RT-PCR amplifiable 83P2H3 mRNA provides an indication of the presence of cancer. RT-PCR assays are well known in the art. RT-PCR detection assays for tumor cells in peripheral blood are currently being evaluated for use in the diagnosis and management of a number of human solid tumors. In the prostate cancer field, these include RT-PCR assays for the detection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res. 25: 373-384; Ghossein et al., 1995, J. Clin. Oncol. 13: 1195-2000; Heston et al., 1995, Clin. Chem 41: 1687-1688).

A further aspect of the invention is an assessment of the susceptibility that an individual has for developing cancer. In one embodiment, a method for predicting susceptibility to cancer comprises detecting 83P2H3 mRNA or 83P2H3 protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of 83P2H3 mRNA expression correlates to the degree of susceptibility. In a specific embodiment, the presence of 83P2H3 in prostate or other tissue is examined, with the presence of 83P2H3 in the sample providing an indication of prostate cancer susceptibility (or the emergence or existence of a prostate tumor). Similarly, one can evaluate the integrity 83P2H3 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 83P2H3 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 83P2H3 mRNA or 83P2H3 protein expressed by tumor cells, comparing the level so determined to the level of 83P2H3

mRNA or 83P2H3 protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of 83P2H3 mRNA or 83P2H3 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 83P2H3 is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors. Another embodiment is the evaluation of the integrity of 83P2H3 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 83P2H3 mRNA or 83P2H3 protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of 83P2H3 mRNA or 83P2H3 protein expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of 83P2H3 mRNA or 83P2H3 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 83P2H3 expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer. Also, one can evaluate the integrity 83P2H3 nucleotide and amino acid sequences in a biological sample in order to identify perturbations in the structure of these molecules such as insertions, deletions, substitutions and the like, where the presence of one or more perturbations indicates a progression of the cancer.

The above diagnostic approaches can be combined with any one of a wide variety of prognostic and diagnostic protocols known in the art. For example, another embodiment of the invention is directed to methods for observing a coincidence between the expression of 83P2H3 gene and 83P2H3 gene products (or perturbations in 83P2H3 gene and 83P2H3 gene products) and a factor that is associated with malignancy, as a means for diagnosing and prognosticating the status of a tissue sample. A wide variety of factors associated with malignancy can be utilized, such as the expression of genes associated with malignancy (e. g. PSA, PSCA and PSM expression for prostate cancer etc.) as well as gross cytological observations (see, e. g., Bocking et al., 1984, Anal. Quant. Cytol. 6 (2): 74-88; Epstein, 1995, Hum. Pathol.

26 (2) : 223-9; Thorson et al., 1998, Mod. Pathol. 11 (6): 543-51; Baisden et al., 1999, Am. J. Surg.

Pathol. 23 (8): 918-24). Methods for observing a coincidence between the expression of 83P2H3 gene and 83P2H3 gene products (or perturbations in 83P2H3 gene and 83P2H3 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 83P2H3 gene and 83P2H3 gene products (or perturbations in 83P2H3 gene and 83P2H3 gene products) and another

factor associated with malignancy entails detecting the overexpression of 83P2H3 mRNA or protein in a tissue sample, detecting the overexpression ofPSA mRNA or protein in a tissue sample (or PSCA or PSM expression), and observing a coincidence of 83P2H3 mRNA or protein and PSA mRNA or protein overexpression (or PSCA or PSM expression). In a specific embodiment, the expression of 83P2H3 and PSA mRNA in prostate tissue is examined, where the coincidence of 83P2H3 and PSA mRNA overexpression in the sample indicates the existence of prostate cancer, prostate cancer susceptibility or the emergence or status of a prostate tumor.

Methods for detecting and quantifying the expression of 83P2H3 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 83P2H3 mRNA include in situ hybridization using labeled 83P2H3 riboprobes, Northern blot and related techniques using 83P2H3 polynucleotide probes, RT-PCR analysis using primers specific for 83P2H3, 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 83P2H3 mRNA expression. Any number of primers capable of amplifying 83P2H3 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 83P2H3 protein can be used in an immunohistochemical assay of biopsied tissue.

IX.) Identification of Molecules That Interact With 83P2H3 The 83P2H3 protein and nucleic acid sequences disclosed herein allow a skilled artisan to identify proteins, small molecules and other agents that interact with 83P2H3, as well as pathways activated by 83P2H3 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. Patent Nos. 5,955,280 issued 21 September 1999,5,925,523 issued 20 July 1999, 5,846,722 issued 8 December 1998 and 6,004,746 issued 21 December 1999. Algorithms are also available in the art for genome-based predictions of protein function (see, e. g., Marcotte, et al., Nature 402: 4 November 1999,83-86).

Alternatively one can screen peptide libraries to identify molecules that interact with 83P2H3 protein sequences. In such methods, peptides that bind to a molecule such as 83P2H3 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 protein of interest.

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 83P2H3 protein sequences are disclosed for example in U. S. Patent Nos. 5,723,286 issued 3 March 1998 and 5,733,731 issued 31 March 1998.

Alternatively, cell lines that express 83P2H3 are used to identify protein-protein interactions mediated by 83P2H3. Such interactions can be examined using immunoprecipitation techniques (see, e. g., Hamilton BJ, et al. Biochem. Biophys. Res. Commun. 1999,261: 646-51). 83P2H3 protein can be immunoprecipitated from 83P2H3-expressing cell lines using anti-83P2H3 antibodies. Alternatively, antibodies against His-tag can be used in a cell line engineered to express 83P2H3 (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 83P2H3 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 83P2H3'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 ion channel, protein pump, or cell communication function of 83P2H3 are identified and used to treat patients that have a cancer that expresses the 83P2H3 antigen (see, e. g., Hille, B., Ionic Channels of Excitable Membranes 2nd Ed., Sinauer Assoc., Sunderland, MA, 1992). Moreover, ligands that regulate 83P2H3 function can be identified based on their ability to bind 83P2H3 and activate a reporter construct. Typical methods are discussed for example in U. S. Patent No. 5,928,868 issued 27 July 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 83P2H3 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 both activators and inhibitors of 83P2H3.

An embodiment of this invention comprises a method of screening for a molecule that interacts with an 83P2H3 amino acid sequence shown in Figure 2 or Figure 3, comprising the steps of contacting a population of molecules with the 83P2H3 amino acid sequence, allowing the population of molecules and the 83P2H3 amino acid sequence to interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the 83P2H3 amino acid sequence, and then separating molecules that do not interact with the 83P2H3 amino acid sequence

from molecules that do. In a specific embodiment, the method further comprises purifying a molecule that interacts with the 83P2H3 amino acid sequence. The identified molecule can be used to modulate a function performed by 83P2H3. In a preferred embodiment, the 83P2H3 amino acid sequence is contacted with a library of peptides.

X.) Therapeutic Methods and Compositions The identification of 83P2H3 as a protein that is normally expressed in a restricted set of tissues, but which is also expressed in prostate and other cancers, opens a number of therapeutic approaches to the treatment of such cancers. As discussed herein, it is possible that 83P2H3 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 83P2H3 protein are useful for patients suffering from a cancer that expresses 83P2H3. These therapeutic approaches generally fall into two classes. One class comprises various methods for inhibiting the binding or association of the 83P2H3 protein with its binding partner or with other proteins. Another class comprises a variety of methods for inhibiting the transcription of the 83P2H3 gene or translation of 83P2H3 mRNA.

X. A.) Anti-Cancer Vaccines The invention further provides cancer vaccines comprising a 83P2H3-related protein or 83P2H3- related nucleic acid. In view of the expression of 83P2H3, cancer vaccines prevent and/or treat 83P2H3- expressing cancers with minimal or no effects on non-target tissues. The use of a tumor antigen in a vaccine that generates humoral and/or cell-mediated immune responses as anti-cancer therapy is well known in the art and has been employed in prostate cancer using human PSMA and rodent PAP immunogens (Hodge et al., 1995, Int. J. Cancer 63 : 231-237 ; Fong et al., 1997, J. Immunol. 159: 3113- 3117).

Such methods can be readily practiced by employing a 83P2H3-related protein, or an 83P2H3- encoding nucleic acid molecule and recombinant vectors capable of expressing and presenting the 83P2H3 immunogen (which typically comprises a number of antibody or T cell epitopes). Skilled artisans understand that a wide variety of vaccine systems for delivery of immunoreactive epitopes are known in the art (see, e. g., Heryln et al., Ann Med 1999 Feb 31 (1) : 66-78; Maruyama et al., Cancer Immunol Immunother 2000 Jun 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 83P2H3 protein shown in SEQ ID NO : 703 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 83P2H3 immunogen contains a biological motif, see e. g., Tables V-XVIII, or a peptide of a size range from 83P2H3 indicated in Figure 14, Figure 15, Figure 16, Figure 17, and Figure 18.

The entire 83P2H3 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., Yaccine 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 BiolTechnology 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, Massachusetts) may also be used.

In patients with 83P2H3-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 83P2H3 protein that bind corresponding HLA alleles (see e. g., Table IV; Eimer and Epimattix, Brown University (URL www. brown. edu/ResearchPTB-HIV Lab/epimatrix/epimatrix. html) ; and, BIMAS, (URL bimas. dcrt. nih. gov/; SYFPEITHI at URL syfpeithi. bmi-heidelberg. com/). In a preferred embodiment, the 83P2H3 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 9,10,1 I, 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 83P2H3 protein) so that an immune response is generated. A typical embodiment consists of a method for generating an immune response to 83P2H3 in a host, by contacting the host with a sufficient amount of at least one 83P2H3 B cell or cytotoxic T-cell epitope or analog thereof; and at least one periodic interval thereafter re-contacting the host with the 83P2H3 B cell or cytotoxic T-cell epitope or analog thereof. A specific embodiment consists of a method of generating an immune response against a 83P2H3-related protein or a man-made multiepitopic peptide comprising: administering 83P2H3 immunogen (e. g. the 83P2H3 protein or a peptide fragment thereof, an 83P2H3 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. Patent No. 6,146,635) or a universal helper epitope such as a PADRETM peptide (Epimmune Inc., San Diego, CA; 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 83P2H3 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 83P2H3 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. Patent 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 : 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 83P2H3. Constructs comprising DNA encoding a 83P2H3-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 83P2H3 protein/immunogen. Alternatively, a vaccine comprises a 83P2H3- related protein. Expression of the 83P2H3-related protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells that bear 83P2H3 protein.

Various prophylactic and therapeutic genetic immunization techniques known in the art can be used (for review, see information and references published at Internet address www. genweb. com). Nucleic acid-based delivery is described, for instance, in Wolff et. al., Science 247: 1465 (1990) as well as U. S.

Patent 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. Patent No. 5,922,687).

For therapeutic or prophylactic immunization purposes, proteins of the invention can be expressed by 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 83P2H3-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 elicit a host immune response. Vaccinia vectors and methods useful in immunization protocols are described in, e. g., U. S. Patent 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, Salniotiella 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 83P2H3-related nucleic acid molecule. In one embodiment, the full-length human 83P2H3 cDNA is employed. In another embodiment, 83P2H3 nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) anc/or antibody opitopes 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 83P2H3 antigen to a patient's immune system Dendritic cells express MHC class I and II molecules, B7 co-stimulator, and

IL-12, and are thus highly specialized antigen presenting cells. In prostate cancer, autologous dendritic cells pulsed with peptides of the prostate-specific membrane antigen (PSMA) are being used in a Phase I clinical trial to stimulate prostate cancer patients'immune systems (Tjoa et al., 1996, Prostate 28: 65- 69; Murphy et al., 1996, Prostate 29: 371-380). Thus, dendritic cells can be used to present 83P2H3 peptides to T cells in the context of MHC class I or II molecules. In one embodiment, autologous dendritic cells are pulsed with 83P2H3 peptides capable of binding to MHC class I and/or class II molecules. In another embodiment, dendritic. cells are pulsed with the complete 83P2H3 protein. Yet another embodiment involves engineering the overexpression of the 83P2H3 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 83P2H3 can also be engineered to express immune modulators, such as GM-CSF, and used as immunizing agents.

X. B.) 83P2H3 as a Target for Antibody-based Therapy 83P2H3 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 83P2H3 is expressed by cancer cells of various lineages relative to corresponding normal cells, systemic administration of 83P2H3-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 83P2H3 are useful to treat 83P2H3-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.

83P2H3 antibodies can be introduced into a patient such that the antibody binds to 83P2H3 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 83P2H3, 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 83P2H3 sequence shown in Figure 2 or Figure 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 (June 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. 83P2H3), 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-83P2H3 antibody) that binds to a marker (e. g. 83P2H3) 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 83P2H3, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a 83P2H3 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-83P2H3 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 Y9' or 1131 to anti-CD20 antibodies (e. g., ZevalinTM, IDEC Pharmaceuticals Corp. or Bexxar, Coulter Pharmaceuticals), while others involve co-administration of antibodies and other therapeutic agents, such as HerceptinTM (trastuzumab) with paclitaxel (Genentech, Inc.). To treat prostate cancer, for example, 83P2H3 antibodies can be administered in conjunction with radiation, chemotherapy or hormone ablation.

Although 83P2H3 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 83P2H3 expression, preferably using immunohistochemical assessments of tumor tissue, quantitative 83P2H3 imaging, or other techniques that reliably indicate the presence and degree of 83P2H3 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-83P2H3 monoclonal antibodies that treat prostate and other cancers include those that initiate a potent immune response against the tumor or those that are directly cytotoxic. In this regard, anti-83P2H3 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-83P2H3 mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express 83P2H3. 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-83P2H3 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 83P2H3 antigen with high affinity but exhibit low or no antigenicity in the patient.

Therapeutic methods of the invention contemplate the administration of single anti-83P2H3 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- 83P2H3 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-83P2H3 mAbs are administered in their"naked"or unconjugated form, or can have a therapeutic agent (s) conjugated to them.

Anti-83P2H3 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-83P2H3 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-83P2H3 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 83P2H3 expression in the patient, the extent of circulating shed 83P2H3 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 83P2H3 in a given sample (e. g. the levels of circulating 83P2H3 antigen and/or 83P2H3 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 (such as serum PSA levels in prostate cancer therapy).

Anti-idiotypic anti-83P2H3 antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a 83P2H3-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-83P2H3 antibodies that mimic an epitope on a 83P2H3-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.) 83P2H3 as a Tareet 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 83P2H3 antigen, or derives at least some therapeutic benefit when the antigen was tumor-associated.

In some embodiments, it may be desirable to combine the class I peptide components with components that induce or facilitate neutralizing antibody and or helper T cell responses directed to the target antigen. A preferred embodiment of such a composition comprises class I and class II epitopes in accordance with the invention. An alternative embodiment of such a composition comprises a class I and/or class II epitope in accordance with the invention, along with a cross reactive HTL epitope such as PADRE (immune, San Diego, CA) molecule (described e. g., in U. S. Patent Number 5,736,142).

A vaccine of the invention can also include antigen-presenting cells (APC), such as dendritic cells (DC), as a vehicle to present peptides of the invention. Vaccine compositions can be created in vitro, following dendritic cell mobilization and harvesting, whereby loading of dendritic cells occurs in vitro. For example, dendritic cells are transfected, e. g., with a minigene in accordance with the invention, or are pulsed with peptides. The dendritic cell can then be administered to a patient to elicit immune responses in vivo. Vaccine compositions, either DNA-or peptide-based, can also be administered in vivo in combination with dendritic cell mobilization whereby loading of dendritic cells occurs in vivo.

Preferably, the following principles are utilized when selecting an array of epitopes for inclusion in a polyepitopic composition for use in a vaccine, or for selecting discrete epitopes to be included in a vaccine and/or to be encoded by nucleic acids such as a minigene. It is preferred that each of the following principles be balanced in order to make the selection. The multiple epitopes to be

incorporated in a given vaccine composition may be, but need not be, contiguous in sequence in the native antigen from which the epitopes are derived.

1.) Epitopes are selected which, upon administration, mimic immune responses that have been observed to, be correlated with tumor clearance. For HLA Class I this includes 3-4 epitopes that come from at least one tumor associated antigen (TAA). For HLA Class II a similar rationale is employed; again 3-4 epitopes are selected from at least one TAA (see, e. g., Rosenberg et al., Science 278: 1447-1450). Epitopes from one TAA may be used in combination with epitopes from one or more additional TAAs to produce a vaccine that targets tumors with varying expression patterns of frequently-expressed TAAs.

2.) Epitopes are selected that have the requisite binding affinity established to be correlated with immunogenicity: for HLA Class I an ICso 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.) In cases where the sequences of multiple variants of the same target protein are available, 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 83P2H3, the PADRE@ universal helper T cell epitope (or multiple HTL epitopes from 83P2H3), 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 tested 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 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. Patent 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, CA). 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-ß) 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, California). 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 (5ICr) labeled and used as target cells for epitope-specific CTL lines; cytolysis, detected by 5tCr 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, 5'Cr-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 evaluated in transgenic mice in an analogous manner.

Alternatively, the nucleic acids can be administered using ballistic delivery as described, for instance, in U. S. Patent 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 (QYIKANSKPIGITE ; SEQ ID NO: 710), Plasmodium falciparum circumsporozoite (CS) protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS ; SEQ ID NO: 711), and Streptococcus

18kD protein at positions 116-131 (GAVDSILGGVATYGAA; SEQ ID NO : 712). 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., PADRE, Epimmune, Inc., San Diego, CA) 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: 713), 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 e-and a-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 s-and a-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 ProgenipoietiriTm (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 83P2H3. 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 83P2H3.

X. D. Adoptive Immunotherapy Antigenic 83P2H3-related peptides are used to elicit a CTL and/or HTL response ex vivo, as well. The resulting CTL or HTL cells, can be used to treat tumors in patients that do not respond to other conventional forms of therapy, or will not respond to a therapeutic vaccine peptide or nucleic acid in accordance with the invention. Ex vivo CTL or HTL responses to a particular antigen are induced by incubating in tissue culture the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of antigen-presenting cells (APC), such as dendritic cells, and the appropriate immunogenic peptide. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused back into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cell (e. g., a tumor cell). Transfected dendritic cells may also be used as antigen presenting cells.

X. E. Administration of Vaccines for Therapeutic or Prophylactic Purposes Pharmaceutical and vaccine compositions of the invention are typically used to treat and/or prevent a cancer that expresses or overexpresses 83P2H3. 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 83P2H3. 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 83P2H3- 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 (ive., 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 83P2H3, a vaccine comprising 83P2H3-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 jUg and the higher value is about 10,000; 20,000; 30,000; or 50,000 Ag. Dosage values for a human typically range from about 500, ug to about 50,000 , ug per 70 kilogram patient. Boosting dosages of between about 1.0 llg to about 50,000 gg of peptide pursuant to a boosting regimen over weeks to months may be administered depending upon the patient's response and condition as determined by measuring the specific activity of CTL and HTL obtained from the patient's blood. Administration should continue until at least clinical symptoms or laboratory tests indicate that the neoplasia, has been eliminated or reduced and for a period thereafter.

The dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.

In certain embodiments, the peptides and compositions of the present invention are employed in serious disease states, that is, life-threatening or potentially life threatening situations. In such cases, as a result of the minimal amounts of extraneous substances and the relative nontoxic nature of the peptides in preferred compositions of the invention, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions relative to these stated dosage amounts.

The vaccine compositions of the invention can also be used purely as prophylactic agents.

Generally the dosage for an initial prophylactic immunization generally occurs in a unit dosage range where the lower value is about 1,5,50,500, or 1000 yg and the higher value is about 10,000; 20,000; 30,000; or 50,000, ug. Dosage values for a human typically range from about 500 jug to about 50,000 Ag per 70 kilogram patient. This is followed by boosting dosages of between about 1.0 jig to about 50,000 llg of peptide administered at defined intervals from about four weeks to six months after the

initial administration of vaccine. The immunogenicity of the vaccine can be assessed by measuring the specific activity of CTL and HTL obtained from a sample of the patient's blood.

The pharmaceutical compositions for therapeutic treatment are intended for parenteral, topical, oral, nasal, intrathecal, or local (e. g. as a cream or topical ointment) administration. Preferably, the pharmaceutical compositions are administered parentally, e. g., intravenously, subcutaneously, intradermally, or intramuscularly. Thus, the invention provides compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.

A variety of aqueous carriers may be used, e. g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well- known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.

The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

The concentration of peptides of the invention in the pharmaceutical formulations can vary widely, i. e., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.

A human unit dose form of 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, 17"' Edition, A. Gennaro, Editor, Mack Publishing Co., Easton, Pennsylvania, 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. Patent 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 0. 01%-20% by weight, preferably 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 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 0.1%-20% by weight of the composition, preferably 0.25-5%. The balance of the composition is ordinarily propellant. A carrier can also be included, as desired, as with, eg., lecithin for intranasal delivery.

XI.) lJiaenostic and Prognostic Embodiments of 83P2H3.

As disclosed herein, 83P2H3 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).

83P2H3 can be analogized to a prostate associated antigen PSA, the archetypal marker that has been used by medical practitioners for years to identify and monitor the presence of prostate cancer (see, e. g., Merrill et al., J. Urol, 163 (2) : 503-5120 (2000) ; Polascik et al., J. Urol. Aug ; 162 (2) : 293-306 (1999) and Fortier et al., J. Nat. Cancer Inst. 91 (19): 1635-1640 (1999)). A variety of other diagnostic

markers are also used in similar contexts including p53 and K-ras (see, e. g., Tulchinsky et al., Int J Mol Med 1999 Jul 4 (1) : 99-102 and Minimoto et al., Cancer Detect Prev 2000; 24 (1) : 1-12). Therefore, this disclosure of the 83P2H3 polynucleotides and polypeptides (as well as the 83P2H3 polynucleotide probes and anti-83P2H3 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 83P2H3 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 83P2H3 polynucleotides described herein can be utilized in the same way to detect 83P2H3 overexpression or the metastasis of prostate and other cancers expressing this gene. Alternatively, just as PSA polypeptides are used to generate antibodies specific for PSA which can then be used to observe the presence and/or the level of PSA proteins in methods to monitor PSA protein overexpression (see, e. g., Stephan et al., Urology 55 (4): 560-3 (2000)) or the metastasis of prostate cells (see, e. g., Alanen et al., Pathol. Res. Pract. 192 (3) : 233-7 (1996)), the 83P2H3 polypeptides described herein can be utilized to generate antibodies for use in detecting 83P2H3 overexpression or the metastasis of prostate cells and cells of other cancers expressing this gene.

Specifically, because metastases involves the movement of cancer cells from an organ of origin (such as the lung or prostate gland etc.) to a different area of the body (such as a lymph node), assays which examine a biological sample for the presence of cells expressing 83P2H3 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 83P2H3-expressing cells (lymph node) is found to contain 83P2H3-expressing cells such as the 83P2H3 expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis.

Alternatively 83P2H3 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 83P2H3 or express 83P2H3 at a different level are found to express 83P2H3 or have an increased expression of 83P2H3 (see, e. g., the 83P2H3 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 83P2H3) such as PSA, PSCA etc. (see, e. g., Alanen et al., Pathol. Res. Pract.

192 (3): 233-237 (1996)).

Just as PSA polynucleotide fragments and polynucleotide variants are employed by skilled artisans for use in methods of monitoring PSA, 83P2H3 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 83P2H3 polynucleotide fragment is used as a probe to show the expression of 83P2H3 RNAs in cancer cells. In addition, variant polynucleotide sequences are typically used as primers and probes for the corresponding mRNAs in PCR and Northern analyses (see, e. g., Sawai et al., Fetal Diagn. Ther. 1996 Nov-Dec 11 (6): 407-13 and Current Protocols In Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubel et al. eds., 1995)). Polynucleotide fragments and variants are useful in this context where they are capable of binding to a target polynucleotide sequence (e. g. the 83P2H3 polynucleotide shown in SEQ ID NO: 701) 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.

83P2H3 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. Patent No. 5,840,501 and U. S.

Patent No. 5,939,533). For example it may be preferable to utilize a polypeptide comprising one of the 83P2H3 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 83P2H3 polypeptide shown in SEQ ID NO: 703).

As shown herein, the 83P2H3 polynucleotides and polypeptides (as well as the 83P2H3 polynucleotide probes and anti-83P2H3 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 83P2H3 gene products, in order to evaluate the presence or onset of a disease condition described herein, such as prostate cancer, are used to identify patients for preventive measures or further monitoring, as has been done so successfully with PSA. Moreover, these materials satisfy a need in the art for molecules having similar or complementary characteristics to PSA in situations where, for example, a definite diagnosis of metastasis of prostatic origin cannot be made on the basis of a test for PSA alone (see, e. g., Alanen et al., Pathol. Res. Pract. 192 (3): 233-237 (1996)), and consequently, materials such as 83P2H3 polynucleotides and polypeptides (as well as the 83P2H3 polynucleotide probes and anti-83P2H3 antibodies used to identify the presence of these molecules) must be employed to confirm metastases of prostatic origin.

Finally, in addition to their use in diagnostic assays, the 83P2H3 polynucleotides disclosed herein have a number of other specific utilities such as their use in the identification of oncogenetic associated chromosomal abnormalities in the chromosomal region to which the 83P2H3 gene maps (see Example 3 below). Moreover, in addition to their use in diagnostic assays, the 83P2H3-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, 83P2H3-related proteins or polynucleotides of the invention can be used to treat a pathologic condition characterized by the over-expression of 83P2H3. For example, the amino acid or nucleic acid sequence of Figure 2 or Figure 3, or fragments of either, can be used to generate an immune response to the 83P2H3 antigen. Antibodies or other molecules that react with 83P2H3 can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.

XII.) Inhibition of 83P2H3 Protein Function The invention includes various methods and compositions for inhibiting the binding of 83P2H3 to its binding partner or its association with other protein (s) as well as methods for inhibiting 83P2H3 function.

XII. A.) Inhibition of 83P2H3 With Intracellular Antibodies In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to 83P2H3 are introduced into 83P2H3 expressing cells via gene transfer technologies.

Accordingly, the encoded single chain anti-83P2H3 antibody is expressed intracellularly, binds to 83P2H3 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 83P2H3 in the nucleus, thereby preventing, its activity within the nucleus. Nuclear targeting signals are engineered into such 83P2H3 intrabodies in order to achieve the desired targeting. Such 83P2H3 intrabodies are designed to bind specifically to a particular 83P2H3 domain. In another embodiment, cytosolic intrabodies that specifically bind to the 83P2H3 protein are used to prevent 83P2H3 from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus (e. g., preventing 83P2H3 from forming transcription complexes with other factors).

In order to specifically direct the expression of such intrabodies to particular cells, the transcription of the intrabody is placed under the regulatory control of an appropriate tumor-specific promoter and/or enhancer. In order to target intrabody expression specifically to prostate, for example, the PSA promoter and/or promoter/enhancer can be utilized (See, for example, U. S. Patent No.

5,919,652 issued 6 July 1999).

XII. B.) Inhibition of 83P2H3 with Recombinant Proteins In another approach, recombinant molecules bind to 83P2H3 and thereby inhibit 83P2H3 function. For example, these recombinant molecules prevent or inhibit 83P2H3 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 83P2H3 specific antibody molecule. In a particular embodiment, the 83P2H3 binding domain of a 83P2H3 binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two 83P2H3 ligand binding domains linked to the Fc portion of a human IgG, such as human IgGl. 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 83P2H3, whereby the dimeric fusion protein specifically binds to 83P2H3 and blocks 83P2H3 interaction with a

binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking technologies.

XII. C.) Inhibition of 83P2H3 Transcription or Translation The present invention also comprises various methods and compositions for inhibiting the transcription of the 83P2H3 gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of 83P2H3 mRNA into protein.

In one approach, a method of inhibiting the transcription of the 83P2H3 gene comprises contacting the 83P2H3 gene with a 83P2H3 antisense polynucleotide. In another approach, a method of inhibiting 83P2H3 mRNA translation comprises contacting the 83P2H3 mRNA with an antisense polynucleotide. In another approach, a 83P2H3 specific ribozyme is used to cleave the 83P2H3 message, thereby inhibiting translation. Such antisense and ribozyme based methods can also be directed to the regulatory regions of the 83P2H3 gene, such as the 83P2H3 promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a 83P2H3 gene transcription factor are used to inhibit 83P2H3 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 83P2H3 by interfering with 83P2H3 transcriptional activation are also useful to treat cancers expressing 83P2H3. Similarly, factors that interfere with 83P2H3 processing are useful to treat cancers that express 83P2H3. 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 83P2H3 (i. e., antisense, ribozyme, polynucleotides encoding intrabodies and other 83P2H3 inhibitory molecules). A number of gene therapy approaches are known in the art. Recombinant vectors encoding 83P2H3 antisense polynucleotides, ribozymes, factors capable of interfering with 83P2H3 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 83P2H3 to a binding partner, etc.

I7t vivo, the effect of a 83P2H3 therapeutic composition can be evaluated in a suitable animal model. For example, xenogenic prostate cancer models can be used, wherein human prostate cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine 3: 402-408). For example, PCT Patent Application W098/16628, Sawyers et al., published April 23,1998, describes various xenograft models of human prostate cancer capable of recapitulating the development of primary tumors, micrometastasis, and the formation of osteoblastic metastases characteristic of late stage disease. Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.

In vivo assays that evaluate the promotion of apoptosis are useful in evaluating therapeutic compositions. In one embodiment, xenografts from tumor bearing mice treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.

The therapeutic compositions used in the practice of the foregoing methods can be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include any material that when combined with the therapeutic composition retains the anti- tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of a number of standard pharmaceutical carriers such as sterile phosphate buffered saline solutions, bacteriostatic water, and the like (see, generally, Remington's Pharmaceutical Sciences 16"'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.) Kits For use in the diagnostic and therapeutic applications described herein, kits are also within the scope of the invention. Such kits can comprise a carrier, package or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the

container (s) comprising one of the separate elements to be used in the method. For example, the container (s) can comprise a probe that is or can be detectably labeled. Such probe can be an antibody or polynucleotide specific for a 83P2H3-related protein or a 83P2H3 gene or message, respectively.

Where the method utilizes nucleic acid hybridization to detect the target nucleic acid, the kit can also have containers containing nucleotide (s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter-means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radioisotope label. The kit can include all or part of the amino acid sequence of Figure 2 or Figure 3 or analogs thereof, or a nucleic acid molecules that encodes such amino acid sequences.

The kit of the invention will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

A label can be present on the container to indicate that the composition is used for a specific therapy or non-therapeutic application, and can also indicate directions for either in vivo or in vitro use, such as those described above. Directions and or other information can also be included on an insert which is included with the kit.

EXAMPLES 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 83P2H3 Gene To isolate genes that are involved in the progression of androgen dependent (AD) prostate cancer to androgen independent (AI) cancer, an experiment was conducted with the LAPC-4 AD xenograft in male SCID mice. Mice that harbored LAPC-4 AD xenografts were castrated when the tumors reached a size of 1 cm in diameter. The tumors regressed in size and temporarily stopped producing the androgen dependent protein PSA. Seven to fourteen days post-castration, PSA levels were detectable again in the blood of the mice. Eventually the tumors develop an AI phenotype and start growing again in the castrated males. Tumors were harvested at different time points after castration to identify genes that are turned on or off during the transition to androgen independence.

Two SSH experiments led to the isolation of numerous candidate gene fragment clones (SSH clones). All candidate clones were sequenced and subjected to homology analysis against all sequences in the major public gene and EST databases in order to provide information on the identity of the corresponding gene and to help guide the decision to analyze a particular gene for differential expression. In general, gene fragments that had no homology to any known sequence in any of the searched databases, and thus considered to represent novel genes, as well as gene fragments showing

homology to previously sequenced expressed sequence tags (ESTs), were subjected to differential expression analysis by RT-PCR and/or northern analysis.

The gene 83P2H3 was derived from an LAPC-4 AD minus LAPC-4 AD (3 days post- castration) subtraction. The SSH DNA sequence of 405 bp (Figure 1A) is 99% (399/400 bp) identical to Homo sapiens calcium transport protein CaTl gene (GenBank accession AF304463). A 83P2H3 cDNA (clone C) of 2,899 bp was isolated from a human placenta library (pEAK8 vector, Pangene) revealing an ORF of 725 amino acids (Figure 2A and 3A). The nucleotide and protein sequences of 83P2H3 shows homology to human mRNA for CaT-like B protein (Figure 4A-E).

Materials and Methods LAPC Xenografts and Human Tissues: LAPC xenografts were obtained from Dr. Charles Sawyers (UCLA) and generated as described (Klein et al, 1997, Nature Med. 3: 402-408; Craft et al., 1999, Cancer Res. 59: 5030-5036).

Androgen dependent and independent LAPC-4 AD and AI xenografts were grown in male SCID mice and were passaged as small tissue chunks in recipient males. LAPC-4 AI xenografts were derived from LAPC-4 AD tumors, respectively. To generate the AI xenografts, male mice bearing AD tumors were castrated and maintained for 2-3 months. After the tumors re-grew, the tumors were harvested and passaged in castrated males or in female SCID mice.

Cell Lines: Human cell lines (e. g., HeLa) were obtained from the ATCC and were maintained in DMEM with 5% fetal calf serum.

RNA Isolation: Tumor tissue and cell lines were homogenized in Trizol reagent (Life Technologies, Gibco BRL) using 10 ml/g tissue or 10 mi 108 cells to isolate total RNA. Poly A RNA was purified from total RNA using Qiagen's Oligotex mRNA Mini and Midi kits. Total and mRNA were quantified by spectrophotometric analysis (O. D. 260/280 nm) and analyzed by gel electrophoresis.

Oligonucleotides : The following HPLC purified oligonucleotides were used.

DPNCDN (cDNA synthesis primer) : 5'TTTTGATCAAGCTT3o3' (SEQ ID NO: 714) Adaptor 1: 5'CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAG3' (SEQ ID NO: 715) 3'GGCCCGTCCTAG5' (SEQ ID NO: 716) Adaptor 2:

5'GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAG3' (SEQ ID NO : 717) 3'CGGCTCCTAG5' (SEQ ID NO: 718) PCR primer 1: 5'CTAATACGACTCACTATAGGGC3' (SEQ ID NO: 719) Nested primer (NP) 1: 5'TCGAGCGGCCGCCCGGGCAGGA3' (SEQ ID NO: 720) Nested primer (nu) 2 : 5'AGCGTGGTCGCGGCCGAGGA3' (SEQ ID NO: 721) Suppression Subtractive Hybridization : Suppression Subtractive Hybridization (SSH) was used to identify cDNAs corresponding to genes that may be differentially expressed in prostate cancer. The SSH reaction utilized cDNA from two LAPC-4 AD xenografts. Specifically, to isolate genes that are involved in the progression of androgen dependent (AD) prostate cancer to androgen independent (AI) cancer, an experiment was conducted with the LAPC-4 AD xenograft in male SCID mice. Mice that harbored LAPC-4 AD xenografts were castrated when the tumors reached a size of 1 cm in diameter. The tumors regressed in size and temporarily stopped producing the androgen dependent protein PSA. Seven to fourteen days post-castration, PSA levels were detectable again in the blood of the mice. Eventually the tumors develop an AI phenotype and start growing again in the castrated males. Tumors were harvested at different time points after castration to identify genes that are turned on or off during the transition to androgen independence.

The gene 83P2H3 was derived from an LAPC-4 AD tumor (grown in intact male mouse) minus an LAPC-4 AD tumor (3 days post-castration) subtraction. The SSH DNA sequence (Figure 1) was identified.

The cDNA derived from an LAPC-4 AD tumor (3 days post-castration) was used as the source of the"driver"cDNA, while the cDNA from the LAPC-9 AD tumor (grown in intact male mouse) was used as the source of the"tester"cDNA. Double stranded cDNAs corresponding to tester and driver cDNAs were synthesized from 2 ug 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 xenograft source (see above) with a mix of digested cDNAs derived from the human cell lines HeLa, 293, A431, Colo205, and mouse liver.

Tester cDNA was generated by diluting 1 ul of Dpn II digested cDNA from the relevant xenograft source (see above) (400 ng) in 5 ul of water. The diluted cDNA (2 J. l, 160 ng) was then ligated to 2 Ill of Adaptor 1 and Adaptor 2 (10 uM), in separate ligation reactions, in a total volume of 10 ul at 16°C overnight, using 400 u of T4 DNA ligase (CLONTECH). Ligation was terminated with 1 1 of 0.2 M EDTA and heating at 72°C for 5 min.

The first hybridization was performed by adding 1.5 ul (600 ng) of driver cDNA to each of two tubes containing 1.5 ul (20 ng) Adaptor 1-and Adaptor 2-ligated tester cDNA. In a final volume of 4 ul, 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 ul of fresh denatured driver cDNA and were allowed to hybridize overnight at 68°C. The second hybridization was then diluted in 200 ul 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 ul of the diluted final hybridization mix was added to 1 gl of PCR primer 1 (10 I1M), 0.5 ul dNTP mix (10 I1M), 2.5 gl 10 x reaction buffer (CLONTECH) and 0.5 ul 50 x Advantage cDNA polymerase Mix (CLONTECH) in a final volume of 25 ul. 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 pl 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 uM) 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 llg of mRNA with oligo (dT) 12-18 priming using the Gibco-BRL Superscript Preamplification system. The manufacturer's protocol was used which included an incubation for 50 min at 42°C with reverse transcriptase followed by RNAse H treatment at 37°C for 20 min. After completing the reaction, the volume can be increased to 200 ul 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: 722) and S'agccacacgcagctcattgtagaagg 3' (SEQ ID NO: 723) to amplify P-actin. First strand cDNA (5 tl) were amplified in a total volume of 50 ul containing 0.4 1M primers, 0.2 uM each dNTPs, 1XPCR buffer (Clontech, 10 mM Tris-HCL, 1.5 mM MgCl2, 50 mM KC1, pH8. 3) and 1X Klentaq DNA polymerase (Clontech). Five gel 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. 8-actin bands from multiple tissues were compared by visual inspection.

Dilution factors for the first strand cDNAs were calculated to result in equal p-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 83P2H3 gene, 5 1ll of normalized first strand cDNA were analyzed by PCR using 26, and 30 cycles of amplification. Semi-quantitative expression analysis can be achieved by comparing the PCR products at cycle numbers that give light band intensities.

A typical RT-PCR expression analysis is shown in Figure 12. 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 83P2H3 was observed in prostate cancer xenografts, prostate cancer tissue pools, and metastatic cancer tissue pools.

Example 1B : 83P2H3 Family Member Identification A degenerate oligo PCR strategy was utilized to identify family members of the calcium transporter, 83P2H3. The family member CaTrF2E 11 was identified (Figure IB).

Materials and Methods A protein alignment between 83P2H3, AJ133128 (rabbit Calcium transporter), and AAD26363.1 (human vanilloid receptor-like protein) revealed at least two conserved regions. The conserved protein sequences listed below were used to design degenerate oligos where (a) represents adenine, (c) cytosine, (g) guanine, (t) thymine, (R) adenine or guanine, (Y) cytosine or thymine, (M) adenine and cytosine and (I) inosine.

Conserved Amino Acid Sequence Degenerate Oligo G (Q/H) (T/S) ALHIA 83P2H3. FMla: 5'ggIcaIWSIgcIYtIcaYatHgc 3' Y (F/Y) GE (H/L) PLS (F/L) AA 83P2H3. FM2.1: 5'aRIgaIaRIggIWgYtcIccRWaRta 3' 83P2H3. FM2.2: 5'aRRctIaRIggYaaYtcIccRWaRta 3' PCR optimization was performed using the Master Amp PCR Optimization Kit from Epicentre Technologies, Madison Wisconsin. (catalogue no. M07201). The kit provides 12 PCR optimization buffers, A through L, that differ in composition. RT-PCR utilized 83P2H3. FM 1 a and an equimolar mix of 83P2H3. FM2.1 and 83P2H3. FM2.2 to amplify CaTrF2El 1 from prostate cancer (1 patient), kidney cancer pool (2 kidney cancers), and bladder cancer pool (3 bladder cancers) first strand cDNAs. The first strand cDNAs were generated from polyA mRNA using Superscript reverse transcriptase (catalogue no. 18089-011 ; Life Technologies, Rockville Maryland). The first strand cDNAs were diluted to 150 1 for each llg of polyA mRNA used in the reverse transcriptase reaction and 5 ul was used in the RT-PCR reaction. Master Amp buffer G was the most optimal buffer for RT-PCR amplification. The sense (83P2H3. FMla) and anti-sense degenerate oligos (83P2H3. FM2.1/FM2.2) were at 1.2 uM and the reaction volume was 50 zip Thermal cycling conditions consisted of a single denaturation step at 92°C for 1 min followed by 35 cycles of 96°C for 30 sec, 50°C for 2 min and 72°C for 1 min. A 10 min, 72°C final extension completed the thermal cycling.

To remove primer-dimer and to prepare the PCR products for cloning, the Qiagen PCR Purification Kit was used (catalogue no. 28104, Valencia California). The purified RT-PCR product was cloned into pCR2.1 using the Invitrogen TA Cloning Kit (catalogue no. K2000-J10, Carlsbad California). White colonies from the transformation were picked into 96-well microtiter plates, grown overnight, and stored at-70°C in 20% glyceroL Clones were sequenced, assembled into contigs, and family members were identified.

Results The CaTrF2El 1 sequences was identified in multiple clones from prostate cancer (3/17), bladder cancer (11/17), and kidney cancer (3/17). The presence of CaTrF2El 1 in all three cancers suggests a role in cancer while the high incidence of CaTrF2El 1 in bladder cancer is indicative of a greater significance. In addition, expression analysis by RT-PCR and Northern blot analysis show expression in bladder, prostate, kidney, and lung cancer (Figure 8, Figure 9, Figure 28, Figure 29, Figure 30).

The nucleic and amino acid sequences and ORFs for CaTrF2El 1 are provided in Figure 1A.

The CaTrF2El 1 sequence is 161 bp in length and codes for a 53 amino acid polypeptide. The highest homology at the DNA and protein level is with the calcium transporter described in the published PCT appliction number W0200032766-A1 (Figure 3B). Other DNA and protein homologies were found with mouse and human vanilloid receptor-related osmotically activated channel (OTRPC4; GenBank Accessions NP 071300 and XP 027181 respectively). CaTrF2El 1 maps to 12q24. 1 (Liedtke et al., Cell 103: 525-535,2000).

Example 2: Full Length Cloning of 83P2H3 & Protein Topotosv A full length 83P2H3 cDNA clone (clone C) of 2899 bp was isolated from a human placenta library, revealing an ORF of 725 amino acids (Figure 2A-B and 3A). The human prostate CaT (PCaT)/83P2H3 ORF encodes a transporter protein with 6 predicted transmembrane domains, and is predicted to be a type IIIa plasma membrane protein using the PSORT program (available at the PSORT WWW Server at URL psort. nibb. ac. jp : 8800/form. html). The protein includes intracellular N- and C-terminal sequences. The hCaT/83P2H3 cDNA sequence is 99% identical to CaT-like B protein (Figure 4A-E).

The 83P2H3 cDNA clone C was deposited on May 19,2000, with the American Type Culture Collection (ATCC; 10801 University Blvd, Manassas, VA 20110) as plasmid p83P2H3-C, and has been assigned Accession No. PTA-1893.

Protein Topology Bioinformatic analysis and homology to ion transporters indicate that 83P2H3 may be expressed at the cell surface in one of two configurations. 83P2H3 may contain either 5 or 6 transmembrane domains that span the cytoplasmic membrane (Figure 13). Both configurations show the amino terminal end to be intracellular, and share the first 3 transmembrane domains (TM). The six TM (TM Pred: http ://www. ch. embnet. org/) model predicts TM1 to span aa 331-349, TM2 aa 390-408, TM3 aa 427-445, TM4 aa 451-469, TM5 aa 490-508, and TM6 aa 554-576, with the C-terminus being intracellular. The five TM model (Sosui : http://www. tuat. ac. jp/~mitaku/adv_sosui) predicts TM1 to span aa 329-351, TM2 aa 384-406, TM3 aa 433-455, TM4 aa 489-506 and TM5 aa 559-576, suggesting that the ion transporter pore is located at the 29 aa long second extracellular loop, and that the C-terminus is extracellular.

Example 3: Chromosomal Mapping of the 83P2H3 Gene Chromosomal localization can implicate genes in disease pathogenesis. Several chromosome mapping approaches are available including fluorescent in situ hybridization (FISH), human/hamster radiation hybrid (RH) panels (Walter et al., 1994; Nature Genetics 7: 22 ; Research Genetics, Huntsville Al), human-rodent somatic cell hybrid panels such as is available from the Coriell Institute (Camden,

New Jersey), and genomic viewers utilizing BLAST homologies to sequenced and mapped genomic clones (NCBI, Bethesda, Maryland).

The chromosomal localization of 83P2H3 was determined using the GeneBridge4 Human/Hamster radiation hybrid (RH) panel (Walter et al., 1994; Nature Genetics 7: 22) (Research Genetics, Huntsville Al).

The following PCR primers were used: 83P2H3.1 5'ACCAGGTTCATGTTCTGGTTCACA 3' 83P2H3.2 5'GCTCAAGTATGAGGATTGCAAGGT 3' The resulting 83P2H3 mapping vector for the 93 radiation hybrid panel DNAs <BR> <BR> <BR> <BR> (00000001100000100010001001001100000001110000001001101011001 00000012000002100000011 01100000100), and the mapping program available at the internet address http://www- genome. wi. mit. edu/cgi-bin/contig/rhmapper. pl, localizes the 83P2H3 gene to chromosome 7q34, a region frequently amplified or rearranged in cancer (Arranz E, et al., Cancer Genet Cytogenet 2000 Feb; 117 (1) : 41-4 ; Ong ST, Le Beau MM. Semin Oncol 1998 Aug; 25 (4): 447-60; Johnson E, Cotter FE.

Blood Rev 1997 Mar ; 1 l (l) : 46-55).

The 83P2H3 family member, CaTrF2El 1, maps to 12q24.1 (Liedtke et al., Cell 103: 525-535, 2000).

Example 4A: Expression analysis of 83P2H3 in normal tissues, cancer cell lines and patient samples 83P2H3 mRNA expression in normal human tissues was analyzed by northern blotting of multiple tissue blots (Clontech; Palo Alto, California), comprising a total of 16 different normal human tissues, using labeled 83P2H3 SSH fragment (Example 1A) as a probe. RNA samples were quantitatively normalized with a p-actin probe. Northern blot analysis using an 83P2H3 SSH fragment probe performed on 16 normal tissues showed predominant expression of a 2.5-3 kb transcript in prostate, placenta, and pancreas (Figure 5).

To analyze 83P2H3 expression in cancer tissues, northern blotting was performed on RNA derived from the LAPC xenografts, and several prostate cancer cell lines. The results show high expression levels in LAPC-4 AD, LAPC-9 AD, LAPC-9 AI, LNCaP and LAPC-4 CL (cell line) (Figure 6). Lower expression was observed in LAPC-4 AI.

Northern analysis also shows that 83P2H3 is expressed in prostate tumor tissues and the normal adjacent prostate tissue derived from prostate cancer patients (Figure 7).

RT-PCR is used to analyze expression of 83P2H3 in various tissues, including patient-derived cancers. First strand cDNAs are generated from 1 llg of mRNA with oligo (dT) 12-18 priming using the Gibco-BRL Superscript Preamplification System. The manufacturer's protocol is preferably

followed, and includes 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 is increased to 200 ul with water prior to normalization. First strand cDNAs are prepared from various tissues of interest.

Normalization can be performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR is performed using primers to 83P2H3.

In the present example, first strand cDNA was prepared from a vital pool 1 (VP1 : liver, lung and kidney), a vital pool 2 (VP2: pancreas, colon and stomach), a LAPC xenograft pool (LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC-9AI), a prostate cancer pool, and a metastatic cancer pool. The metastatic cancer pool consisted of metastatic tissues from cancers of the following organs: breast, ovarian, pancreas, colon, prostate and bladder. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to 83p2H3, was performed at 30 cycles of amplification. Results show expression of 83P2H3 in VP2, xenograft pool, prostate cancer pool and metastatic cancer pool (Figure 12).

These data indicate that 83P2H3 represents a suitable cancer target for diagnosis and therapy.

Example 4B : Expression analysis of CaTrF2Ell in normal tissues and patien specimens Analysis of CaTrF2El 1 by RT-PCR is shown in Figure 8 and Figure 9. Normal tissue expression is restricted to kidney and prostate. Analysis of human patient cancer RNA pools shows expression in bladder and kidney cancer pools (Figure 8 and Figure 9), and in lung and ovarian cancer pools (Figure 9).

Extensive northern blot analysis of CaTrF2E11 in 16 human normal tissues confirms the expression observed by RT-PCR (Figure 10). An approximately 4 kb transcript is detected in kidney, placenta, and to lower levels in prostate.

Northern blot analysis of CaTrF2E 11 on patient tumor specimens shows expression in bladder tumor tissues, kidney tumor tissues and lung tumor tissues derived from cancer patients (Figure 28).

Northern blot analysis of individual bladder cancer patient specimens shows expression of CaTrF2E 11 in all 4 bladder tumors tested and in one bladder cancer cell line SCaBER (Figure 29). The expression detected in normal adjacent tissue (isolated from a patient) but not in normal tissue (isolated from a healthy donor) may indicate that this tissue is not fully normal and that CaTrF2El 1 may be expressed in early stage tumors.

Expression of CaTrF2El l is also detected in 2 of 3 kidney cancer cell lines, and in all normal and kidney cancer tissues tested (Figure 30). In lung cancer samples, CaTrF2El 1 expression is observed in the CALU-1 cancer cell line and in 2 lung tumor tissues isolated from lung cancer patients (Figure 11). The expression detected in normal adjacent tissues (isolated from a patient) but not in

normal tissues (isolated from a healthy donor) may indicate that these tissues are not fully normal and that CaTrF2El 1 may be expressed in early stage tumors.

The restricted expression of CaTrF2El 1 in normal tissues and the expression detected in bladder cancer, lung cancer, ovarian cancer, and kidney cancer suggest that CaTrF2El 1 is a potential therapeutic target and a diagnostic marker for human cancers.

Example 5A : Production of Recombinant 83P2H3 in Prokarvotic Systems A. In vitro transcription and translation constructs: pCRII : To generate 83P2H3 sense and anti-sense RNA probes for RNA in situ investigations, pCRII constructs (Invitrogen, Carlsbad CA) are generated encoding either all or fragments of the 83P2H3 cDNA. The pCRII vector has Sp6 and T7 promoters flanking the insert to drive the. transcription of 83P2H3 RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze the cell and tissue expression of 83P2H3 at the RNA level. Transcribed 83P2H3 RNA representing the cDNA amino acid coding region of the 83P2H3 gene is used in in vitro translation systems such as the Tint Coupled Reticulolysate Sytem (Promega, Corp., Madison, WI) to synthesize 83P2H3 protein.

B. Bacterial Constructs: pGEX Constructs: To generate recombinant 83P2H3 proteins in bacteria that are fused to the Glutathione S-transferase (GST) protein, all or parts of the 83P2H3 cDNA protein coding sequence are fused to the GST gene by cloning into pGEX-6P-1 or any other GST-fusion vector of the pGEX family (Amersham Pharmacia Biotech, Piscataway, NJ). The constructs allow controlled expression of recombinant 83P2H3 protein sequences with GST fused at the amino-terminus and a six histidine epitope (6X His) at the carboxyl-terminus. The GST and 6X 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 His antibodies. The six histidine epitope tag is generated by adding 6 histidine codons to the cloning primer at the 3'end of the open reading frame (ORF). A proteolytic cleavage site, such as the PreScission recognition site in pGEX-6P-l, may be employed such that it permits cleavage of the GST tag from 83P2H3-related protein. The ampicillin resistance gene and pBR322 origin permits selection and maintenance of the pGEX plasmids in E. coli.

For example, constructs are made utilizing pGEX-6P-1 such that the following regions of 158P1D7 are expressed as an amino-terminal fusions to GST: amino acids 1 to 725; or any 8,9,10,11,12,13,14,15, or more contiguous amino acids from 83P2H3 or analogs thereof.

In one embodiment, amino acids 615-725 of 83P2H3 was cloned into pGEX-6P-l vector and the fusion protein was purified from induced bacteria. The fusion protein was subjected to proteolytic digestion with PreScission protease and the cleavage product free of GST sequences were used as an immunogen to generate polyclonal and monoclonal antibodies (see sections entitled"Generation of Polyclonal Antibodies"and"Generation of Monoclonal Antibodies", examples 6 and 7 respectively).

pMAL Constructs: To generate recombinant 83P2H3 proteins that are fused to maltose- binding protein (MBP) in bacterial cells, all or parts of the 83P2H3 cDNA protein coding sequence are fused to the MBP gene by cloning into the pMAL-c2X and pMAL-p2X vectors (New England Biolabs, Beverly, MA). The constructs allow controlled expression of recombinant 83P2H3 protein sequences with MBP fused at the ammo-terminus and a 6X His epitope at the carboxyl-terminus. The MBP and 6X 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 6X His is generated by adding the histidine codons to the 3'cloning primer. A Factor Xa recognition site permits cleavage of the pMAL tag from 83P2H3. 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. For example, constructs are made utilizing pMAL-c2X and pMAL-p2X such that the following regions of the 83P2H3 protein are expressed as amino-terminal fusions to MBP: amino acids 1 to 725; or any 8,9,10,11,12,13,14,15, or more contiguous amino acids from 83P2H3 or analogs thereof. pET Constructs: To express 83P2H3 in bacterial cells, all or parts of the 83P2H3 cDNA protein coding sequence is cloned into the pET family of vectors (Novagen, Madison, WI). These vectors allow tightly controlled expression of recombinant 83P2H3 protein in bacteria with and without fusion to proteins that enhance solubility, such as NusA and thioredoxin (Trx), and epitope tags, such as 6X His and S-Tag IM that aid purification and detection of the recombinant protein. For example, constructs are made utilizing pET NusA fusion system 43.1 such that the following regions of the 83P2H3 protein are expressed as an amino-terminal fusions to NusA: amino acids 1 to 725; or any 8, 9,10,11,12,13,14,15, or more contiguous amino acids from 83P2H3 or analogs thereof.

C. Yeast Constructs : pESC Constructs: To express 83P2H3 in the yeast species Saccharomyces cerevisiae for generation of recombinant protein and functional studies, all or parts of the 83P2H3 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, CA). These vectors allow controlled expression from the same plasmid of up to 2 different genes or cloned sequences containing either Flag or Myc epitope tags in the same yeast cell. This system is useful to confirm protein-protein interactions of 83P2H3. In addition, expression in yeast yields similar post-translational modifications, such as glycosylations and phosphorylations, that are found when expressed in eukaryotic cells. For example, constructs are made utilizing pESC-HIS such that the following regions of the 83P2H3 protein are expressed: amino acids 1 to 725; or any 8,9,10,11,12,13,14,15, or more contiguous amino acids from 83P2H3 or analogs thereof.

'pESP Constructs: To express 83P2H3 in the yeast species Saccharomycespombe, all or parts of the 83P2H3 cDNA protein coding sequence are cloned into the pESP family of vectors. These vectors allow controlled high level of expression of a 83P2H3 protein sequence that is fused at either

the amino terminus or at the carboxyl terminus to GST which aids purification of the recombinant protein. A Flag epitope tag allows detection of the recombinant protein with anti-FlagTM antibody.

For example, constructs are made utilizing pESP-1 vector such that the following regions of the 83P2H3 protein are expressed as amino-terminal fusions to GST: amino acids 1 to 725; or any 8,9,10, 11,12,13,14,15, or more contiguous amino acids from 83P2H3 or analogs thereof Example 5B : Production of Recombinant CaTrF2Ell in Prokarvotic Systems A. Bacterial Constructs: pGEX Constructs: To generate recombinant CaTrF2El 1 proteins in bacteria that are fused to the Glutathione S-transferase (GST) protein, all or parts of the CaTrF2Ell nucleic acid sequence are fused to the GST gene by cloning into pGEX-6P-l or any other GST-fusion vector of the pGEX family (Amersham Pharmacia Biotech, Piscataway, NJ). The constructs allow controlled expression of recombinant CaTrF2El 1 protein sequences with GST fused at the N-terminus and a six histidine epitope at the C-terminus. The GST and 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 HIS antibodies. The six histidine epitope tag is generated by adding the histidine codons to the cloning primer at the 3'end of the open reading frame (ORF). A proteolytic cleavage site, such as the PreScission recognition site in pGEX-6P-l, may be employed such that it permits cleavage of the GST tag from CaTrF2El l-related protein. The ampicillin resistance gene and pBR322 origin permits selection and maintenance of the pGEX plasmids in E. coli. For example, constructs are made utilizing pGEX-6P-1 such that the following regions of 158P1D7 are expressed as an amino- terminal fusions to GST: amino acids 1 to 963 ; or any 8, 9,10,11,12,13,14,15, or more contiguous amino acids from CaTrF2El 1 or analogs thereof. pMAL Constructs: To generate recombinant CaTrF2E 11 proteins that are fused to maltose- binding protein (MBP) in bacterial cells, all or parts of the CaTrF2El 11 nucleic acid sequence are fused to the MBP gene by cloning into the pMAL-c2X and pMAL-p2X vectors (New England Biolabs, Beverly, MA). The constructs allow controlled expression of recombinant CaTrF2El 1 protein sequences with MBP fused at the N-terminus and a six histidine epitope at the C-terminus. The MBP and 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 six histidine epitope tag is generated by adding the histidine codons to the 3'cloning primer. A Factor Xa recognition site permits cleavage of the pMAL tag from CaTrF2El 1. 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. For example, constructs are made utilizing pMAL-c2X and pMAL-p2X such that the following regions of the CaTrF2Ell protein are expressed as amino-terminal fusions to MBP: amino acids 1 to 963; or any 8, 9, 10,11,12,13,14,15, or more contiguous amino acids from CaTrF2Ell or analogs thereof.

pET Constructs: To express CaTrF2El 1 in bacterial cells, all or parts of the CaTrF2El 1 sequence is cloned into the pET family of vectors (Novagen, Madison, WI). These vectors allow tightly controlled expression of recombinant CaTrF2El 1 protein in bacteria with and without fusion to proteins that enhance solubility, such as NusA and thioredoxin (Trx), and epitope tags, such as 6X His and S-Tag TM that aid purification and detection of the recombinant protein. For example, constructs are made utilizing pET NusA fusion system 43.1 such that the following regions of the CaTrF2El 1 protein are expressed as an amino-terminal fusions to NusA: amino acids 1 to 963; or any 8, 9,10,11, 12,13,14,15, or more contiguous amino acids from CaTrF2El 1 or analogs thereof.

B. Yeast Constructs : pESC : To express CaTrF2El 1 in the yeast species Saccharomyces cerevisiae for generation of recombinant protein and functional studies, all or parts of the CaTrF2E 11 sequence is cloned into the pESC family of vectors each of which contain 1 of 4 selectable markers, HIS3, TRP 1, LEU2, and URA3 (Stratagene, La Jolla, CA). These vectors allow controlled expression from the same plasmid of up to 2 different genes or cloned sequences containing either Flag or Myc epitope tags in the same yeast cell. This system is useful to study protein-protein interactions of CaTrF2El 1. In addition, expression in yeast yields similar post-translational modifications, such as glycosylations and phosphorylations, that are found when expressed in eukaryotic cells. For example, constructs are made utilizing pESC-HIS such that the following regions of the CaTrF2El 11 protein are expressed: amino acids 1 to 963; or any 8,9,10,11,12,13,14,15, or more contiguous amino acids from CaTrF2El 1 or analogs thereof. pESP : To express CaTrF2El 1 in the yeast species Saccharomyces pombe, all or parts of the CaTrF2El 1 sequence is cloned into the pESP family of vectors. These vectors allow controlled high level of expression of a CaTrF2El 1 protein sequence that is fused at either the amino terminus or at the carboxyl terminus to GST which aids purification of the recombinant protein. A Flag epitope tag allows detection of the recombinant protein with anti-Flag antibody. For example, constructs are made utilizing pESP-1 vector such that the following regions of the CaTrF2El 1 protein are expressed as amino-terminal fusions to GST: amino acids 1 to 963; or any 8, 9,10,11,12,13,14,15, or more contiguous amino acids from CaTrF2Ell or analogs thereof. pCRII : To generate CaTrF2Ell sense and anti-sense riboprobes for RNA in situ investigations, pCRII constructs (Invitrogen, Carlsbad CA) are generated using cDNA sequence encoding all or fragments of the cDNA. The pCRII vector has Sp6 and T7 promoters flanking the insert to drive the production of CaTrF2El 1 RNA riboprobes for use in RNA in situ hybridization experiments.

Example 6A: Production of Recombinant 83P2H3 in Eukarvotic Systems A. Mammalian Constructs : To express recombinant 83P2H3 in eukaryotic cells, the full or partial length 83P2H3 cDNA sequences can be cloned into any one of a variety of expression vectors known in the art. 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-83P2H3 polyclonal serum, described above. pcDNA4/HisMax Constructs: To express 83P2H3 in mammalian cells, the 83P2H3 ORF is cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter and the SP 163 translational enhancer. The recombinant protein has XpressTM and six histidine epitopes fused to the N-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 ColEl origin permits selection and maintenance of the plasmid in E. coli. The following regions of 83P2H3 are expressed in this construct, amino acids 1 to 725; or any 8, 9,10,11,12,13,14,15, or more contiguous amino acids from 83P2H3, variants, or analogs thereof. pcDNA3. 1/MvcHis Constructs: To express 83P2H3 in mammalian cells, the ORFs with consensus Kozak translation initiation site arecloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the myc epitope and six histidines fused to the C-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 ColEl origin permits selection and maintenance of the plasmid in E. coli. The following regions of 83P2H3 are expressed in this construct, amino acids 1 to 725; or any 8,9,10,11,12,13,14,15, or more contiguous amino acids from 83P2H3, variants, or analogs thereof. pcDNA3. 1 Construct: To express 83P2H3 in mammalian cells the ORF with consensus Kozak translation initiation site was cloned into pcDNA3.1 (Invitrogen, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The pcDNA3.1 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 ColEl origin permits selection and maintenance of the plasmid in E. coli. The following regions of 83P2H3 are expressed in this

construct, amino acids 1 to 725; or any 8,9,10,11,12,13,14,15, or more contiguous amino acids from 83P2H3, variants, or analogs thereof. pcDNA3. 1/CT-GFP-TOPO Construct: To express 83P2H3 in mammalian cells and to allow detection of the recombinant proteins using fluorescence, the ORFs with consensus Kozak translation initiation site are cloned into pcDNA3.1CT-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 C-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 ColEl origin permits selection and maintenance of the plasmid in E. coli. An additional construct with a N-terminal GFP fusion is made in pcDNA3.1/NT-GFP-TOPO spanning the entire length of the 83P2H3 protein. The following regions of 83P2H3 are expressed in this construct, amino acids 1 to 725; or any 8,9,10,11,12,13,14,15, or more contiguous amino acids from 83P2H3, variants, or analogs thereof.

PAPtas : The 83P2H3 ORFs are cloned into pAPtag-5 (GenHunter Corp. Nashville, TN).

This construct generates an alkaline phosphatase fusion at the C-terminus of the 83P2H3 proteins while fusing the IgGK signal sequence to N-terminus. The resulting recombinant 83P2H3 proteins are optimized for secretion into the media of transfected mammalian cells and can be used to identify proteins such as ligands or receptors that interact with the 83P2H3 proteins. Protein expression is driven from the CMV promoter and the recombinant proteins also contain myc and six histidines fused to the C-terminus of alkaline phosphatase. The Zeocin resistance gene allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene permits selection of the plasmid in E. coli. The following regions of 83P2H3 are expressed in this construct, amino acids 1 to 725; or any 8,9,10,11,12,13,14,15, or more contiguous amino acids from 83P2H3, variants, or analogs thereof. ptag5 : The 83P2H3 ORFs are also cloned into pTag-5. This vector is similar to pAPtag but without the alkaline phosphatase fusion. This construct generates an immunoglobulin Gl Fc fusion at the C-terminus of the 83P2H3 protein while fusing the IgGK signal sequence to the N-terminus. The resulting recombinant 83P2H3 proteins are optimized for secretion into the media of transfected mammalian cells, and can be used to identify proteins such as ligands or receptors that interact with the 83P2H3 proteins. Protein expression is driven from the CMV promoter and the recombinant protein also contains myc and six histidines fused to the C-terminus of alkaline phosphatase. The Zeocin resistance gene allows for selection of mammalian cells expressing the protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli. The following regions of 83P2H3 are

expressed in this construct, amino acids 1 to 725; or any 8,9,10,11,12,13,14,15, or more contiguous amino acids from 83P2H3, variants, or analogs thereof.

PsecFc: The 83P2H3 ORFs are also cloned into psecFc. The psecFc vector was assembled by cloning immunoglobulin Gl Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen, California).

This construct generates an immunoglobulin Gl Fc fusion at the C-terminus of the 83P2H3 proteins, while fusing the IgG-kappa signal sequence to N-terminus. The resulting recombinant 83P2H3 protein is optimized for secretion into the media of transfected mammalian cells, and can be used to identify proteins such as ligands or receptors that interact with the 83P2H3 protein. Protein expression is driven from the CMV promoter and the recombinant protein also contain myc and six histidines fused to the C-terminus of alkaline phosphatase. The Zeocin resistance gene allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli. The following regions of 83P2H3 are expressed in this construct, amino acids 1 to 725; or any 8, 9,10,11,12,13,14,15, or more contiguous amino acids from 83P2H3, variants, or analogs thereof. pSRa Constructs : To generate mammalian cell lines that express 83P2H3 constitutively, the 83P2H3 ORF was cloned into pSRa construct. Amphotropic and ecotropic retroviruses were generated by transfection of pSRa constructs into the 293T-lOA1 packaging line or co-transfection of pSRa and a helper plasmid (containing deleted packaging sequences) into the 293 cells, respectively.

The retrovirus was used to infect a variety of mammalian cell lines, resulting in the integration of the cloned gene, 83P2H3, into the host cell-lines. Protein expression is driven from a long terminal repeat (LTR). The Neomycin resistance gene allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColEl 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, SCaBER, NIH 3T3, TsuPrl, 293 or rat-1 cells.

Additional pSRa constructs are made that fuse an epitope tag such as the FLAG tag to the C- terminus of 83P2H3 sequences to allow detection using anti-epitope tag antibodies. For example, the FLAG sequence 5'gat tac aag gat gac gac gat aag 3'is added to cloning primer at the 3'end of the ORF. Additional pSRa constructs are made to produce both N-terminal and C-terminal GFP and myc/6 HIS fusion proteins of the full-length 83P2H3 proteins. The following regions of 83P2H3 are expressed in such constructs, amino acids 1 to 725; or any 8,9,10,11,12,13,14,15, or more contiguous amino acids from 83P2H3, variants, or analogs thereof.

Additional Viral Vectors: Additional constructs are made for viral-mediated delivery and expression of 83P2H3. High virus titer leading to high level expression of 83P2H3 is achieved in viral delivery systems such as adenoviral vectors and herpes amplicon vectors. The 83P2H3 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. Altenatively, 83P2H3 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 SCaBER, NIH 3T3,293 or rat-1 cells. The following regions of 83P2H3 are expressed in this construct, amino acids 1 to 725; or any 8,9,10,11, 12,13,14,15, or more contiguous amino acids from 83P2H3, variants, or analogs thereof.

Regulated Expression Systems : To control expression of 83P2H3 in mammalian cells, coding sequences of 83P2H3 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 83P2H3. These vectors are thereafter used to control expression of 83P2H3 in various cell lines such as SCaBER, NIH 3T3,293 or rat-1 cells. The following regions of 83P2H3 are expressed in these constructs, amino acids 1 to 725; or any 8,9,10,11,12,13,14,15, or more contiguous amino acids from 83P2H3, variants, or analogs thereof.

B. Baculovirus Expression Systems To generate recombinant 83P2H3 proteins in a baculovirus expression system, 83P2H3 ORFs are cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag at the N-terminus. Specifically, pBlueBac-83P2H3 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 83P2H3 protein is then generated by infection of HighFive insect cells (Invitrogen) with purified baculovirus. Recombinant 83P2H3 protein can be detected using anti- 83P2H3 or anti-His-tag antibody. 83P2H3 protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for 83P2H3.

The following regions of 83P2H3 are expressed in this construct, amino acids 1 to 725; or any 8,9,10,11,12,13,14,15, or more contiguous amino acids from 83P2H3, variants, or analogs thereof.

Example 6B: Production of Recombinant CaTrF2E11 in Eukaryotic Systems A. Mammalian Constructs: To express recombinant CaTrF2El 1 in eukaryotic cells, the full or partial length CaTrF2El 1 cDNA sequences can be cloned into any one of a variety of expression vectors known in the art. The constructs can be transfected into any one of a wide variety of mammalian cells such as 293T cells.

Transfected 293T cells can be screened for recombinant CaTrF2El 1 as described above. pcDNA4/HisMax Constructs : To express CaTrF2El 1 in mammalian cells, the CaTrF2E11 ORF is cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter and the SP163 translational enhancer. The recombinant protein has XpressTM and six histidine epitopes fused to the N-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 ColEl origin permits selection and maintenance of the plasmid in E. coli. pcDNA3. 1/MvcHis Constructs: To express CaTrF2El 1 in mammalian cells, the ORFs with consensus Kozak translation initiation site are cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the myc epitope and six histidines fused to the C-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 ColEl origin permits selection and maintenance of the plasmid in E. coli. pcDNA3. 1/CT-GFP-TOPO Construct: To express CaTrF2El 1 in mammalian cells and to allow detection of the recombinant proteins using fluorescence, the ORFs with consensus Kozak translation initiation site are cloned into pcDNA3.1CT-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 C-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 ColEl origin permits selection and maintenance of the plasmid in E. coli. An additional construct with a N-terminal GFP fusion is made in pcDNA3.1/NT-GFP-TOPO spanning the entire length of the CaTrF2E 11 protein.

PAPtae : The CaTrF2El 1 sequences are cloned into pAPtag-5 (GenHunter Corp. Nashville, TN). This construct generates an alkaline phosphatase fusion at the C-terminus of the CaTrF2El 1 proteins while fusing the IgGK signal sequence to N-terminus. The resulting recombinant CaTrF2El 1 proteins are optimized for secretion into the media of transfected mammalian cells and can be used to identify proteins such as ligands or receptors that interact with the CaTrF2El 1 proteins. Protein expression is driven from the CMV promoter and the recombinant proteins also contain myc and six histidines fused to the C-terminus of alkaline phosphatase. The Zeocin resistance gene allows for selection of mammalian cells expressing the protein and the ampicillin resistance gene permits selection of the plasmid in E. coli.

tP a5 : The CaTrF2El 1 sequences are also cloned into pTag-5. This vector is similar to pAPtag but without the alkaline phosphatase fusion. This construct generates an immunoglobulin Gl Fc fusion at the C-terminus of the CaTrF2El 1 protein while fusing the IgGK signal sequence to the N- terminus. The resulting recombinant CaTrF2El 1 proteins are optimized for secretion into the media of transfected mammalian cells, and can be used to identify proteins such as ligands or receptors that interact with the CaTrF2El 1 proteins. Protein expression is driven from the CMV promoter and the recombinant protein also contains myc and six histidines fused to the C-terminus of alkaline phosphatase. The Zeocin resistance gene allows for selection of mammalian cells expressing the protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli.

PsecFc : The CaTrF2Ell sequences are also cloned into psecFc. The psecFc vector was assembled by cloning immunoglobulin G1 Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen, California). This construct generates an immunoglobulin G1 Fc fusion at the C-terminus of the CaTrF2El 1 proteins, while fusing the IgGK signal sequence to N-terminus. The resulting recombinant CaTrF2El 1 protein is optimized for secretion into the media of transfected mammalian cells, and can be used to identify proteins such as ligands or receptors that interact with the CaTrF2El 1 protein.

Protein expression is driven from the CMV promoter and the recombinant proteins also contain myc and six histidines fused to the C-terminus of alkaline phosphatase. The Zeocin resistance gene allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene permits selection of the plasmid in E. coli. uSRa Constructs: To generate mammalian cell lines that express CaTrF2E 11 constitutively, the sequences are cloned into pSRa constructs. Amphotropic and ecotropic retroviruses are generated by transfection of pSRa constructs into the 293T-10A1 packaging line or co-transfection of pSRa and a helper plasmid (containing deleted packaging sequences) into the 293 cells, respectively. The retrovirus can be used to infect a variety of mammalian cell lines, resulting in the integration of the cloned gene, CaTrF2El 1, into the host cell-lines. Protein expression is driven from a long terminal repeat (LTR). The Neomycin resistance gene allows for selection of mammalian cells that express the protein, and the ampicillin resistance gene and ColEl 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, SCaBER, NIH 3T3, TsuPrl, 293 or rat-1 cells.

Additional pSRa constructs are made that fuse an epitope tag such as the FLAG tag to the C- terminus of CaTrF2E 11 sequences to allow detection using anti-epitope tag antibodies. For example, the FLAG sequence 5'gat tac aag gat gac gac gat aag 3'is added to cloning primer at the 3'end of the ORF. Additional pSRa constructs are made to produce both N-terminal and C-terminal GFP and myc/6 HIS fusion proteins of the full-length CaTrF2El 1 proteins.

Additional Viral Vectors: Additional constructs are made for viral-mediated delivery and expression of CaTrF2El 1. High virus titer leading to high level expression of CaTrF2El l is achieved

in viral delivery systems such as adenoviral vectors and herpes amplicon vectors. The CaTrF2E11 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. Altenatively, CaTrF2E11 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 SCaBER, NIH 3T3,293 or rat-1 cells.

Regulated Expression Systems : To control expression of CaTrF2El 1 in mammalian cells, coding sequences of CaTrF2El 1 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 CaTrF2El 1. These vectors are thereafter used to control expression of CaTrF2E11 in various cell lines such as SCaBER, NIH 3T3, 293 or rat-1 cells.

B. Baculovirus Expression Systems To generate recombinant CaTrF2EI I proteins in a baculovirus expression system, CaTrF2El 1 ORFs are cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a His- tag at the N-terminus. Specifically, pBlueBac-CaTrF2E11 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 CaTrF2El 1 protein is then generated by infection of HighFive insect cells (Invitrogen) with purified baculovirus. Recombinant CaTrF2El 1 protein can be detected using anti-CaTrF2El 1 or anti-His-tag antibody. CaTrF2E 11 protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for CaTrF2E 11.

Example 7A: Antisenicitv Profiles of 83P2H3 Figure 14A, Figure 15A, Figure 16A, Figure 17A, and Figure 18A depict graphically five amino acid profiles of the 83P2H3 amino acid sequence, each assessment available by accessing the ProtScale website (URL www. expasy. ch/cgi-bin/protscale. pl) on the ExPasy molecular biology server.

These profiles: Figure 14A, Hydrophilicity, (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad.

Sci. U. S. A. 78 : 3824-3828); Figure 15A, Hydropathicity, (Kyte J., Doolittle R. F., 1982. J. Mol. Biol.

157: 105-132); Figure 16A, Percentage Accessible Residues (Janin J., 1979 Nature 277: 491-492); Figure 17A, Average Flexibility, (Bhaskaran R., and Ponnuswamy P. K., 1988. Int, J. Pept. Protein Res.

32: 242-255); Figure 18A, Beta-turn (Deleage, G., Roux B. 1987 Protein Engineering 1: 289-294); and optionally others available in the art, such as on the ProtScale website, were used to identify antigenic regions of the 83P2H3 protein. Each of the above amino acid profiles of 83P2H3 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 (Figure 14A), Hydropathicity (Figure 15A) and Percentage Accessible Residues (Figure 16A) 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 (Figure 17A) and Beta-turn (Figure 18A) 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 83P2H3 protein indicated, e. g., by the profiles set forth in Figure 14A, Figure 1 SA, Figure 16A, Figure 17A, or Figure 18A are used to prepare immunogens, either peptides or nucleic acids that encode them, to generate therapeutic and diagnostic anti-83P2H3 antibodies : The immunogen can be any 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22, 23,25,25,30,35,40,45,50 or more than 50 contiguous amino acids, or the corresponding nucleic acids that encode them, from the 83P2H3 protein. In particular, peptide immunogens of the invention can comprise, a peptide region of at least 5 amino acids of Figure 2A-B in any whole number increment up to 725 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of Figure 14A; a peptide region of at least 5 amino acids of Figure 2A-B in any whole number increment up to 725 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of Figure 15A ; a peptide region of at least 5 amino acids of Figure 2A-B in any whole number increment up to 725 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 16A; a peptide region of at least 5 amino acids of Figure 2A-B in any whole number increment up to 725 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile on Figure 17A ; and, a peptide region of at least 5 amino acids of Figure 2A-B in any whole number increment up to 725 that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of Figure 18A. 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.

Example 7B: Antisenicitv Profiles of CaTrF2E11 Figure 14B, Figure 15B, Figure 16B, Figure 17B, and Figure 18B depict graphically five amino acid profiles of the CaTrF2El 1 amino acid sequence, each assessment available by accessing the ProtScale website (URL www. expasy. ch/cgi-bin/protscale. pl) on the ExPasy molecular biology server.

These profiles: Figure 14B, Hydrophilicity, (Hopp T. P., Woods K. R., 1981. Proc. Natl. Acad.

Sci. U. S. A. 78: 3824-3828) ; Figure 15B, Hydropathicity, (Kyte J., Doolittle R. F., 1982. J. Mol. Biol.

157: 105-132); Figure 16B, Percentage Accessible Residues (Janin J., 1979 Nature 277: 491-492); Figure 17B, Average Flexibility, (Bhaskaran R., and Ponnuswamy P. K., 1988. Int. J. Pept. Protein Res.

32: 242-255); Figure 18B, Beta-turn (Deleage, G., Roux B. 1987 Protein Engineering 1: 289-294); and optionally others available in the art, such as on the ProtScale website, were used to identify antigenic regions of the CaTrF2El 1 protein. Each of the above amino acid profiles of CaTrF2El 1 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 (Figure 14B), Hydropathicity (Figure 15B) and Percentage Accessible Residues (Figure 16B) 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 (Figure 17B) and Beta-turn (Figure 18B) 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 CaTrF2E 11 protein indicated, e. g., by the profiles set forth in Figure 14B, Figure 15B, Figure 16B, Figure 17B, or Figure 18B are used to prepare immunogens, either peptides or nucleic acids that encode them, to generate therapeutic and diagnostic anti- CaTrF2E1l antibodies. The immunogen can be any 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19, 20,21,22,23,25,25,30,35,40,45,50 or more than 50 contiguous amino acids, or the corresponding nucleic acids that encode them, from the CaTrF2Ell protein. In particular, peptide immunogens of the invention can comprise, a peptide region of at least 5 amino acids of Figure 2C-D in any whole number increment up to 963 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of Figure 14B; a. peptide region of at least 5 amino acids of Figure 2C-D in any whole number increment up to 963 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of Figure 15B ; a. peptide region of at least 5 amino acids of Figure 2C-D in any whole number increment up to 963 that includes an amino acid position having a value greater than

0.5 in the Percent Accessible Residues profile of Figure 16B; a peptide region of at least 5 amino acids of Figure 2C-D in any whole number increment up to 963 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile on Figure 17B; and, a peptide region of at least 5 amino acids of Figure 2C-D in any whole number increment up to 963 that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of Figure 18B. 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.

Example 8A: Generation of 83P2H3 Polvclonal Antibodies Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. In addition to immunizing with the full length 83P2H3 protein, computer algorithms are employed in design of immunogens that, based on amino acid sequence analysis contain characteristics of being antigenic and available for recognition by the immune system of the immunized host (see the Example entitled "Antigenicity Profiles"). Such regions would be predicted to be hydrophilic, flexible, in beta-turn conformations, and be exposed on the surface of the protein (see, e. g., Figure 14A, Figure 15A, Figure 16A, Figure 17A, or Figure 18A for amino acid profiles that indicate such regions of 83P2H3).

For example, 83P2H3 recombinant bacterial fusion proteins or peptides encoding hydrophilic, flexible, beta-turn regions of the 83P2H3 sequence, such as amino acids 350-389 are used, and amino acids 615-725 of 83P2H3 were used as antigens to generate polyclonal antibodies in New Zealand White rabbits. 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 367-385 of 83P2H3 is conjugated to KLH and used to immunize the rabbit. Alternatively the immunizing agent may include all or portions of the 83P2H3 protein, analogs or fusion proteins thereof. For example, the 83P2H3 amino acid sequence can be fused using recombinant DNA techniques to any one of a variety of fusion protein partners that are well known in the art such as glutathione-S-transferase (GST) and HIS tagged fusion proteins. Such fusion proteins are purified from induced bacteria using the appropriate affinity matrix.

Other recombinant bacterial fusion proteins that may be employed include maltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see e. g. the section entitled "Expression of PHORlFSD6 in Prokaryotic Systems"Current and Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, P. S., Brady, W., Urnes, M., Grosmaire, L., Damle, N., and Ledbetter, L. (1991) J. Exp. Med. 174,561-566).

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 g, typically 100-200 gg, 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 jug, typically 100-200 u. g, of the immunogen in incomplete Freund's adjuvant (IFA). Test bleeds are taken approximately 7-10 days following each immunization and used to monitor the titer of the antiserum by ELISA.

To test serum, such as rabbit serum, for reactivity with 83P2H3 proteins, the full-length 83P2H3 cDNA can be cloned into an expression vector such as one that provides a 6 His tag at the carboxyl-terminus (pCDNA 3.1 myc-his, Invitrogen, see the Example herein entitled"Production of Recombinant 83P2H3 in Eukaryotic Systems"). After transfection of the constructs into 293T cells, cell lysates are probed with the anti-83P2H3 serum and with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) to determine specific reactivity to denatured 83P2H3 protein using the Western blot technique. In addition, recognition of native protein by the antiserum can be determined by flow cytometric analysis of 293T or other recombinant 83P2H3-expressing cells.

Alternatively, specificity of the antiserum is tested by Western blot, immunoprecipitation, and flow cytometric techniques using lysates of cells that endogenously express 83P2H3.

Sera from rabbits immunized with fusion proteins, such as GST and MBP fusion proteins, are purified by depletion of antibodies reactive to GST, MBP, or other fusion partner sequence by passage over an affinity column containing the fusion partner either alone or in the context of an irrelevant fusion protein. Sera from His-tagged protein and peptide immunized rabbits as well as fusion partner depleted sera are further purified by passage over an affinity column composed of the original protein immunogen or free peptide coupled to Affigel matrix (BioRad).

In one embodiment, a GST-fusion protein encoding amino acids 615-725 of 83P2H3 was produced and purified and a cleavage product was generated in which GST sequences were removed by proteolytic cleavage. This cleavage protein was used to generate a polyclonal antibody by immunization of a rabbit. The rabbit immune serum was partially purified by removal of anti-bacterial and anti-GST reactive antibodies by passage over an irrelevant GST-fusion protein column and then further purified by protein G column chromatography. This polyclonal antibody specifically recognized 83P2H3 protein on 293T cells by Western blotting and immunohistochemistry, and stained the surface of 293T-83P2H3 and PC3-83P2H3 cells demonstrating that the 83P2H3 protein resides in the plasma membrane (Figure 19 and Figure 31).

Example 8B : Generation of CaTrF2Ell Polvelonal Antibodies Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. In addition to immunizing with the full length CaTrF2El 1 protein, computer algorithms are employed in design of immunogens that, based on amino acid sequence analysis contain characteristics of being antigenic and available for recognition by the immune system of the immunized host (see the Example entitled "Antigenicity Profiles"). Such regions would be predicted to be hydrophilic, flexible, in beta-turn conformations, and be exposed on the surface of the protein (see, e. g., Figure 14B, Figure 15B, Figure 16B, Figure 17B, or Figure 18B for amino acid profiles that indicate such regions of CaTrF2El 1).

For example, CaTrF2Ell recombinant bacterial fusion proteins or peptides encoding hydrophilic, flexible, beta-turn regions of the CaTrF2Ell sequence, such as amino acids 586-606,733- 758, and amino acids 812-963 of CaTrF2El 1 are used as antigens to generate polyclonal antibodies in New Zealand White rabbits. 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 586-606 of CaTrF2Ell is conjugated to KLH and used to immunize the rabbit. Alternatively the immunizing agent may include all or portions of the CaTrF2El 1 protein, analogs or fusion proteins thereof. For example, the CaTrF2El 1 amino acid sequence can be fused using recombinant DNA techniques to any one of a variety of fusion protein partners that are well known in the art such as glutathione-S-transferase (GST) and HIS tagged fusion proteins. Such fusion proteins are purified from induced bacteria using the appropriate affinity matrix. Other recombinant bacterial fusion proteins that may be employed include maltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see e. g. the section entitled"Expression of ! F5D6 in Prokaryotic Systems"and Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995 ; Linsley, P. S., Brady, W., Urnes, M., Grosmaire, L., Damle, N., and Ledbetter, L. (1991) J. Exp. Med. 174,561-566; Novagen, Madison, WI). In one embodiment, a GST-fusion protein encoding amino acids 816-963 of CaTrF2El 1 is produced and purified and a cleavage product is generated in which GST sequences are removed by proteolytic cleavage. This cleavage protein is used to generate a polyclonal antibody by immunization of a rabbit.

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 g, typically 100-200 ug, 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 ug, typically 100-200 ug, 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 serum, such as rabbit serum, for reactivity with CaTrF2El 1 proteins, the full-length CaTrF2El 11 cDNA can be cloned into an expression vector such as one that provides a 6 His tag at the carboxyl-terminus (pCDNA 3.1 myc-his, Invitrogen, see the Example entitled"Production of Recombinant CaTrF2El 1 in Eukaryotic Systems"). After transfection of the constructs into 293T cells, cell lysates are probed with the anti-CaTrF2El 1 serum and with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) to determine specific reactivity to denatured CaTrF2El 1 protein using the Western blot technique. In addition, recognition of native protein by the antiserum can be determined by flow cytometric analysis of 293T or other recombinant CaTrF2El 1-expressing cells.

Alternatively, specificity of the antiserum is tested by Western blot, immunoprecipitation, and flow cytometric techniques using lysates of cells that endogenously express CaTrF2El 1.

Sera from rabbits immunized with fusion proteins, such as GST and MBP fusion proteins, are purified by depletion of antibodies reactive to GST, MBP, or other fusion partner sequence by passage over an affinity column containing the fusion partner either alone or in the context of an irrelevant fusion protein. Sera from His-tagged protein and peptide immunized rabbits as well as fusion partner depleted sera are further purified by passage over an affinity column composed of the original protein immunogen or free peptide coupled to Affigel matrix (BioRad).

Example 9A: Generation of 83P2H3 Monoclonal Antibodies (mAbs) In one embodiment, therapeutic mAbs to 83P2H3 comprise those that react with epitopes of the protein that would disrupt or modulate the biological function of 83P2H3, for example those that disrupt the Ca2+ transport function of 83P2H3. Therapeutic mAbs also comprise those which specifically bind epitopes of 83P2H3 exposed on the cell surface and thus are useful in targeting mAb- toxin conjugates. Immunogens for generation of such mAbs include those designed to encode or contain the entire 83P2H3 protein or regions of the 83P2H3 protein predicted to be antigenic from computer analysis of the amino acid sequence (see, e. g., Figure 14A, Figure 15A, Figure 16A, Figure 17A, or Figure 18A, and the Example entitled"Antigenicity Profiles").

Immunogens include peptides, recombinant bacterial proteins, and mammalian expressed Tag 5 proteins and human and murine IgG Fc fusion proteins. In addition, cells expressing high levels of 83P2H3, such as 293T-83P2H3 cells, are used to immunize mice. To generate mAbs to 83P2H3, mice are first immunized intraperitoneally (IP) with, typically, 10-50 ig of protein immunogen or 107

83P2H3-expressing cells mixed in complete Freund's adjuvant. Mice are then subsequently immunized IP every 2-4 weeks with, typically, 10-50 gg of protein immunogen or 107 cells mixed in incomplete Freund's adjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations.

Alternatively, a DNA-based immunization protocol is employed in which a mammalian expression vector encoding 83P2H3 sequence is used to immunize mice by direct injection of the plasmid DNA. For example, either pCDNA 3.1 encoding the full length 83P2H3 cDNA, or amino acids 615-725 of 83P2H3 (predicted to contain ntigenic sequences from analysis, see, e. g., Figure 14A, Figure 15A, Figure 16A, Figure 17A, or Figure 18A) fused at the N-terminus to an IgK leader sequence and at the C-terminus to the coding sequence of the murine or human IgG Fc region, is used.

This protocol is used alone or in combination with protein or cell-based immunogens. Test bleeds are taken 7-10 days following immunization to monitor titer and specificity of the immune response. Once appropriate reactivity and specificity is obtained as determined by ELISA, Western blotting, immunoprecipitation, 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 83P2H3 monoclonal antibodies, a glutathione-S-transferase (GST) fusion protein encoding amino acids 615-725 of 83P2H3 protein was expressed and purified.

An 83P2H3 amino acid-specific cleavage fragment of the immunogen in which GST was removed by site-specific proteolysis was then used as immunogen. Balb C mice were initially immunized intraperitoneally with 25 ug of the 83P2H3 cleavage protein mixed in complete Freund's adjuvant.

Mice were subsequently immunized every two weeks with 25 pg of 83P2H3 cleavage protein mixed in incomplete Freund's adjuvant for a total of three immunizations. The titer of serum from immunized mice was determined by ELISA using the full length GST-fusion protein and the cleaved immunogen.

Reactivity and specificity of serum to full length 83P2H3 protein was monitored by Western blotting and flow cytometry using 293T cells transfected with an expression vector encoding the 83P2H3 cDNA (see e. g., the Example entitled"Production of Recombinant 83P2H3 in Eukaryotic Systems").

As can be seen in Figure 19A-F, serum from a representative immunized mouse specifically recognized 83P2H3 on the surface of 293T cells as determined by flow cytometry and in 293T cell lysates by Western blotting. Two mice showing the strongest reactivity were rested and given a final injection of GST-83P2H3 fusion protein in PBS and then sacrificed four days later. The spleens of the sacrificed mice were then harvested and fused to SPO/2 myeloma cells using standard procedures (Harlow and Lane, 1988). Supernatants from growth wells following HAT selection were screened by ELISA, Western blot, and flow cytometry to identify 83P2H3 specific antibody-producing clones, As shown in Figure 20A-F, two hybridoma supernatants, #4 and #8A, specifically recognized 83P2H3 protein by Western blotting and stained the surface of 293T-83P2H3 cells.

The binding affinity of a 83P2H3 monoclonal antibody is determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and are used

to help define which 83P2H3 monoclonal antibodies preferred for diagnostic or therapeutic use, as appreciated by one of skill in the art. The BIAcore system (Uppsala, Sweden) is a preferred method for determining binding affinity. The BIAcore system uses surface plasmon resonance (SPR, Welford K.

1991, Opt. Quant. Elect. 23: 1; Morton and Myszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecular interactions in real time. BIAcore analysis conveniently generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants.

Example 9B: Generation of CaTrF2Ell Monoclonal Antibodies (ni. Abs) In one embodiment, therapeutic mAbs to CaTrF2El 1 comprise those that react with epitopes of the protein that would disrupt or modulate the biological function of CaTrF2E 11, for example those that disrupt the ion transport function of CaTrF2E 11. Therapeutic mAbs also comprise those which specifically bind epitopes of CaTrF2El 1 exposed on the cell surface and thus are useful in targeting mAb-toxin conjugates. Immunogens for generation of such mAbs include those designed to encode or contain the entire CaTrF2El 11 protein or regions of the CaTrF2El 1 protein predicted to be antigenic from computer analysis of the amino acid sequence (see, e. g., Figure 14B, Figure 15B, Figure 16B, Figure 17B, or Figure 18B, and the Example entitled"Antigenicity Profiles").

Immunogens include peptides, recombinant bacterial proteins, and mammalian expressed Tag 5 proteins and human and murine IgG Fc fusion proteins. In addition, cells expressing high levels of 83P2H3, such as 293T-83P2H3 cells, are used to immunize mice. To generate mAbs to 83P2H3, mice are first immunized intraperitoneally (IP) with, typically, 10-50 ug of protein immunogen or 107 83P2H3-expressing cells mixed in complete Freund's adjuvant. Mice are then subsequently immunized IP every 2-4 weeks with, typically, 10-50 ug of protein immunogen or 107 cells mixed in incomplete Freund's adjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations.

Alternatively, a DNA-based immunization protocol is employed in which a mammalian expression vector encoding CaTrF2El 1 sequence is used to immunize mice by direct injection of the plasmid DNA. For example, either pCDNA 3.1 encoding the full length CaTrF2El 1 cDNA, or amino acids 816-963 of CaTrF2El 1 (predicted to be antigenic from sequence analysis, see, e. g., Figure 14B, Figure 15B, Figure 16B, Figure 17B, or Figure 18B) fused at the N-terminus to an IgK leader sequence and at the C-terminus to the coding sequence of the murine or human IgG Fc region, is used. This protocol is used alone or in combination with protein or cell-based immunogens. Test bleeds are taken 7-10 days following immunization to monitor titer and specificity of the immune response. Once appropriate reactivity and specificity is obtained as determined by ELISA, Western blotting, immunoprecipitation, 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 CaTrF2El 1 monoclonal antibodies, a peptide is synthesized encoding amino acids 733-758 and is coupled to KLH. Balb C mice are initially immunized

intraperitoneally with 25 u. g of the peptide conjugate mixed in complete Freund's adjuvant. Mice are subsequently immunized every two weeks with 25 llg of peptide conjugate mixed in incomplete Freund's adjuvant for a total of three immunizations. The titer of serum from immunized mice is determined by ELISA using non-conjugated free peptide. Reactivity and specificity of serum to full length CaTrF2El 1 protein is monitored by Western blotting and flow cytometry using 293T cells transfected with an expression vector encoding the CaTrF2El 1 cDNA (see e. g., the Example entitled "Production ofRecombinant CaTrF2El 1 in Eukaryotic Systems"). Mice showing the strongest reactivity are rested and given a final injection of peptide conjugate in PBS and then sacrificed four days later. The spleens of the sacrificed mice are then harvested and fused to SPO/2 myeloma cells using standard procedures (Harlow and Lane, 1988). Supernatants from growth wells following HAT selection are screened by ELISA, Western blot, and flow cytometry to identify CaTrF2El 1 specific antibody-producing clones.

The binding affinity of a CaTrF2El 1 monoclonal antibody is determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which CaTrF2El 1 monoclonal antibodies preferred for diagnostic or therapeutic use, as appreciated by one of skill in the art. The BIAcore system (Uppsala, Sweden) is a preferred method for determining binding affinity. The BIAcore system uses surface plasmon resonance (SPR, Welford K.

1991, Opt. Quant. Elect. 23: 1; Morton and Myszka, 1998, Methods in Enzymology 295: 268) to monitor biomolecular interactions in real time. BIAcore analysis conveniently generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants.

Example 10: HLA Class I and Class II Bindine Assays HLA class I and class II binding assays using purified HLA molecules are performed in accordance with disclosed protocols (e. g., PCT publications WO 94/20127 and WO 94/03205; Sidney et al., Current Protocols in Immunology 18.3.1 (1998) ; Sidney, et al., J. Immunol. 154 : 247 (1995); Sette, et al., Mol. Immunol. 31: 813 (1994)). Briefly, purified MHC molecules (5 to 500 nM) are incubated with various unlabeled peptide inhibitors and 1-10 nM l25I-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 ICso> [HLA], the measured ICso values are reasonable approximations of the true KD values. Peptide inhibitors are typically tested at concentrations ranging from 120 ug/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 ICso of a positive control for inhibition by the ICso 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 ICso nM values by dividing the ICso 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-Bearine 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 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 employ the protein sequence data from the gene product of 83P2H3 set forth in Figure 2 and Figure 3.

Computer searches for epitopes bearing HLA Class I or Class II supermotifs or motifs are performed as follows. All translated 83P2H3 protein sequences are analyzed using a text string search software program to identify potential peptide sequences containing appropriate HLA binding motifs; such programs are readily produced in accordance with information in the art in view of known motif/supermotif disclosures. Furthermore, such calculations can be made mentally.

Identified A2-, A3-, and DR-supermotif sequences are scored using polynomial algorithms to predict their capacity to bind to specific HLA-Class I or Class II molecules. These polynomial algorithms account for the impact of different amino acids at different positions, and are essentially based on the premise that the overall affinity (or AG) of peptide-HLA molecule interactions can be approximated as a linear polynomial function of the type: "AG"= all x a2 x a3 x a" 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 amountj 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 j. 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 sunertype cross-reactive peptides Complete protein sequences from 83P2H3 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 83P2H3 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 83P2H3 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 ICso 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 83P2H3 protein can also be performed to identify HLA- Al-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 Immunoeenicitv Cross-reactive candidate CTL A2-supermotif-bearing peptides that are identified as described herein are selected for in vitro immunogenicity testing. Testing is performed using the following methodology: Target Cell Lines for Cellular Screening : The. 221A2. 1 cell line, produced by transferring the HLA-A2.1 gene into the HLA-A,-B,-C null mutant human B-lymphoblastoid cell line 721.221, is used as the peptide-loaded target to measure activity of HLA-A2. 1-restricted CTL. This cell line is grown in RPMI-1640 medium supplemented with antibiotics, sodium pyruvate, nonessential amino acids and 10% (v/v) heat inactivated FCS. Cells that express an antigen of interest, or transfectants comprising the gene encoding the antigen of interest, can be used as target cells to test 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 llg/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 x 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. TNFa 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 (DynabeadsX M-450) and the detacha-beadX reagent. Typically about 200-250x106 PBMC are processed to obtain 24xl06 CD8+ T-cells (enough for a 48-well plate culture).

Briefly, the PBMCs are thawed in RPMI with 30, ug/ml DNAse, washed once with PBS containing 1% human AB serum and resuspended in PBS/1% AB serum at a concentration of 20xl06cells/ml. The magnetic beads are washed 3 times with PBS/AB serum, added to the cells (140µl beads/20x106 cells) and incubated for 1 hour at 4°C with continuous mixing. The beads and cells are washed 4x with PBS/AB serum to remove the nonadherent cells and resuspended at 100X106 cells/ml (based on the original cell number) in PBS/AB serum containing 10011vol detacha-beadX reagent and 30 llg/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 4011g/ml of peptide at a cell concentration of 1-2xlO6/ml in the presence of3ug/ml 82- 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 1x105 cells/ml) are co- cultured with 0. 25ml of CD8+ T-cells (at 2x106 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 5X106 cells/ml and irradiated at-4200 rads. The PBMCs are plated at 2x106 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 lOgg/ml of peptide in the presence of 3 fig/Ml B2 microglobulin in 0.25ml 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 mol with fresh media. The cells are then transferred to the wells containing the peptide-pulsed adherent cells. Twenty four hours later recombinant human IL-10 is added at a final concentration of 10 ng/ml and recombinant human IL2 is added the next day and again 2-3 days later at 50IU/ml (Tsai et al., Critical Reviews in Immunology 18 (1-2): 65-75,1998). Seven days later, the cultures are assayed for CTL activity in a 5'Cr 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 lvtic activity by 5lCr release.

Seven days after the second restimulation, cytotoxicity is determined in a standard (5 hr) 5'Cr release assay by assaying individual wells at a single E: T. Peptide-pulsed targets are prepared by incubating the cells with lOug/ml peptide overnight at 37°C.

Adherent target cells are removed from culture flasks with trypsin-EDTA. Target cells are labeled with 200gCi of 5'Cr sodium chromate (Dupont, Wilmington, DE) 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. 3x106/ml (an NK-sensitive erythroblastoma cell line used to reduce non-specific lysis). Target cells (100 ul) and effectors (100111) are plated in 96 well round-bottom plates and incubated for 5 hours at 37°C. At that time, 100 ul 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 S'cor release sample)/ (cpm of the maximal S'Cr release sample-cpm of the spontaneous S'Cr release sample)] x 100.

Maximum and spontaneous release are determined by incubating the labeled targets with 1% Triton X-100 and media alone, respectively. A positive culture is defined as one in which the specific lysis (sample-background) is 10% or higher in the case of individual wells and is 15% or more at the two highest E: T ratios when expanded cultures are assayed.

In situ Measurement of Human IFNv Production as an Indicator of Peptide-specific and Endogenous Recognition Immulon 2 plates are coated with mouse anti-human IFNy monoclonal antibody (4 ug/ml 0. 1M NaHCO3, pH8.2) overnight at 4°C. The plates are washed with Ca 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 jul/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 1x106 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 1200pg/100 microliter/well and the plate incubated for two hours at 37°C. The plates are washed and 100 111 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 6x with wash buffer, 100 microliter/well developing solution (TMB 1: 1) are added, and the plates allowed to develop for 5-15 minutes. The reaction is stopped with 50 microliter/well 1M H3PO4 and read at OD450. A culture is considered positive if it measured at least 50 pg of IFN-gamma/well above background and is twice the background level of expression.

CTL Expansion.

Those cultures that demonstrate specific lytic activity against peptide-pulsed targets and/or tumor targets are expanded over a two week period with anti-CD3. Briefly, 5x104 CD8+ cells are added to a T25 flask containing the following: 1x106 irradiated (4,200 rad) PBMC (autologous or allogeneic) per ml, 2x105 irradiated (8, 000 rad) EBV-transformed cells per ml, and OKT3 (anti-CD3)

at 30ng per ml in RPMI-1640 containing 10% (v/v) human AB serum, non-essential amino acids, sodiumpyruvate, 2511M 2-mercaptoethanol, L-glutamine and penicillin/streptomycin. Recombinant human IL2 is added 24 hours later at a final concentration of 200IU/ml and every three days thereafter with fresh media at 50IU/ml. The cells are split if the cell concentration exceeds lxlO6/rnl and the cultures are assayed between days 13 and 15 at E: T ratios of 30,10,3 and 1: 1 in the 'Cr release assay or at 1x106/ml in the in situ IFNy 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 5x104 CD8+ cells are added to a T25 flask containing the following : 1x106 autologous PBMC per ml which have been peptide-pulsed with 10 u. g/ml peptide for two hours at 37°C and irradiated (4,200 rad); 2x105 irradiated (8,000 rad) EBV-transformed cells per ml RPMI-1640 containing 10% (v/v) human AB serum, non-essential AA, sodium pyruvate, 25mM 2-ME, L-glutamine and gentamicin.

Immuno e, g nicitv 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 83P2H3. Briefly, PBMCs are isolated from patients, re-stimulated with peptide-pulsed monocytes and assayed for the ability to recognize peptide-pulsed target cells as well as transfected cells endogenously expressing the antigen.

Evaluation of A*03/Al l immunogenicitv 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 evaluated in a manner analogous to the evaluation of A2-and A3-supermotif- bearing peptides.

Peptides bearing other supermotifs/motifs, e. g., HLA-A1, HLA-A24 etc. are also evaluated using similar methodology Example 13: Implementation of the Extended Supermotif to Improve the Binding Capacity of Native Epitopes by Creatine Analogs HLA motifs and supermotifs (comprising primary and/or secondary residues) are useful in the identification and preparation of highly cross-reactive native peptides, as demonstrated herein.

Moreover, the definition of HLA motifs and supermotifs also allows one to engineer highly cross- reactive epitopes by identifying residues within a native peptide sequence which can be analoged to confer upon the peptide certain characteristics, e. g. greater cross-reactivity within the group of HLA molecules that comprise a supertype, and/or greater binding affinity for some or all of those HLA molecules. Examples of analoging peptides to exhibit modulated binding affinity are set forth in this example.

Analoging at Primarv 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 tested for binding to one or all supertype members and then analoged to modulate binding affinity to any one (or more) of the supertype members to add population coverage.

The selection of analogs for immunogenicity in a cellular screening analysis is typically further restricted by the capacity of the parent wild type (WT) peptide to bind at least weakly, i. e., bind at an ICso of 5000nM 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 demonstrate 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 tested for 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 tested for immunogenicity, typically in a cellular screening assay. Again, it is generally important to demonstrate that analog-specific CTLs are also able to recognize the wild-type peptide and, when possible, targets that endogenously express the epitope.

Analoging at Secondary Anchor Residues Moreover, HLA supermotifs are of value in engineering highly cross-reactive peptides and/or peptides that bind HLA molecules with increased affinity by identifying particular residues at secondary anchor positions that are associated with such properties. For example, the binding capacity of a B7 supermotif-bearing peptide with an F residue at position 1 is analyzed. The peptide is then analoged to, for example, substitute L for F at position 1. The analoged peptide is evaluated for increased binding affinity, binding half life and/or increased cross-reactivity. Such a procedure identifies analoged peptides with enhanced properties.

Engineered analogs with sufficiently improved binding capacity or cross-reactivity can also be tested for immunogenicity in HLA-B7-transgenic mice, following for example, IFA immunization or lipopeptide immunization. Analoged peptides are additionally tested for the ability to stimulate a recall response using PBMC from patients with 83P2H3-expressing tumors.

Other analoging strategies Another form of peptide analogizing, unrelated to anchor positions, involves the substitution of a cysteine with a-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 a-amino butyric acid for cysteine not only alleviates this problem, but has been shown to improve binding and crossbinding capabilities in some instances (see, e. g., the review by Sette et al., In: Persistent Viral Infections, Eds. R. Ahmed and I. Chen, John Wiley & Sons, England, 1999).

Thus, by the use of single amino acid substitutions, the binding properties and/or cross- reactivity of peptide ligands for HLA supertype molecules can be modulated.

Example 14: Identification of 83P2H3/CaTrF2Ell-derived sequences with HLA-DR binding motifs Peptide epitopes bearing an HLA class II supermotif or motif are identified as outlined below using methodology similar to that described for HLA Class I peptides.

Selection of HLA-DR-supermotif-bearing epitopes.

To identify 83P2H3-derived, HLA class II HTL epitopes, the 83P2H3 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 83P2H3-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 P 1, DR2w2 P2, DR6wl 9, 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, DR5wl 1, 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. 83P2H3-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 83P2H3 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 tested for the ability to bind DR3 with an affinity of luM or better, i. e., less than 1 uM. 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: Immunosenicitv of 83P2H3/CaTrF2E11-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 evaluated 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 83P2H3- expressing tumors.

Example 16: Calculation of phenotvpic 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=l-(SQRT (l-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- zu 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* (l-A)).

Confirmed members of the A3-like supertype are A3, All, A31, A*3301, andA*6801. Althoughthe 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 Al 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 Al 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.

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 determines 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 5'Cr labeled Jurkat-A2.1/Kb target cells in the absence or presence of peptide, and also tested on 5'Cr labeled target cells bearing the endogenously synthesized antigen, i. e. cells that are stably transfected with 83P2H3 expression vectors.

The results demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized 83P2H3 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 Al 1, 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 Coniueated Epitopes In Transgenic Mice This example illustrates the induction of CTLs and HTLs in transgenic mice, by use of a 83P2H3-derived CTL and HTL peptide vaccine compositions. The vaccine composition used herein comprise peptides to be administered to a patient with a 83P2H3-expressing tumor. The peptide composition. can comprise multiple CTL and/or HTL epitopes. The epitopes are identified using methodology as described herein. This example also illustrates that enhanced immunogenicity can be achieved by inclusion of one or more HTL epitopes in a CTL vaccine composition; such a peptide composition can comprise an HTL epitope conjugated to a CTL epitope. The CTL epitope can be one that binds to multiple HLA family members at an affinity of 500 nM or less, or analogs of that epitope.

The peptides may be lipidated, if desired.

Immunization procedures : Immunization of transgenic mice is performed as described (Alexander et al., J. Immunol. 159: 4753-4761,1997). For example, A2/Kb mice, which are transgenic for the human HLA A2.1 allele and are used to assess 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 syngeneic 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/Kbchimeric gene (e. g., Vitiello et al., J. Exp. Med. 173: 1007,1991) In vitro CTL activation : One week after priming, spleen cells (30x106 cells/flask) are co- cultured at 37°C with syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (10x10" 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. 5x106) are incubated at 37°C in the presence of 200 ul of 5'Cr. After 60 minutes, cells are washed three times and resuspended in R10 medium. Peptide is added where required at a concentration of 1 ug/ml. For the assay, 104 Cr- labeled target cells are added to different concentrations of effector cells (final volume of 200 p1) 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 x (experimental release-spontaneous release)/ (maximum release-spontaneous release). To facilitate comparison between separate CTL assays run under the same conditions, % 5'Cr 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 5'Cr release assay. To obtain specific lytic units/106, the lytic units/106 obtained in the absence of peptide is subtractedfromthelyticunits/106 obtainedinthe presence of peptide. For example, if 30% 'Cr release is obtained at the effector (E): target (T) ratio of

50: 1 (i. e., 5x105 effector cells for 10,000 targets) in the absence of peptide and 5: 1 (i. e., sXlo4 effector cells for 10,000 targets) in the presence of peptide, the specific lytic units would be: [ (1/50, 000)- (1/500, 000)] x 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 evaluate 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 83P2H3/CaTrF2Ell-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 83P2H3 clearance. The number of epitopes used depends on observations of patients who spontaneously clear 83P2H3. For example, if it has been observed that patients who spontaneously clear 83P2H3 generate an immune response to at least three (3) from 83P2H3 antigen, then three or four (3-4) epitopes should be included for HLA class I. A similar rationale is used to determine HLA class II epitopes.

Epitopes are often selected that have a binding affinity of an ICso of 500 nM or less for an HLA class I molecule, or for class II, an ICso of 1000 nM or less; or HLA Class I peptides with high binding scores form 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 83P2H3, 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 83P2H3.

Example 20: Construction of"Minisene"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 83P2H3, are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from 83P2H3 to provide broad population coverage, i. e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct. The selected CTL and HTL epitopes are then incorporated into a minigene for expression in an expression vector.

Such a construct may additionally include sequences that direct the HTL epitopes to the endoplasmic reticulum. For example, the Ii protein may be fused to one or more HTL epitopes as described in the art, wherein the CLIP sequence of the Ii protein is removed and replaced with an HLA class II epitope sequence so that HLA class II epitope is directed to the endoplasmic reticulum, where the epitope binds to an HLA class II molecules.

This example illustrates the methods to be used for construction of a minigene-bearing expression plasmid. Other expression vectors that may be used for minigene compositions are available and known to those of skill in the art.

The minigene DNA plasmid of this example contains a consensus Kozak sequence and a consensus murine kappa Ig-light chain signal sequence followed by CTL and/or HTL epitopes selected in accordance with principles disclosed herein. The sequence encodes an open reading frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.

Overlapping oligonucleotides that can, for example, average about 70 nucleotides in length with 15 nucleotide overlaps, are synthesized and HPLC-purified. The oligonucleotides encode the selected peptide epitopes as well as appropriate linker nucleotides, Kozak sequence, and signal sequence. The final multiepitope minigene is assembled by extending the overlapping oligonucleotides in three sets of reactions using PCR. A Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are performed using the following conditions: 95°C for 15 sec, annealing temperature (5° below the lowest calculated Tm of each primer pair) for 30 sec, and 72°C for 1 min.

For example, a minigene is prepared as follows. For a first PCR reaction, 5 ag 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 1ll reactions containing Pfu polymerase buffer (lx= 10 mM KCL, 10 mM (NH4) 2SO4, 20 mM Tris-chloride, pH 8.75,2 mM MgS04, 0.1% Triton X-100,100 zg/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 Decree to Which It Induces Immunosenicity The degree to which a plasmid construct, for example a plasmid constructed in accordance with the previous Example, is able to induce immunogenicity is evaluated in vitro by testing for 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 evaluated 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 assess 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 jug 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 5'Cr 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 assess the capacity of a class II epitope-encoding minigene to induce HTLs in vivo, DR transgenic mice, or for those epitopes that cross react with the appropriate mouse MHC molecule, I-Ab- restricted mice, for example, are immunized intramuscularly with 100 llg 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 evaluated 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 jig 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 ug of DNA or recombinant vaccinia without the minigene sequence, or with DNA encoding the minigene, but without the vaccinia boost. After an additional incubation period of two weeks, splenocytes from the mice are immediately assayed for peptide-specific activity in an ELISPOT assay. Additionally, splenocytes are stimulated in vitro with the A2-restricted peptide epitopes encoded in the minigene and recombinant vaccinia, then assayed for peptide-specific activity in an alpha, beta and/or gamma IFN ELISA.

It is found that the minigene utilized in a prime-boost protocol elicits greater immune responses toward the HLA-A2 supermotif peptides than with DNA alone. Such an analysis can also be performed using HLA-Al l 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 83P2H3 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 83P2H3-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 Freund's Adjuvant. The dose of peptide for the initial immunization is from about 1 to about 50,00011g, generally 100-5,000 Mg, 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 83P2H3- 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: Polvepitopic Vaccine Compositions Derived from Native 83P2H3/CaTrF2Ell Sequences A native 83P2H3 polyprotein sequence is screened, 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 83P2H3 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 83P2H3, 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 83P2H3 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 83P2H3 and such other antigens. For example, a vaccine composition can be provided as a single polypeptide that incorporates multiple epitopes from 83P2H3 as well as tumor-associated antigens that are often expressed with a target cancer associated with 83P2H3 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 83P2H3. 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, 83P2H3 HLA-A*0201-specific CTL frequencies from HLA A*0201-positive individuals at different stages of disease or following immunization comprising an 83P2H3 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 ß2-microglobulm 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, ß2- 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, Missouri), 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 300g for 5 minutes and resuspended in 50 rut 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 83P2H3 epitope, and thus the status of exposure to 83P2H3, 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 83P2H3-associated disease or who have been vaccinated with an 83P2H3 vaccine.

For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any 83P2H3 vaccine. PBMC are collected from vaccinated individuals and HLA typed. Appropriate peptide epitopes of the invention that, optimally, bear supermotifs to provide cross-reactivity with multiple HLA supertype family members, are then used for analysis of samples derived from individuals who bear that HLA type.

PBMC from vaccinated individuals are separated on Ficoll-Histopaque density gradients (Sigma Chemical Co., St. Louis, MO), washed three times in HBSS (GIBCO Laboratories), resuspended in RPMI-1640 (GIBCO Laboratories) supplemented with L-glutamine (2mM), penicillin (SOU/ml), streptomycin (50 gg/ml), and Hepes (lOrnM) 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 llg/rnl to each well and HBV core 128-140 epitope is added at 1 gg/ml to each well as a source of T cell help during the first week of stimulation.

In the microculture format, 4 x 105 PBMC are stimulated with peptide in 8 replicate cultures in 96-well round bottom plate in 100 ul/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 'Cr release, based on comparison with non-diseased control subjects as previously described (Rehermann, et al., Nature Med. 2 : 1104,1108,1996; Rehermannetal., J. Clin. Invest. 97: 1655-1665, 1996; andRehermannetal. J. Clin. Invest. 98: 1432-1440,1996).

Target cell lines are autologous and allogeneic EBV-transformed B-LCL that are either purchased from the American Society for Histocompatibility and Immunogenetics (ASHI, Boston,

MA) 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 uM, and labeled with 100 IlCi of 'Cr (Amersham Corp., Arlington Heights, IL) for 1 hour after which they are washed four times with HBSS.

Cytolytic activity is determined in a standard 4-h, split well 5'Cr 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 x [ (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 83P2H3 or an 83P2H3 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. 5x105 cells/well and are stimulated with 10 pg/ml synthetic peptide of the invention, whole 83P2H3 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 lOU/ml IL-2. Two days later, 1 uCi 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 ug of peptide composition; Group II : 3 subjects are injected with placebo and 6 subjects are injected with 50 ug peptide composition; Group III : 3 subjects are injected with placebo and 6 subjects are injected with 500 llg 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 83P2H3 Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients having cancer that expresses 83P2H3. The main objectives of the trial are to determine an effective dose and regimen for inducing CTLs in cancer patients that express 83P2H3, 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 83P2H3.

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 83P2H3-associated disease.

Example 29: Induction of CTL Responses Usine a Prime Boost Protocol A prime boost protocol similar in its underlying principle to that used to evaluate 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 jug) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-107 to 5x109 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 83P2H3 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 83P2H3 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 Progenipoietin (Monsanto, St. Louis, MO) or GM-CSF/IL-4. After pulsing the DC with peptides, and prior to reinfusion into patients, the DC are washed to remove unbound peptides.

As appreciated clinically, and readily determined by one of skill based on clinical outcomes, the number of DC reinfused into the patient can vary (see, e. g., Nature Med. 4: 328,1998; Nature Med.

2: 52,1996 and Prostate 32: 272,1997). Although 2-50 x 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 ProgenipoietinT are injected into patients without purification of the DC. The total number of PBMC that are administered often ranges from 108 to 101°. Generally, the cell doses injected into patients is based on the percentage of DC in the blood of each patient, as determined, for example, by immunofluorescence analysis with specific anti-DC antibodies. Thus, for example, if Progenipoietin mobilizes 2% DC in the peripheral blood of a given patient, and that patient is to receive 5 x 106 DC, then the patient will be injected with a total of 2.5 x 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 83P2H3 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 Identifvine Motif-Bearing Peptides Another method of identifying 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. 83P2H3. 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 83P2H3 to isolate peptides corresponding to 83P2H3 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 Polvnucleotides Sequences complementary to the 83P2H3-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring 83P2H3. 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 83P2H3. 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 83P2H3-encoding transcript.

Example 33: Purification of Naturallv-occurrinr or Recombinant 83P2H3/CaTrF2Ell Using 83P2H3/CaTrF2Ell Specific Antibodies Naturally occurring or recombinant 83P2H3 is substantially purified by immunoaffinity chromatography using antibodies specific for 83P2H3. An immunoaffinity column is constructed by covalently coupling anti-83P2H3 antibody to an activated chromatographic resin, such as CNBr- activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.

Media containing 83P2H3 are passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of 83P2H3 (e. g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/83P2H3 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 83P2H3/CaTrF2Ell 83P2H3, 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 83P2H3, washed, and any wells with labeled 83P2H3 complex are assayed. Data obtained using different concentrations of 83P2H3 are used to calculate values for the number, affinity, and association of 83P2H3 with the candidate molecules.

Example 35A: In Vivo Assav for 83P2H3 Tumor Growth Promotion The effect of the 83P2H3 protein on tumor cell growth is evaluated in vivo by gene overexpression in tumor-bearing mice. For example, SCID mice are injected subcutaneously on each flank with 1 x 106 of either PC3, TSUPR1, or DU145 cells containing tkNeo empty vector or 83P2H3.

At least two strategies may be used: (1) Constitutive 83P2H3 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 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or from heterologous mammalian promoters, e. g., the actin promoter or an immunoglobulin promoter, provided such promoters are compatible with the host cell systems, and (2) Regulated expression under control of an inducible vector system, such as ecdysone, tet, etc., provided such promoters are compatible with the host cell systems. Tumor volume is then monitored at the appearance of palpable tumors and followed over time to determine if 83P2H3-expressing cells grow at a faster rate and whether tumors produced by 83P2H3-expressing cells demonstrate characteristics of altered aggressiveness (e. g. enhanced metastasis, vascularization, reduced responsiveness to chemotherapeutic drugs).

Additionally, mice can be implanted with 1 x 105 of the same cells orthotopically to determine if 83P2H3 has an effect on local growth in the prostate or on the ability of the cells to metastasize, specifically to lungs, lymph nodes, and bone marrow.

The assay is also useful to determine the 83P2H3 inhibitory effect of candidate therapeutic compositions, such as for example, 83P2H3 intrabodies, 83P2H3 antisense molecules and ribozymes.

Example 35B: In Vivo Assay for CaTr F2E11 Tumor Growth Promotion The effect of the CaTr F2E11 protein on tumor cell growth is evaluated in vivo by gene overexpression in tumor-bearing mice. For example, SCID mice are injected subcutaneously on each flank with 1 x 106 of cells containing tkNeo empty vector or CaTr F2E11. At least two strategies may be used: (1) Constitutive CaTr F2E11 expression under regulation constitutive promoter such as those obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), or from heterologous mammalian promoters, e. g., the actin promoter or an immunoglobulin promoter, provided such promoters are compatible with the host cell systems, and (2) Regulated expression under control of an inducible vector system, such as ecdysone, tet, etc., provided such promoters are compatible with the host cell systems. Tumor volume is then monitored at the appearance of palpable tumors and followed over time to determine if CaTr F2E11-expressing cells grow at a faster rate and whether tumors produced by CaTr F2E11-expressing cells demonstrate characteristics of altered aggressiveness (e. g. enhanced metastasis, vascularization, reduced responsiveness to chemotherapeutic drugs).

Additionally, mice can be implanted with 1 x 105 of the same cells orthotopically to determine if CaTr F2E11 has an effect on local growth in the prostate or on the ability of the cells to metastasize, specifically to lungs, lymph nodes, and bone marrow.

The assay is also useful to determine the CaTr F2E 11 inhibitory effect of candidate therapeutic compositions, such as for example, CaTr F2E11 intrabodies, CaTr F2E11 antisense molecules and ribozymes.

Example 36A: 83P2H3 Monoclonal Antibody-mediated Inhibition of Prostate Tumors In Vivo The significant expression of 83P2H3, in cancer tissues, together with its restrictive expression in normal tissues along with its expected cell surface expression makes 83P2H3 an excellent target for antibody therapy. Similarly, 83P2H3 is a target for T cell-based immunotherapy. Thus, the therapeutic efficacy of anti-83P2H3 mAbs in human prostate cancer xenograft mouse models is evaluated by using androgen-independent LAPC-4 and LAPC-9 xenografts (Craft, N., et al.,. Cancer Res, 1999.59 (19): p. 5030-6) and the androgen independent recombinant cell line PC3-83P2H3 (see, e. g., Kaighn, M. E., et al., Invest Urol, 1979.17 (1) : p. 16-23).

Antibody efficacy on tumor growth and metastasis formation is studied, e. g., in a mouse orthotopic prostate 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. Anti-83P2H3 mAbs inhibit formation of both the androgen-dependent LAPC-9 and androgen-independent PC3-83P2H3 tumor xenografts. Anti-83P2H3 mAbs also retard the growth of established orthotopic tumors and prolonged survival of tumor-bearing mice. These results indicate the utility of anti-83P2H3 mAbs in the treatment of local and advanced stages of prostate cancer. (See, e. g., (Saffran, D., et al., PNAS 10: 1073-1078 or www. pnas. org/cgi/doi/10. 1073/pnas. 051624698) Administration of the anti-83P2H3 mAbs led to retardation of established orthotopic tumor growth and inhibition of metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies indicate that 83P2H3 as an attractive target for immunotherapy and demonstrate the therapeutic potential of anti-83P2H3 mAbs for the treatment of local and metastatic prostate cancer, This example demonstrates that unconjugated 83P2H3 monoclonal antibodies are effective to inhibit the growth of human prostate tumor xenografts grown in SCID mice; accordingly a combination of such efficacious monoclonal antibodies is also effective.

Tumor inhibition using multiple unconjugated 83P2H3 mAbs Materials and Methods 83P2H3 Monoclonal Antibodies: Monoclonal antibodies are raised against 83P2H3 as described in the Example entitled "Generation of 83P2H3 Monoclonal Antibodies (mAbs)."The antibodies are characterized by ELISA,

Western blot, FACS, and immunoprecipitation for their capacity to bind 83P2H3. Epitope mapping data for the anti-83P2H3 mAbs, as determined by ELISA and Western analysis, recognize epitopes on the 83P2H3 protein. Immunohistochemical analysis of prostate cancer tissues and cells with these antibodies is performed.

The monoclonal antibodies are purified from ascites or hybridoma tissue culture supernatants by Protein-G Sepharose chromatography, dialyzed against PBS, filter sterilized, and stored at-20°C.

Protein determinations are performed by a Bradford assay (Bio-Rad, Hercules, CA). A therapeutic monoclonal antibody or a cocktail comprising a mixture of individual monoclonal antibodies is prepared and used for the treatment of mice receiving subcutaneous or orthotopic injections of LAPC-9 prostate tumor xenografts.

Prostate Cancer Xenografts and Cell Lines The LAPC-9 xenograft, which expresses a wild-type androgen receptor and produces prostate- specific antigen (PSA), is passaged in 6-to 8-week-old male ICR-severe combined immunodeficient (SCID) mice (Taconic Farms) by s. c. trocar implant (Craft, N., et al., supra). Single-cell suspensions of LAPC-9 tumor cells are prepared as described in Craft, et al. The prostate carcinoma cell line PC3 (American Type Culture Collection) is maintained in DMEM supplemented with L-glutamine and 10% (vol/vol) FBS.

A PC3-83P2H3 cell population is 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 U S A, 1999.96 (25): p. 14523-8. Anti-83P2H3 staining is detected by using an FITC-conjugated goat anti-mouse antibody (Southern Biotechnology Associates) followed by analysis on a Coulter Epics-XL f low cytometer.

XenoRraft Mouse Models.

Subcutaneous (s. c.) tumors are generated by injection of 1 x 10 6 LAPC-9, PC3, or PC3- 83P2H3 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 x width x height. Mice with s. c. tumors greater than 1.5 cm in diameter are sacrificed. PSA levels are determined by using a PSA ELISA kit (Anogen, Mississauga, Ontario).

Circulating levels of anti-83P2H3 mAbs are determined by a capture ELISA kit (Bethyl Laboratories, Montgomery, TX). (See, e. g., (Saffran, D., et al., PNAS 10: 1073-1078 or www. pnas. orjz/cjzi/ doi/10. 1073/pnas. 051624698)

Orthotopic injections are performed under anesthesia by using ketamine/xylazine. An incision is made through the abdominal muscles to expose the bladder and seminal vesicles, which then are delivered through the incision to expose the dorsal prostate. LAPC-9 cells (5 x 105) mixed with Matrigel are injected into each dorsal lobe in a 10-1 volume. To monitor tumor growth, mice are bled on a weekly basis for determination of PSA levels. Based on the PSA levels, the mice are segregated into groups for the appropriate treatments. To test the effect of anti-83P2H3 mAbs on established orthotopic tumors, i. p. antibody injections are started when PSA levels reach 2-80 ng/ml.

Anti-83P2H3 mAbs Inhibit Growth of 83P2H3-Expressing Prostate-Cancer Tumors The effect of anti-83P2H3 mAbs on tumor formation is tested by using the LAPC-9 orthotopic model. As compared with the s. c. tumor model, the orthotopic model, which requires injection of tumor cells directly in the mouse prostate, results in a local tumor growth, development of metastasis in distal sites, deterioration of mouse health, and subsequent death (Saffran, D., et al., PNAS supra; Fu, X., et al., Int J Cancer, 1992.52 (6): p. 987-90; Kubota, T., J Cell Biochem, 1994.56 (1) : p. 4-8). The features make the orthotopic model more representative of human disease progression and allowed us to follow the therapeutic effect of mAbs on clinically relevant end points.

Accordingly, LAPC-9 tumor cells are injected into the mouse prostate, and 2 days later, the mice are segregated into two groups and treated with either: a) 50-200011g, usually 200-50011g, of anti- 83P2H3 Ab, or b) PBS three times per week for two to five weeks. Mice are monitored weekly for circulating PSA levels as an indicator of tumor growth.

A major advantage of the orthotopic prostate-cancer model is the ability to study the development of metastases. Formation of metastasis in mice bearing established orthotopic tumors is studies by IHC analysis on lung sections using an antibody against a prostate-specific cell-surface protein STEAP expressed at high levels in LAPC-9 xenografts (Hubert, R. S., et a/., Proc Natl Acad Sci U S A, 1999.96 (25): p. 14523-8).

Mice bearing established orthotopic LAPC-9 tumors are administered 1000lig injections of either anti-83P2H3 mAb or PBS over a 4-week period. Mice in both groups are allowed to establish a high tumor burden (PSA levels greater than 300 ng/ml), to ensure a high frequency of metastasis formation in mouse lungs. Mice then are killed and their prostate and lungs are analyzed for the presence of LAPC-9 cells by anti-STEAP IHC analysis.

These studies demonstrate a broad anti-tumor efficacy of anti-83P2H3 antibodies on initiation and progression of prostate cancer in xenograft mouse models. Anti-83P2H3 antibodies inhibit tumor formation of both androgen-dependent and androgen-independent tumors as well as retarding the growth of already established tumors and prolong the survival of treated mice. Moreover, anti-83P2H3 mAbs demonstrate a dramatic inhibitory effect on the spread of local prostate tumor to distal sites, even in the presence of a large tumor burden. Thus, anti-83P2H3 mAbs are efficacious on major clinically relevant end points/PSA levels (tumor growth), prolongation of survival, and health.

Example 36B: CaTr F2E11 Monoclonal Antibody-mediated Inhibition of Prostate Tumors In Vivo The significant expression of CaTr F2E11, in cancer tissues along with its expected cell surface expression makes CaTr F2E11 an excellent target for antibody therapy. Similarly, CaTr F2E11 is a target for T cell-based immunotherapy. Thus, the therapeutic efficacy ofanti-CaTr F2E11 mAbs in human prostate cancer xenograft mouse models is evaluated by using androgen-independent LAPC-4 and LAPC-9 xenografts (Craft, N., et al.,. Cancer Res, 1999.59 (19): p. 5030-6) and the androgen independent recombinant cell line PC3-CaTr F2E11 (see, e. g., Kaighn, M. E., et al., Invest Urol, 1979.

17 (1) : p. 16-23). Similarly the therapeutic effect of anti-CaTr F2E11 Ab in human bladder and lung cancer will be evaluated using xenograft animal models of bladder (UM-UC3, Scaber, etc) and lung (A427, SK-Lu, etc) cancer that lack or express CaTr F2E11.

Antibody efficacy on tumor growth and metastasis formation is studied, e. g., in a mouse orthotopic prostate 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. Anti-CaTr F2E11 mAbs can inhibit formation of tumors in xenografts. Anti-CaTr F2E11 can retard the growth of established orthotopic tumors and prolonged survival of tumor-bearing mice. These results indicate the utility ofanti-CaTr F2E11 mAbs in the treatment of local and advanced stages of prostate cancer.

(Saffran, D., et al., PNAS 10: 1073-1078 or www. pnas. org/cgi/doi/10. 1073/pnas. 051624698) Tumor inhibition using multiple unconjugated CaTr F2Ell mAbs Materials and Methods CaTr F2E11 Monoclonal Antibodies: Monoclonal antibodies are raised against CaTr F2E11 as described in the Example entitled "Generation of CaTr F2E11 Monoclonal Antibodies (mAbs),"The antibodies are characterized by ELISA, Western blot, FACS, and immunoprecipitation for their capacity to bind CaTr F2E11. Epitope mapping data for the anti-CaTr F2E11 mAbs, as determined by ELISA and Western analysis, recognize epitopes on the CaTr F2E11 protein. Immunohistochemical analysis of prostate cancer tissues and cells with these antibodies is performed.

The monoclonal antibodies are purified from ascites or hybridoma tissue culture supernatants by Protein-G Sepharose chromatography, dialyzed against PBS, filter sterilized, and stored at-20°C.

Protein determinations are performed by a Bradford assay (Bio-Rad, Hercules, CA). A therapeutic monoclonal antibody or a cocktail comprising a mixture of individual monoclonal antibodies is prepared and used for the treatment of mice receiving subcutaneous or orthotopic injections of LAPC-9 prostate tumor xenografts.

Prostate Cancer Xenografts and Cell Lines The LAPC-9 xenograft, which expresses a wild-type androgen receptor and produces prostate- specific antigen (PSA), is passaged in 6-to 8-week-old male ICR-severe combined immunodeficient (SCID) mice (Taconic Farms) by s. c. trocar implant (Craft, N., et a/., supra). Single-cell suspensions of LAPC-9 tumor cells are prepared as described in Craft, et al. The prostate carcinoma cell line PC3 (American Type Culture Collection) is maintained in DMEM supplemented with L-glutamine and 10% (vol/vol) FBS.

A PC3-CaTr F2E 11 cell population is 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 U S A, 1999.96 (25): p. 14523-8. Anti-CaTr F2E11 staining is detected by using an FITC-conjugated goat anti-mouse antibody (Southern Biotechnology Associates) followed by analysis on a Coulter, Epics-XL f low cytometer.

Xenograft Mouse Models.

Subcutaneous (s. c.) tumors are generated by injection of 1 x 10 6 LAPC-9, PC3, or PC3-CaTr F2E11 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 x width x height. Mice with s. c. tumors greater than 1.5 cm in diameter are sacrificed. PSA levels are determined by using a PSA ELISA kit (Anogen, Mississauga, Ontario).

Circulating levels ofanti-CaTr F2E11 mAbs are determined by a capture ELISA kit (Bethyl Laboratories, Montgomery, TX). (See, e. g., (Saffran, D., et al., PNAS 10: 1073-1078 or www. pnas. orc/cEi/ doi/10. 1073/pnas. Q51624698 Orthotopic injections are performed under anesthesia by using ketamine/xylazine. An incision is made through the abdominal muscles to expose the bladder and seminal vesicles, which then are delivered through the incision to expose the dorsal prostate. LAPC-9 cells (5 x 105) mixed with Matrigel are injected into each dorsal lobe in a 10-111 volume. To monitor tumor growth, mice are bled on a weekly basis for determination of PSA levels. Based on the PSA levels, the mice are segregated into groups for the appropriate treatments. To test the effect ofanti-CaTr F2E11 mAbs on established orthotopic tumors, i. p. antibody injections are started when PSA levels reach 2-80 ng/ml.

Anti-CaTr F2E11 mAbs Inhibit Growth of CaTr F2El l-Expressing Prostate-Cancer Tumors The effect ofanti-CaTr F2E11 mAbs on tumor formation is tested by using the LAPC-9 orthotopic model. As compared with the s. c. tumor model, the orthotopic model, which requires injection of tumor cells directly in the mouse prostate, results in a local tumor growth, development of metastasis in distal sites, deterioration of mouse health, and subsequent death (Saffran, D., et al., PNAS

supra; Fu, X., et al., Int J Cancer, 1992.52 (6): p. 987-90; Kubota, T., J Cell Biochem, 1994.56 (1) : p.

4-8). The features make the orthotopic model more representative of human disease progression and allowed us to follow the therapeutic effect of mAbs on clinically relevant end points.

Accordingly, LAPC-9 tumor cells are injected into the mouse prostate, and 2 days later, the mice are segregated into two groups and treated with either: a) 50-20001lg, usually 200-500ug, of anti- CaTr F2E11 Ab, or b) PBS three times per week for two to five weeks. Mice are monitored weekly for circulating PSA levels as an indicator of tumor growth.

A major advantage of the orthotopic prostate-cancer model is the ability to study the development of metastases. Formation of metastasis in mice bearing established orthotopic tumors is studies by IHC analysis on lung sections using an antibody against a prostate-specific cell-surface protein STEAP expressed at high levels in LAPC-9 xenografts (Hubert, R. S., et al., Proc Natl Acad Sci U S A, 1999.96 (25): p. 14523-8).

Mice bearing established orthotopic LAPC-9 tumors are administered lOOOug injections of either anti-CaTr F2E11 mAb or PBS over a 4-week period. Mice in both groups are allowed to establish a high tumor burden (PSA levels greater than 300 ng/ml), to ensure a high frequency of metastasis formation in mouse lungs. Mice then are killed and their prostate and lungs are analyzed for the presence of LAPC-9 cells by anti-STEAP IHC analysis.

These studies demonstrate a broad anti-tumor efficacy of anti-CaTr F2E11 antibodies on initiation and progression of prostate cancer in xenograft mouse models. Anti-CaTr F2El l antibodies inhibit tumor formation of both androgen-dependent and androgen-independent tumors as well as retarding the growth of already established tumors and prolong the survival of treated mice. Moreover, anti-CaTr F2E11 mAbs demonstrate a dramatic inhibitory effect on the spread of local prostate tumor to distal sites, even in the presence of a large tumor burden. Thus, anti-CaTr F2E11 mAbs are efficacious on major clinically relevant end points/PSA levels (tumor growth), prolongation of survival, and health.

Example 37A : Comparison of 83P2H3 to Known Genes '83P2H3 hCaT is a 725 amino acid protein with a calculated MW of 83.2kDa, and PI of 7.56..

83P2H3 is predicted to be a cell surface protein that functions as an ion transporter. 83P2H3 shows 84% identity and 90% homology to a mouse calcium transporter (gi 9081801). 83P2H3 show 99% identity to the recently cloned human calcium transporter CaTl (gp: AF304463).

As disclosed in the priority application (USSN 60/226,329, filed August 17,2000), 83P2H3 PcaT (also referred to as hCaT) participates in calcium signaling as well as tumor initiation and progression, can be expressed in 293T cells, and functions as a calcium transporter. Recent studies published in a peer-reviewed journal have validated these disclosures. These studies have shown that the human CaTl functions as a calcium transporter when expressed inXenopus laevis and 293T human

kidney cells (J. Biol Chem 2001,276: 29461). In addition, the study confirms, by in situ hybridization, that CaTl is highly expressed in prostate cancer.

The following show the alignment of PcaT/83P2H3 with these similar human and mouse calcium transporters: Alignment with hCaT JBC 2001, 276: 19461 >gp: AF304463_1 calcium transport protein CaTl [Homo sap (725 aa) initn: 4862 initl : 4862 opt: 4862 Z-score: 5671.1 bits: 1059.9 E () : 0 Smith-Waterman score: 4862 ; 99.724% identity (99.724% ungapped) in 725 aa overlap (1-725: 1-725) 10 20 30 40 50 60 query MGLSLPKEKGLILCLWSKFCRWFQRRESWAQSRDEQNLLQQKRIWESPLLLAAKDNDVQA gp : AF3 MGLSLPKEKGLILCLWSKFCRWFQRRESWAQSRDEQDLLQQKRIWESPLLLAAKDNDVQA 10 20 30 40 50 60 70 80 90 100 110 120 query LNKLLKYEDCKVHQRGAMGETALHIAALYDNLEAAMVLMEAAPELVFEPMTSELYEGQTA gp : AF3 LNKLLKYEDCKVHHRGAMGETALHIAALYDNLEAAMVLMEAAPELVFEPMTSELYEGQTA 70 80 90 100 110 120 130 140 150 160 170 180 query LHIAVVNQNMNLVRALLARRASVSARATGTAFRRSPCNLIYFGEHPLSFAACVNSEEIVR gp : AF3 LHIAWNQNMNLVRALLARRASVSARATGTAFRRSPCNLIYFGEHPLSFAACVNSEEIVR 130 140 150 160 170 180 190 200 210 220 230 240 <BR> <BR> <BR> query LLIEHGADIRAQDSLGNTVLHILILQPNKTFACQMYNLLLSYDRHGDHLQPLDLVPNHQG <BR> <BR> <BR> <BR> gp AF3 LLIEHGADIRAQDSLGNTVLHILILQPNKTFACQMYNLLLSYDRHGDHLQPLDLVPNHQG 190 200 210 220 230 240 250 260 270 280 290 300 query LTPFKLAGVEGNTVMFQHLMQKRKHTQWTYGPLTSTLYDLTEIDSSGDEQSLLELIITTK gp AF3 LTPFKLAGVEGNTVMFQHLMQKRKHTQWTYGPLTSTLYDLTEIDSSGDEQSLLELIITTK 250 260 270 280 290 300 310 320 330 340 350 360 query KREARQILDQTPVKELVSLKWKRYGRPYFCMLGAIYLLYIICFTMCCIYRPLKPRTNNRT gp AF3 KREARQILDQTPVKELVSLKWKRYGRPYFCMLGAIYLLYIICFTMCCIYRPLKPRTNNRT 310 320 330. 340 350 360

370 380 390 400 410 420 query SPRDNTLLQQKLLQEAYMTPKDDIRLVGELVTVIGAIIILLVEVPDIFRMGVTRFFGQTI gp: AF3 SPRDNTLLQQKLLQEAYMTPKDDIRLVGELVTVIGAIIILLVEVPDIFRMGVTRFFGQTI 370 380 390 400 410 420 430 440 450 460 470 480 query LGGPFHVLIITYAFMVLVTMVMRLISASGEWPMSFALVLGWCNVMYFARGFQMLGPFTI gp: AF3 LGGPFHVLIITYAFMVLVTMVMRLISASGEWPMSFALVLGWCNVMYFARGFQMLGPFTI 430 440 450 460 470 480 490 500 510 520 530 540 query MIQKMIFGDLMRFCWLMAVVILGFASAFYIIFQTEDPEELGHFYDYPMALFSTFELFLTI gp: AF3 MIQKMIFGDLMRFCWLMAWILGFASAFYIIFQTEDPEELGHFYDYPMALFSTFELFLTI 490 500 510 520 530 540 550 560 570 580 590 600 query IDGPANYNVDLPFMYSITYAAFAIIATLLMLNLLIAMMGDTHWRVAHERDELWRAQIVAT gp: AF3 IDGPANYNVDLPFMYSITYAAFAIIATLLMLNLLIAMMGDTHWRVAHERDELWRAQIVAT 550 560 570 580 590 600 610 620 630 640 650 660 query TVMLERKLPRCLWPRSGICGREYGLGDRWFLRVEDRQDLNRQRIQRYAQAFHTRGSEDLD gp: AF3 TVMLERKLPRCLWPRSGICGREYGLGDRWFLRVEDRQDLNRQRIQRYAQAFHTRGSEDLD 610 620 630 640 650 660 670 680 690 700 710 720 query KDSVEKLELGCPFSPHLSLPMPSVSRSTSRSSANWERLRQGTLRRDLRGIINRGLEDGES gp: AF3 KDSVEKLELGCPFSPHLSLPMPSVSRSTSRSSANWERLRQGTLRRDLRGIINRGLEDGES 670 680 690 700 710 720 query WEYQI gp : AF3 WEYQI Mouse Catl >gi 9081801 calcium transporting protein homolog [Mus musculus] Score = 1189 bits (3077), Expect = 0.0 Identities = 622/732 (84%), Positives = 668/732 (90%), Gaps = 10/732 (1%) Query: 1 MGLSLPKEKGLILCLWSKFCRWFQRRESWAQSRDEQNLLQQKRIWESPLLLAAKDNDVQA 60

MG SLPKEKGLILCLW+KFCRWF R+ESWAQSRDEQNLLQQKRIWESPLLLAAK+NDVQA Sbjct: 1 MGWSLPKEKGLILCLWNKFCRWFHRQESWAQSRDEQNLLQQKRIWESPLLLAAKENDVQA 60 Query: 61 LNKLLKYEDCKVHQRGAMGETALHIAALYDNLEAAMVLMEAAPELVFEPMTSELYEGQTA 120 L+KLLK+E C+VHQRGAMGETALHIAALYDNLEAAMVLMEAAPELVFEPMTSELYEGQTA Sbjct : 61 LSKLLKFEGCEVHQRGAMGETALHIAALYDNLEAAMVLMEAAPELVFEPMTSELYEGQTA 120 Query: 121 LHIAWNQNMNLVRALLARRASVSARATGTAFRRSPCNLIYFGEHPLSFAACVNSEEIVR 180 LH+AV+NQN+N TG+ F P Y+GEHPLSFAACV SE R Sbjct: 121 LHMAVINQNVNLVRALLARRASVSARATGSVFTTGPYKPHYYGEHPLSFAACVGSEGDGR 180 . Query : 181 LLIEHGADIRAQDSLGN-TVLHILILQPNKTFACQMYNLLLSYDRHGDHLQPLDLVPNHQ 239 LLIEHGADIRAQ G +ILILQPNKTFACQMYNLLLSYD GDHL+ L+LVPN+Q Sbjct: 181 LLIEHGADIRAQGLSGKYEYYNILILQPNKTFACQMYNLLLSYDG-GDHLKSLELVPNNQ 239 Query: 240 GLTPFKLAGVEGNTVMFQHLMQKRKHTQWTYGPLTSTLYDLTEIDSSGDEQSLLELIITT 299 GLTPFKLAGVEGN VMFQHLMQKRKH QWTYGPLTSTLYDLTEIDSSGD+QSLLELI+TT Sbjct: 240 GLTPFKLAGVEGNIVMFQHLMQKRKHIQWTYGPLTSTLYDLTEIDSSGDDQSLLELIVTT 299 Query: 300 KKREARQILDQTPVKELVSLKWKRYGRPYFCMLGAIYLLYIICFTMCCIYRPLKPRTNNR 359 KKREARQILDQTPVKELVSLKWKRYGRPYFC+LGAIY+LYIICFTMCC+YRPLKPR NR Sbjct : 300 KKREARQILDQTPVKELVSLKWKRYGRPYFCVLGAIYVLYIICFTMCCVYRPLKPRITNR 359 Query: 360 TSPRDNTLLQQKLLQEAYMTPKDDIRLVGELVTVIGAIIILLVEVPDIFRMGVTRFFGQT 419 T+PRDNTL+QQKLLQEAY+TPKDD+RLVGELV+++GA+IILLVE+PDIFR+GVTRFFGQT Sbjct: 360 TNPRDNTLMQQKLLQEAYVTPKDDLRLVGELVSIVGAVIILLVEIPDIFRLGVTRFFGQT 419 Query: 420 ILGGPFHVLIITYAFMVLVTMVMRLISASGEWPMSFALV-LGWCNVMYFARGFQMLGPF 478 ILGGPFHV+IITYAFMVLVTMVMRL + GEWPMSFA L C+ FARGFQMLGPF Sbjct: 420 ILGGPFHVIIITYAFMVLVTMVMRLTNVDGEWPMSFARCWLVQCH--DFARGFQMLGPF 477 Query: 479 TIM-IQKMIFGDLMR-FCWLMAWILGFASAFYIIFQTEDPEELGHFYDYPMALFSTFEL 536 T+ +++IFGDL FCWLMAWILGFASAFYIIFQTEDP+ELGHFYDYPMALFSTFEL Sbjct: 478 TLHDSRRLIFGDLNAIFCWLMAWILGFASAFYIIFQTEDPDELGHFYDYPMALFSTFEL 537 Query: 537 FLTIIDGPANYNVDLPFMYSITYAAFAIIATLLMLNLLIAMMGDTHWRVAHERDELWRAQ 596 FLTIIDGPANY+VDLPFMYS+TYAAFAIIATLLMLNLLIAMMGDTHWRVAHERDELWRAQ Sbjct: 538 FLTIIDGPANYDVDLPFMYSVTYAAFAIIATLLMLNLLIAMMGDTHWRVAHERDELWRAQ 597 Query: 597 IVATTVMLERKLPRCLWPRSGICGREYGLGDRWFLRVEDRQDLNRQRIQRYAQAFHTRG- 655 +VATTVMLERKLPRCLWPRSGICGREYGLGDRWFLRVEDRQDLNRQRI+RYAQAF +

Sbjct : 598 WATTVMLERKLPRCLWPRSGICGREYGLGDRWFLRVEDRQDLNRQRIRRYAQAFQQQDG 657 Query: 656--SEDLDKDSVEKLELGCPFSPHLSLPMPSVSRSTSRSSANWERLRQGTLRRDLRGI INR 713 SEDL+KDS EKLE PF +LS P P NWERLRQG LR+DLRGIINR Sbjct: 658 LYSEDLEKDSGEKLETARPFGAYLSFPTPSVSRSTSRSSTNWERLRQGALRKDLRGIINR 717 Query: 714 GLEDGESWEYQI 725 GLEDGE WEYQI Sbjct: 718 GLEDGEGWEYQI 729 Example 37B: Comparison of CaTr F2E11 to Known Genes CaTr F2E11 is a 963 amino acid protein with a calculated MW of 107.7kDa, and PI of 8.23.

CaTr F2E11 is predicted to be a cell surface protein that functions as an ion transporter. CaTr F2E11 shows 91% identity and 93% homology to a mouse osmosensitive receptor potential channel (PubMed cite: gi 11528502) (http://www. ncbi. nlm. nih. gov/). CaTr F2El show 96% identity to human vanilloid receptor-related osmotically activated channel (PubMed cite: gi 14767872).

The following shows the alignment of CaTr F2E11 with human vallinoid receptor-related channel.

Alignment with of CaTr F2E 11 with human Vanilloid receptor Query = CaTr F2E11 Subject = gi|14767872 vanilloid receptor-related osmotically activated channel Query: 276 EFREPSTGKTCLPKALLNLSNGRNDTIPVLLDIAERTGNMREFINSPFRDIYYRGQTALH 335 E EPSTGKTCLPKALLNLSNGRNDTIPVLLDIAERTGNMREFINSPFRDIYYRGQTALH Sbjct : 5 EVLEPSTGKTCLPKALLNLSNGRNDTIPVLLDIAERTGNMREFINSPFRDIYYRGQTALH 64 Query: 336 IAIERRCKHYVELLVAQGADVHAQARGRFFQPKDEGGYFYFGELPLSLAACTNQPHIVNY 395 IAIERRCKHYVELLVAQGADVHAQARGRFFQPKDEGGYFYFGELPLSLAACTNQPHIVNY Sbjct : 65 IAIERRCKHYVELLVAQGADVHAQARGRFFQPKDEGGYFYFGELPLSLAACTNQPHIVNY 124 Query : 396 LTENPHKKADMRRQDSRGNTVLHALVAIADNTRENTKFVTKMYDLLLLKCARLFPDSNLE 455 LTENPHKKADMRRQDSRGNTVLHALVAIADNTRENTKFVTKMYDLLLLKCARLFPDSNLE Sbjct : 125 LTENPHKKADMRRQDSRGNTVLHALVAIADNTRENTKFVTKMYDLLLLKCARLFPDSNLE 184 Query: 456 AVLNNDGLSPLMMAAKTGKIGIFQHIIRREVTDEDTRHLSRKSKDWAYGPVXXXXXXXXX 515 AVLNNDGLSPLMMAAKTGKIG+FQHIIRREVTDEDTRHLSRKKDWAYGPV Sbjct : 185 AVLNNDGLSPLMMAAKTGKIGVFQHIIRREVTDEDTRHLSRKFKDWAYGPVYSSLYDLSS 244

Query: 516 XXTCGEEASVLEILVYNSKIENRHEMLAVEPINELLRDKWRKFGAVSFYINVVSYLCAMV 575 TCGEEASVLEILVYNSKIENRHEMLAVEPINELLRDKWRKFGAVSFYINVVSYLCAMV Sbjct : 245 LDTCGEEASVLEILVYNSKIENRHEMLAVEPINELLRDKWRKFGAVSFYINVVSYLCADI V 304 Query: 576 IFTLTAYYQPLEGTPPYPYRTTVDYLRLAGEVITLFTGVLFFFTNIKDLFMKKCPGVNSL 635 IFTLTAYYQPLEGTPPYPYRTTVDYLRLAGEVITLFTGVLFFFTNIKDLFMKKCPGVNSL Sbjct: 305 IFTLTAYYQPLEGTPPYPYRTTVDYLRLAGEVITLFTGVLFFFTNIKDLFMKKCPGVNSL 364 Query: 636 FIDGSFQLLYFIYSVLVIVSAALYLAGIEAYLAMMVFALVLGWMNALYFTRGLKLTGTYS 695 FIDGSFQLLYFIYSVLVIVSAALYLAGIEAYLA+MVFALVLGWMNALYFTRGLKLTGTYS Sbjct: 365 FIDGSFQLLYFIYSVLVIVSAALYLAGIEAYLAVMVFALVLGWMNALYFTRGLKLTGTYS 424 Query: 696 IMIQKILFKDLFRFLLVYLLFMIGYASALVSLLNPCANMKVCNEDQTNCTVPTYPSCRDS 755 IMIQKILFKDLFRFLLVYLLFMIGYASALVSLLNPCANMKVCNEDQTNCTVPTYPSCRDS Sbjct : 425 IMIQKILFKDLFRFLLVYLLFMIGYASALVSLLNPCANMKVCNEDQTNCTVPTYPSCRDS 484 Query: 756 ETFSTFLLDLFKLTIGMGDLEMLSSTKYPWFIILLVTYIILTSVLLLNMLIALMGETVG 815 ETFSTFLLDLFKLTIGMGDLEMLSSTKYPWFIILLVTYIILT VLLLNMLIALMGETVG Sbjct : 485 ETFSTFLLDLFKLTIGMGDLEMLSSTKYPWFIILLVTYIILTFVLLLNMLIALMGETVG 544 Query: 816 QVSKESKHIWKLQWATTILDIERSFPVFLRKAFRSGEMVTVGKSSDGTPDRRWCFRVDEV 875 QVSKESKHIWKLQWATTILDIERSFPVFLRKAFRSGEMVTVGKSSDGTPDRRWCFRV+EV Sbjct: 545 QVSKESKHIWKLQWATTILDIERSFPVFLRKAFRSGEMVTVGKSSDGTPDRRWCFRVNEV 604 Query: 876 NWSHWNQNLGIINEDPGKNETYQYY 900 NWSHWNQNLGIINEDPGKNE +QYY Sbjct : 605 NWSHWNQNLGIINEDPGKNEXHQYY 629 Vallinoid receptors are mostly ligand-gated ion channels that can be activated by a variety of stimuli including capsaicin, vanilloids, protons and heat. A well-studied vallinoid receptor is VR1 which transmits pain sensations and induces muscle contraction in a variety of tissues (Szallasi A, Di Marzo V. Trends Neurosci. 2000,23: 491; Yiangou Y. BJU Int. 2001,87: 774). VR1 mediates calcium responsiveness in ganglia, terminals of neurons and muscles (Caterina MJ. Annu Rev Neurosci.

2001; 24: 487;). The ion channel activity of VR1 is regulated by ligands as well as post-translational modification including phosphorylation (Vellani V et al. J Physiol. 2001,534: 813). VR1 is proposed to play a role in increasing cell proliferation and blood flow in the stomach and gut (Nozawa Y et al.

Neurosci Lett. 2001,309: 33).

Based on its significant homology to vallinoid receptors, CaTr F2E11 also participates in calcium. signaling, cation transport, as well as tumor initiation and progression and angiogenesis.

Moreover, CaTr F2E11 contains several protein motifs with known functional significance, including an ion channel motif at aa 608-810 and two ankyrin motifs starting at aa 329 and aa 376 (http ://www. sanger. ac. uk).

Example 38A: Identification of Potential Siena ! Transduction Pathwavs Many mammalian proteins have been reported to interact with signaling molecules and to participate in regulating signaling pathways (J Neurochem. 2001; 76: 217-223). Using immunoprecipitation and Western blotting techniques, proteins are identified that associate with 83P2H3 and mediate signaling events. Several pathways known to play a role in cancer biology can be regulated by several of these genes, 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.). Using Western blotting techniques, the ability of 83P2H3 to regulate these pathways is examined. Cells expressing 83P2H3 and cells lacking these genes are either left untreated or stimulated with ions, channel activators, or 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.

Figure 21, Figure 22, and Figure 23 show that expression of 83P2H3 regulates the phosphorylation of several proteins in NIH 3T3 cells, and induces the activation of the ERK pathway in prostate cancer cells. Figure 26 shows that expression of hCaT induces the phosphorylation of calmodulin kinase. The transport of ions across membranes is regulated by calmodulin and cahnodulin kinases (CaMK). Since the phosphorylation of CamK reflects its activation, the effect of hCaT on the phosphorylation of CaMK was investigated. Control and 83P2H3-expressing PC3 cell lines were compared for their ability to alter the phosphorylation state of CaMKII. Cells were grown in 0.1% FBS and either left untreated or stimulated with 10% FBS, ionomycin or calcium. Whole cell lysates were separated by SDS-PAGE and analyzed by Western blotting using an anti-phospho-CaMKII antibody.

The results indicate that expression of hCaT was sufficient to enhance the phosphorylation and activation of CaMKII in PC3 cells. When 83P2H3 play a role in the regulation of signaling pathways, whether individually or communally, it is used as a target for diagnostic, preventative and therapeutic purposes.

To determine whether 83P2H3 directly or indirectly activates known signal transduction pathways in cells, luciferase (luc) based transcriptional reporter assays are carried out in cells expressing individual genes. These transcriptional reporters contain consensus-binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways. The

reporters and examples of these associated transcription factors, signal transduction pathways, and activation stimuli are listed below.

1. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK ; growth/apoptosis/stress 2. SRE-luc, SRF/TCF/ELK1 ; MAPK/SAPK ; growth/differentiation 3. AP-1-luc, FOS/JUN ; MAPK/SAPK/PKC; growth/apoptosis/stress 4. ARE-luc, androgen receptor; steroids/MAPK ; growth/differentiation/apoptosis 5. p53-luc, p53; SAPK; growth/differentiation/apoptosis 6. CRE-luc, CREB/ATF2; PKA/p38; growth/apoptosis/stress Gene-mediated effects are assayed in cells showing mRNA expression. Luciferase reporter plasmids can be introduced by lipid-mediated transfection (TFX-50, Promega). Luciferase activity, an indicator of relative transcriptional activity, is measured by incubation of cell extracts with luciferin substrate and luminescence of the reaction is monitored in a luminometer. Signaling pathways activated by 83P2H3 are mapped and used for the identification and validation of therapeutic targets.

When these genes are involved in cell signaling, they are used as targets for diagnostic, preventative and therapeutic purposes.

Example 38B: Identification of Potential Signal Transduction Pathwavs Many mammalian proteins have been reported to interact with signaling molecules and to participate in regulating signaling pathways (J Neurochem. 2001; 76: 217-223). Vanilloid receptors have been documented to activate calcium-mediated signaling as well as protein kinases (Vellani V et al. J Physiol. 2001, 534: 813; Szallasi A et al. Mol Pharmacol. 1999,56 : 581). Using immunoprecipitation and Western blotting techniques, proteins are identified that associate with CaTr F2E11 and mediate signaling events. Several pathways known to play a role in cancer biology can be regulated by several of these genes, 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.). Using Western blotting techniques, CaTr F2E11's regulation of these pathways is determined. Cells expressing CaTr F2E11 and cells lacking these genes are either left untreated or stimulated with ions, channel activators, or 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.

It is found that CaTr F2El l plays a role in the regulation of signaling pathways, individually or communally, it is used as a target for diagnostic, preventative and therapeutic purposes.

To determine that CaTr F2E11 directly or indirectly activates known signal transduction pathways in cells, luciferase (luc) based transcriptional reporter assays are carried out in cells

expressing individual genes. These transcriptional reporters contain consensus-binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways. The reporters and examples of these associated transcription factors, signal transduction pathways, and activation stimuli are listed below.

7. NFkB-luc, NFkB/Rel; Ik-kinase/SAPK ; growth/apoptosis/stress 8. SRE-luc, SRF/TCF/ELK1 ; MAPK/SAPK; growth/differentiation 9. AP-1-luc, FOS/JLTN ; MAPK/SAPK/PKC; growth/apoptosis/stress 10. ARE-luc, androgen receptor; steroids/MAPK; growth/differentiation/apoptosis 11. p53-luc, p53 ; SAPK; growth/differentiation/apoptosis 12. CRE-luc, CREB/ATF2; PKA/p38 ; growth/apoptosis/stress Gene-mediated effects are assayed in cells showing mRNA expression. Luciferase reporter plasmids can be introduced by lipid-mediated transfection (TFX-50, Promega). Luciferase activity, an indicator of relative transcriptional activity, is measured by incubation of cell extracts with luciferin substrate and luminescence of the reaction is monitored in a luminometer. Signaling pathways activated by CaTr F2E11 are mapped and used for the identification and validation of therapeutic targets. Thus, this gene is used as targets for diagnostic, prognistic, preventative and therapeutic purposes.

Example 39A: Involvement in Tumor Progression 83P2H3 can contribute to the growth of cancer cells. The role of 83P2H3 in tumor growth is investigated 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 83P2H3. Parental cells lacking our 83P2H3 and cells expressing the gene are evaluated for cell growth using a well-documented proliferation assay (Fraser SP, Grimes JA, Djnmgoz Mi. Prostat, 2000; 44: 61, Johnson DE, Ochieng J, Evans SL. Anticancer Drugs. 1996,7: 288). Figure 24 shows that expression of 83P2H3 in NIH- 3T3 enhances the proliferation of these cells relative to control 83P2H3 negative cells. These results indicate that 83P2H3 plays a critical role in tumor cell growth.

To determine the role of 83P2H3/hCaT in the transformation process, the effect of 83P2H3 in colony forming assays is evaluated. Parental NIH3T3 cells lacking 83P2H3 are compared to NHI-3T3 cells expressing 83P2H3, using a soft agar assay under stringent and more permissive conditions (Song Z. et al. Cancer Res. 2000 ; 60: 6730).

To determine the role of 83P2H3 in invasion and metastasis of cancer cells, a well-established Transwell Insert System assay (Becton Diekinson) (Cancer Res. 1999; 59: 6010) is used. Control cells, including prostate, colon, bladder and kidney cell lines lacking 83P2H3 are compared to cells expressing 83P2H3. 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.

83P2H3 can also play a role in cell cycle and apoptosis. Parental cells and cells expressing 83P2H3 are compared for differences in cell cycle regulation using a well-established BrdU assay (Abdel-Malek ZA. J Cell Physiol. 1988,136: 247). In short, cells are grown under both optimal (full serum) and limiting (low serum) conditions are labeled with BrdU and stained with anti-BrdU Ab and propidium iodide. Cells are analyzed for entry into the Gl, S, and G2M phases of the cell cycle.

Alternatively, the effect of stress on apoptosis is evaluated in control parental cells and cells expressing genes under consideration, including normal and tumor prostate, colon and lung cells. Engineered and parental cells are treated with various chemotherapeutic agents, such as etoposide, flutamide, etc, and protein synthesis inhibitors, such as cycloheximide. Cells are stained with annexin V-FITC and cell death is measured by FACS analysis.

The function of 83P2H3 is evaluated using anti-sense RNA technology coupled to the various functional assays described above, e. g. growth, invasion and migration. Anti-sense RNA oligonucleotides can be introduced into 83P2H3 expressing cells, thereby preventing the expression of 83P2H3. Control and anti-sense containing cells are analyzed for proliferation, invasion, migration, apoptotic and transcriptional potential. The local as well as systemic effect of the loss of 83P2H3 expression is evaluated.

When 83P2H3 plays a role in cell growth, transformation, invasion or apoptosis, it is used as a target for diagnostic, preventative and therapeutic purposes.

Example 39B: Involvement in Tumor Progression Based on its homology to vallinoid receptors and transient receptor potential (Trp) family of ion channels (Wissenbach U et al. FEBS Lett. 2000 485: 127), CaTr F2E11 contributes to the growth of cancer cells. The role of CaTr F2E11 in tumor growth is investigated 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 CaTr F2E11. Parental cells lacking our CaTr F2E11 and cells expressing the gene are evaluated for cell growth using a well-documented proliferation assay (Fraser SP, Grimes JA, Djamgoz MB. Prostate. 2000; 44: 61, Johnson DE, Ochieng J, Evans SL. Anticancer Drugs. 1996, 7: 288). Figure 24 shows that expression of CaTr F2E11 in NIH-3T3 enhances the proliferation of these cells relative to control CaTr F2E11 negative cells. These results indicate that CaTr F2E11 plays a critical role in tumor cell growth.

To determine CaTr F2E11's role in transformation, the effect of CaTr F2E11 in colony forming assays is evaluated. Parental NIH3T3 cells lacking CaTr F2E 11 are compared to NHI-3T3 cells expressing CaTr F2E11, using a soft agar assay under stringent and more permissive conditions (Song Z. et al. Cancer Res. 2000; 60: 6730).

To determine the role of CaTr F2E11 in invasion and metastasis of cancer cells, a well- established Transwell Insert System assay (Becton Dickinson) (Cancer Res. 1999; 59: 6010) is used.

Control cells, including prostate, colon, bladder and kidney cell lines lacking CaTr F2E11 are compared to cells expressing CaTr F2E 11. 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.

CaTr F2E11 also plays a role in cell cycle and apoptosis. Parental cells and cells expressing CaTr F2E11 are compared for differences in cell cycle regulation using a well-established BrdU assay (Abdel-Malek ZA. J Cell Physiol. 1988,136: 247). In short, cells 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 Gl, S, and G2M phases of the cell cycle.

Alternatively, the effect of stress on apoptosis is evaluated in control parental cells and cells expressing genes under consideration, including normal and tumor prostate, colon and lung cells. Engineered and parental cells are treated with various chemotherapeutic agents, such as etoposide, flutamide, etc, and protein synthesis inhibitors, such as cycloheximide. Cells are stained with annexin V-FITC and cell death is measured by FACS analysis.

The function of CaTr F2E11 is evaluated using anti-sense RNA technology coupled to the various functional assays described above, e. g. growth, invasion and migration. Anti-sense RNA oligonucleotides can be introduced into CaTr F2E11 expressing cells, thereby preventing the expression of CaTr F2E11. Control and anti-sense containing cells are analyzed for proliferation, invasion, migration, apoptotic and transcriptional potential. The local as well as systemic effect of the loss of CaTr F2E11 expression is evaluated.

Thus, CaTr F2E11 plays a role in cell growth, transformation, invasion and/or apoptosis, and is a target for diagnostic, prognostic preventative and therapeutic purposes.

Example 40A: Regulation of Transcription Several ion transporters have been shown to play a role in transcriptional regulation of eukaryotic genes. Regulation of gene expression can be evaluated by studying gene expression in cells expressing or lacking 83P2H3. For this purpose, two types of experiments are performed. In the first set of experiments, RNA from parental and gene-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 ions, 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 (see, e. g., Chen K et al. Thyroid. 2001.

11: 41.).

In the second set of experiments, specific transcriptional pathway activation is evaluated using commercially available (Stratagene) luciferase reporter constructs including: NFkB-luc, SRE-luc, ELKl-luc, ARE-Iuc, 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 83P2H3 plays a role in gene regulation, it is used as a target for diagnostic, prognostic, preventative and therapeutic purposes.

Example 40B: Regulation of Transcription Several ion transporters, including vanilloid receptors, have been shown to play a role in transcriptional regulation of eukaryotic genes (Int Immunopharmacol. 2001,1: 777). Regulation of gene expression can be evaluated by studying gene expression in cells expressing or lacking CaTr F2E11. For this purpose, two types of experiments are performed.

In the first set of experiments, RNA from parental and gene-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 ions, 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 (see, e. g., Chen K et al. Thyroid.

2001.11: 41.).

In the second set of experiments, specific transcriptional pathway activation is evaluated using commercially available (Stratagene) luciferase reporter constructs including: NFkB-luc, SRE-luc, ELKl-luc, ARE-luc, p53-luc, and CRE-luc. These transcriptional reporters contain consensus binding sites for known transcription factors that lie downstream of well-characterized signal transduction pathways, and represent a good tool to ascertain pathway activation and screen for positive and negative modulators of pathway activation.

Thus, CaTr F2E11 plays a role in gene regulation, and it is used as a target for diagnostic, prognostic, preventative and therapeutic purposes.

Example 41A: Subcellular Localization and Cell Bindin Based on bioinformatic analysis and hypothesized function, 83P2H3 is proposed to be located at the cell surface. The cellular location of 83P2H3 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 can be 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 can be tested using Western blotting techniques.

Alternatively, 293T cells can be transfected with an expression vector encoding 83P2H3 HIS- tagged (PCDNA 3.1 MYC/HIS, Invitrogen) as shown in figure 27A-F, and the subcellular localization of 83P2H3 is determined by immunofluorescence. Alternatively, the location of the HIS-tagged 83P2H3 is followed by Western blotting.

When 83P2H3 is localized to specific subcellular locale, such as the cell surface, it is used as a target for diagnostic, preventative and therapeutic purposes as appreciated by one of ordinary skill in the art.

Example 41B: Subcellular Localization and Cell Binding Based on bioinformatic analysis and disclosed function, CaTr F2E 11 is located at the cell surface. The cellular location of CaTr F2E11 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 can be 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 can be tested using Western blotting techniques.

Alternatively, 293T cells can be transfected with an expression vector encoding CaTr F2E11 HIS-tagged (PCDNA 3.1 MYC/HIS, Invitrogen), and the subcellular localization of CaTr F2E11 determined by immunofluorescence. Alternatively, the location of the HIS-tagged CaTr F2E11 is followed by Western blotting.

Thus, CaTr F2E11 is localized to specific subcellular locale, namely the cell surface, and it is used as a target for diagnostic, preventative and therapeutic purposes as appreciated by one of ordinary skill in the art.

Example 42A: Protein and Ion Transporter Function Based on bioinformatic analysis, 83P2H3 is likely to function as a transporter. To determine whether 83P2H3 functions as an ion channel, FACS analysis and electrophysiology techniques are used (Gergely L, Cook L, Agnello V. Clin Diagn Lab Immunol. 1997; 4: 70; Skryma R, et al. J Physiol.

2000,527: 71). Using FACS analysis and commercially available indicators (Molecular Probes), parental cells and cells expressing 83P2H3 are compared for their ability to transport calcium, sodium and potassium. Prostate, colon, bladder and kidney normal and tumor cell lines are used in these studies. For example cells loaded with calcium responsive indicators such as Fluo4 and Fura red are incubated in the presence or absence of ions and analyzed by flow'cytometry.

Information derived from these experiments provides a data regarding important mechanisms by which cancer cells are regulated. This is particularly true in the case of calcium, as calcium channel inhibitors have been reported to induce the death of certain cancer cells, including prostate cancer cell lines (Batra S, Popper LD, Hartley-Asp B. Prostate. 1991,19: 299). Figure 25 shows that 83P2H3 mediates calcium transport in the prostate cancer cell line PC3, and as such, may regulate prostate cancer growth by regulating intracellular levels of calcium.

Using a modified rhodamine retention assay (Davies J et al. Science 2000,290: 2295; Leith C et al. Blood 1995,86: 2329) it is determined whether 83P2H3 functions as a protein transporter. Cell lines, such as prostate, colon, bladder and kidney cancer and normal cells, expressing or lacking 83P2H3 are loaded with Calcein AM (Molecular Probes). Cells are examined over time for dye transport using a fluorescent microscope or fluorometer. Quantitation is performed using a fluorometer (Hollo Z. et al., Biochim. Biophys. Acta. 1994.1191: 384). Information obtained from such experiments is used to determine whether 83P2H3 serves to extrude chemotherapeutic drugs, such as doxorubicin, paclitaxel, etoposide, etc, from tumor cells, thereby lowering drug content and reducing tumor responsiveness to treatment. Such a system is also used to determine whether 83P2H3 functions in transporting small molecules.

When 83P2H3 functions as a transporter, it is used as a target for preventative and therapeutic purposes as well as drug sensitivity/resistance.

Using electrophysiology, uninjected oocytes and oocytes injected with gene-specific cRNA are compared for ion channel activity. Patch/voltage clamp assays are performed on oocytes in the presence or absence of selected ions, including calcium, potassium, sodium, etc. Ion channel activators (such as cAMP/GMP, forskolin, TPA, etc) and inhibitors (such as calcicludine, conotoxin, TEA, tetrodotoxin, etc) are used to evaluate the function of 83P2H3 as an ion channel (Schweitz H. et al.

Proc. Natl. Acad. Sci. 1994.91: 878; Skryma R. et al. Prostate. 1997.33: 112). Using similar techniques, it was recently demonstrated that hCaT induces calcium flux in 293T cells (Wissenbach, U., et al. J. Biol. Chem. 2001,276: 19461). The magnitude of the flux shown in this paper was similar to the one observed in figure A, where hCaT was expressed in prostate cancer cells.

When 83P2H3 functions as an ion channel, it is used as a target for diagnostic, preventative and therapeutic purposes.

Example 42: Protein and Ion Transporter Function CaTr F2E11 is disclosed herein to function as a transporter. To conform that CaTr F2E11 functions as an ion channel, FACS analysis and electrophysiology techniques are used (Gergely L, Cook L, Agnello V. Clin Diagn Lab Immunol. 1997; 4: 70; Skryma R, et al. J Physiol. 2000,527: 71).

Using FACS analysis and commercially available indicators (Molecular Probes), parental cells and cells expressing CaTr F2E11 are compared for their ability to transport calcium, sodium and potassium.

Prostate, colon, bladder and kidney normal and tumor cell lines are used in these studies. For example cells loaded with calcium responsive indicators such as Fluo4 and Fura red are incubated in the presence or absence of ions and analyzed by flow cytometry.

Information derived from these experiments provides a data regarding important mechanisms by which cancer cells are regulated. This is particularly true in the case of calcium, as calcium channel inhibitors have been reported to induce the death of certain cancer cells, including prostate cancer cell lines (Batra S, Popper LD, Hartley-Asp B. Prostate. 1991,19: 299). Figure 25 shows that CaTrF2Ell mediates calcium transport in the prostate cancer cell line PC3, and as such, can regulate prostate cancer growth by regulating intracellular levels of calcium.

Using a modified rhodamine retention assay (Davies J et al. Science 2000,290: 2295; Leith C et al. Blood 1995,86: 2329) it is determined that CaTr F2E11 functions as a protein transporter. Cell lines, such as prostate, colon, bladder and kidney cancer and normal cells, expressing or lacking CaTr F2E11 are loaded with Calcein AM (Molecular Probes). Cells are examined over time for dye transport using a fluorescent microscope or fluorometer. Quantitation is performed using a fluorometer (Hollo Z. et al., Biochim. Biophys. Acta. 1994.1191: 384). Information obtained from such experiments is used to determine that CaTr F2E11 serves to extrude chemotherapeutic drugs, such as doxorubicin, paclitaxel, etoposide, etc, from tumor cells, thereby lowering drug content and reducing tumor responsiveness to treatment. Such a system is also used to determine that CaTr F2E11 functions in transporting small molecules.

Thus, CaTr F2E11's function as a transporter, and it is a target for preventative, prognostic, diagnostic and therapeutic purposes as well as drug sensitivity/resistance.

Using electrophysiology, uninjected oocytes and oocytes injected with gene-specific cRNA are compared for ion channel activity. Patch/voltage clamp assays are performed on oocytes in the presence or absence of selected ions, including calcium, potassium, sodium, etc. Ion channel activators (such as cAMP/GMP, forskolin, TPA, etc) and inhibitors (such as calcicludine, conotoxin, TEA, tetrodotoxin, etc) are used to evaluate the function of CaTr F2E11 as an ion channel (Schweitz H. et al.

Proc. Natl. Acad. Sci. 1994.91: 878; Skryma R. et al. Prostate. 1997.33: 112). Using similar techniques, it was recently demonstrated that hCaT induces calcium flux in 293T cells (Wissenbach, U., et al. J. Biol. Chem. 2001,276: 19461). The magnitude of the flux shown in this paper was similar to the one observed in Figure 25A-C, where hCaT was expressed in prostate cancer cells.

Example 43A: Involvement in Cell-Cell Communication Cell-cell communication is essential in maintaining organ integrity and homeostasis, both of which become dysregulated during tumor formation and progression. Intercellular communications can be measured using two types of assays (J. Biol. Chem. 2000,275: 25207). In the first assay, cells loaded with a fluorescent dye are incubated in the presence of unlabeled recipient cells and the cell populations are examined under fluorescent microscopy. This qualitative assay measures the exchange

of dye between adjacent cells. In the second assay system, donor and recipient cell populations are treated as above and quantitative measurements of the recipient cell population are performed by FACS analysis. Using these two assay systems, cells expressing or lacking 83P2H3 are compared and it is determines whether 83P2H3 enhances or suppresses cell communications. This assay is used to identify small molecules and/or specific antibodies that modulate cell-cell communication.

When 83P2H3 functions in cell-cell communication, it is used as a target for diagnostic, preventative and therapeutic purposes Example 43B: Involvement in Cell-Cell Communication Cell-cell communication is essential in maintaining organ integrity and homeostasis, both of which become dysregulated during tumor formation and progression. Intercellular communications can be measured using two types of assays (J. Biol. Chem. 2000,275: 25207). In the first assay, cells loaded with a fluorescent dye are incubated in the presence of unlabeled recipient cells and the cell populations are examined under fluorescent microscopy. This qualitative assay measures the exchange of dye between adjacent cells. In the second assay system, donor and recipient cell populations are treated as above and quantitative measurements of the recipient cell population are performed by FACS analysis. Using these two assay systems, cells expressing or lacking CaTr F2E 11 are compared and it is determined that CaTr F2E11 enhances or suppresses cell communications. This assay is used to identify small molecules and/or specific antibodies that modulate cell-cell communication.

Thus, as CaTr F2E11 functions in cell-cell communication, it is used as a target for diagnostic, preventative and therapeutic purposes Example 44A: Protein-Protein Interaction Several ion transporters have been shown to interact with other proteins, thereby forming a protein complex that can regulate ion transport, cell division, gene transcription, and cell transformation (Biochem Biophys Res Commun. 2000,277: 611; J Biol Chem. 1999; 274: 20812).

Using immunoprecipitation techniques as well as two yeast hybrid systems, we can identify proteins that associate with 83P2H3. Immunoprecipitates from cells expressing 83P2H3 and cells lacking 83P2H3 are compared for specific protein-protein associations. 83P2H3 may also associate with, for example, effector molecules, such as adaptor proteins, SNARE proteins, signaling molecules, syntaxins, ATPase subunits, etc (J Biol Chem. 1999; 274: 20812; Proc Natl Acad Sci U S A 1998, 95: 14523). Studies comparing 83P2H3 positive and 83P2H3 negative cells as well as studies comparing unstimulated/resting cells and cells treated with epithelial cell activators, such as cytokines, growth factors, androgen and anti-integrin Ab reveal unique interactions.

In addition, protein-protein interactions are studied using two yeast hybrid methodologies (see, e. g., 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 83P2H3-DNA-binding

domain fusion protein and a reporter construct. Protein-protein interaction is detected by colorimetric reporter activity. Specific association with effector molecules and transcription factors directs one of skill to the mode of action of 83P2H3, and thus identifies therapeutic, preventative and/or diagnostic targets for cancer. This and similar assays are also used to identify and screen for small molecules that interact with 83P2H3.

When 83P2H3 associates with proteins or small molecules is used as a target for diagnostic, prognostic, preventative and therapeutic purposes.

Example 44B: Protein-Protein Interaction Several ion transporters have been shown to interact with other proteins, thereby forming a protein complex that can regulate ion transport, cell division, gene transcription, and cell transformation (BiochemBiophysRes Commun. 2000,277: 611; J Biol Chem. 1999; 274: 20812). In addition to forming multimers of VR1 molecules, VR1 has been shown to associate with other ion channels including (Kedei N et al J Biol Chem. 2001,276: 28613;: Premkumar LS Proc Natl Acad Sci U S A. 2001,98: 6537.) Using immunoprecipitation techniques as well as two yeast hybrid systems, proteins that associate with CaTr F2E11 are identified. Immunoprecipitates from cells expressing CaTr F2E11 and cells lacking CaTr F2E11 are compared for specific protein-protein associations. CaTr F2E 11 associates with, for example, effector molecules, such as adaptor proteins, SNARE proteins, signaling molecules, syntaxins, ATPase subunits, etc (J Biol Chem. 1999; 274: 20812; Proc Natl Acad Sci U S A 1998,95: 14523). Studies comparing CaTr F2E11 positive and CaTr F2E11 negative cells as well as studies comparing unstimulated/resting cells and cells treated with epithelial cell activators, such as cytokines, growth factors, androgen and anti-integrin Ab reveal unique interactions.

In addition, protein-protein interactions are studied using two yeast hybrid methodologies (see, e. g., 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 CaTr F2E11-DNA- binding domain fusion protein and a reporter construct. Protein-protein interaction is detected by colorimetric reporter activity. Specific association with effector molecules and transcription factors directs one of skill to the mode of action of CaTr F2E11, and thus identifies therapeutic, preventative and/or diagnostic targets for cancer. This and similar assays are also used to identify and screen for small molecules that interact with CaTr F2E11.

Thus, CaTr F2E11 associates with proteins or small molecules and is used as a target for diagnostic, prognostic, preventative and therapeutic purposes.

Example 45: Splice Variants Splice variants are also called alternative transcripts. When a gene is transcribed from genomic DNA, the initial RNA is generally 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 alternatively spliced mRNA products. Alternative transcripts each have a unique exon

makeup, and can have different coding and ! or non-coding (5'or 3'end) portions, from the original transcript. Alternative transcripts can code for similar proteins with same or similar function or may encode proteins with different functions, and may be expressed in the same tissue at the same time, or at different tissue at different times, proteins encoded by alternative transcripts can have similar or different cellular or extracellular localizations, e. g., be secreted.

Splice variants are identified by a variety of art-accepted methods. For example, splice variants are identified by use of EST data. 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 starting gene is compared to the consensus sequence (s). Each consensus sequence is a potential splice variant for that gene. Even when a variant is identified that is not a full-length clone, that portion of the variant is very useful for antigen generation and for further cloning of the full-length splice variant, using techniques known in the art.

Computer programs that predicted genes based on genomic sequence, such as Grail (http://compbio. ornl. gov/Grail-bin/EmptyGrailForm) and GenScan (http://genes. mit. edu/GENSCAN. html), also predict transcripts that can be splice variants (also see., e. g., Southan C.,"A genomic perspective on human proteases,"FEBS Lett. 2001 Jun 8; 498 (2-3): 214-8; de Souza SJ, et al.,"Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags,"Proc. Natl Acad Sci U S A. 2000 Nov 7; 97 (23): 12690-3; Jia HP, et al., Discovery of new human beta-defensins using a genomics-based approach,"Gene. 2001 Jan 24 ; 263 (1- 2): 211-8.) Using the EST assembly method, we identified three splice variants (designated as A, B and C), as shown below. Table XXI shows the nucleotide sequences of the splice variants. Table XXII shows the alignment of the splice variants with the PCaT nucleic acid sequence. Table XXIII displays the single longest alignment of an amino acid sequence encoded by a splice variant, out of all six potential reading frames with PCaT. Thus, for each splice variant, a variant's reading frame that encodes the longest single contiguous peptide homology between PCaT and the variant is the proper reading frame orientation for the variant. Due to the possibility of sequencing errors in EST or genomic data, other peptides in the relevant reading frame orientation (5'to 3'or 3'to 5') can also be encoded by the variant. Table XXIV lays out all amino acid translations of the splice variants for their respective reading frame orientations in each of the three reading frames. Tables XXI through XXIV are set forth herein on a variant-by-variant basis.

To further conform the parameters of the splice variants a variety of techniques are available in the art, such as proteomic validation, PCR-based validation, and 5'RACE validation, etc. (see e. g., Proteomic Validation: Brennan SO, Fellowes AP, George PM.;"Albumin banks peninsula: a new termination variant characterised by electrospray mass spectrometry."Biochim 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 (sl)-casein." Eur J Biochem. 1997 Oct 1; 249 (1) : 1-7; 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 Apr; 47 (4): 654-60; Jia HP, et al., Discovery of new human beta-defensins using a genomics-based approach,"Gene. 2001 Jan 24 ; 263 (1-2): 211-8; PCR-based and 5'RACE Validation: Brigle KE, et al., "Organization of the murine reduced folate carrier gene and identification of variant splice forms," Biochim Biophys Acta. 1997 Aug 7 ; 1353 (2): 191-8.

It is known in the art that genomic regions are upregulated in cancers. When the genomic region to which PCaT maps is upregulated in a particular cancer, the splice variants of PCaT are upregulated as well. Disclosed herein is that PCaT has a particular expression profile. Splice variants of PCaT that are structurally and/or functionally similar to PCaT share this expression pattern, thus serving as tumor-associated markers/antigens.

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 references are hereby incorporated by reference herein in their entireties.

The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any that are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention.

TABLES TABLE IA: Tissues that Express 83P2H3 When Malignant Prostate TABLE IB : Tissues that Express CaTrF2E11 When Malignant Prostate Bladder Kidney Lung Ovary TABLE II : AMINO ACID ABBREVIATIONS SINGLE LETTER THREE LETTER FULL NAME F Phe phenylalanine L Leu leucine S Ser serine Y Tyr tyrosine Cys cysteine W Trp tryptophan Pro prolrne 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 glutmaic 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 web site for Molecular Biology Laboratory, Dept. of Clinical Pharmacology, University of Berne, Switzerland, at URL www. ikp. unibe. ch/manual/blosum62. html) <BR> <BR> <BR> <BR> <BR> <BR> <BR> A C D E F G H I K L M N P Q R S T V W Y.

4 0-2-1-2 0-2-1-1-1-1-2-1-1-1 1 0 0-3-2 A 9-3-4-2-3-3-1-3-1-1-3-3-3-3-1-1-1-2-2 C 6 2-3-1-1-3-1-4-3 1-1 0-2 0-1-3-4-3 D 5-3-2 0-3 1-3-2 0-1 2 0 0-1-2-3-2 E 6-3-1 0-3 0 0-3-4-3-3-2-2-1 1 3 F 6-2-4-2-4-3 0-2-2-2 0-2-3-2-3 G 8-3-1-3-2 1-2 0 0-1-2-3-2 2 H 4-3 2 1-3-3-3-3-2-1 3-3-1 I 5-2-1 0-1 1 2 0-1-2-3-2 K 4 2-3-3-2-2-2-1 1-2-1 L 5-2-2 0-1-1-1 1-1-1 M 6-2 0 0 1 0-3-4-2 N 7-1-2-1-1-2-4-3 P 5 1 0-1-2-2-1 Q 5-1-1-3-3-2 R 4 1-2-3-2 S 5 0-2-2 T 4-3-1 V 11 2 W 7 Y TABLE IV (A) SUPERMOTIFS POSITION POSITION POSITION 2 (Primary Anchor) 3 (Primary Anchor) C Terminus (Primary Anchor) 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 LMVOIAT VLIMAT A3 LMVISATFCGD KYRHFA Al l 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<BR> MOTIFS 1° anchor 1 2 3 4 5 1° anchor 6 7 8 9<BR> DR4 preferred FMYLIVW M T I VSTCPALIM MH MH<BR> deleterious W R WDE<BR> DR1 preferred MELIVWY PAMQ VMATSPLIC M AVM<BR> deleterious C CH FD CWD GDE D<BR> DR7 preferred MELVWY M W A IVMSACTPL M IV<BR> deleterious C G GRD N G<BR> DR3 MOTIFS 1° anchor 1 2 3 1° anchor 4 5 1° anchor 6<BR> motif a LIVMFY D<BR> prefered<BR> motif b LIVMFAY DNQEST KRH<BR> preferred<BR> DR Supermotif MFLIVWY VMSTACPLI<BR> Italicized residues indicate less preferred or "tolerated" residues.

TABLE IV D<BR> POSITION<BR> 1 2 3 4 5 6 7 8 C-terminus<BR> SUPERMOTIFS<BR> A 1° Anchor 1° Anchor<BR> TILVMS FWY<BR> A2 1° Anchor 1° Anchor<BR> LIVMATQ LIVMAT<BR> A3 preferred 1° Anchor YFW(4/5) YFW (3/5) YFW (4/5) P (4/5) 1° Anchor<BR> VSMATLI RK.<BR> deleterious DE (3/5); DE (4/5)<BR> P (5/5)<BR> A24 1° Anchor 1° Anchor<BR> YFWVLMT FIYWLM<BR> B7 preferred FWY (5/5) 1° Anchor FWY(4/5) FWY (3/5) 1°Anchor<BR> LIVM (3/5) P VILFMWYA<BR> deleterious DE (3/5); P(5/5); DE(3/5) G(4/5) QN(4/5) DE(4/5)<BR> G(4/5); A(3/5);<BR> ON(3/5)<BR> B27 1° Anchor 1°Anchor<BR> RH FYLWMIVA<BR> B44 1° Anchor 1° Anchor<BR> ED FWYLIMVA<BR> B58 1° Anchor 1° Anchor<BR> ATS FWYLIVAM B62 1° Anchor 1° Anchor<BR> QLIVMP FWYMIVLA<BR> TABLE IV E<BR> POSITION<BR> 1 2 3 4 5 6 7 8 9 C-ter-<BR> minus<BR> or<BR> C-terminus<BR> A1 peferred GFYW 1° Anchor DEA YFW P DEQN YFW 1° Anchor<BR> 9-mer STM Y<BR> deleterious DE RHKLIVMP A G A<BR> A1 preferred GRHK ASTCLIVM 1° Anchor GSTC ASTC LIVM DE 1° Anchor<BR> 9-mer DEAS Y<BR> deleterious A RHKDEPYFW DE PQN RHK PG GP<BR> A1 preferred YFW 1° Anchor DEAQN A YFWQN PASTC GDE P 1° Anch<BR> 10-mer STM or<BR> Y<BR> deleterious GP RHKGLIVM DE RHK QNA RHKYFW RHK A<BR> A1 preferred YFW STCLIVM 1° Anchor A YFW PG G YFW 1° Anch<BR> or 1 2 3 4 5 6 7 8 9 C-ter-<BR> minus<BR> 10-mer Y<BR> deleterous RHK RHKDEPY P G PRHK QN<BR> FW<BR> A2.1 perferred YFW 1° Anchor YFW STC YFW A P 1° Anchor<BR> 9-mer LMIVQAT VLIMAT<BR> deleterious DEP DERKH RKH DERKH<BR> A2.1 10- preferred AYFW 1° Anchor LVIM G G FYWL 1° Anch<BR> mer LMIVQAT or<BR> VLIMA<BR> T<BR> deleterious DEP DE RKHA P RKH DERKH RKH<BR> A3 preferred RHK 1° Anchor YFW PRHKYFW A YFW P 1° Anchor<BR> LMVISATCG KYRHFA<BR> D<BR> deleterious DEP DE<BR> A11 preferred A 1° Anchor YFW YFW A YFW YFW P 1° Anchor<BR> VILMISAGN KRYH<BR> CDF<BR> deleterious DEP A G<BR> A24 preferred YFWRHK 1° Anchor STC YFW YFW 1° Anchor<BR> 9-mer YFWM FLIW 1 2 3 4 5 6 7 8 9 C-ter-<BR> minus<BR> deleterious DEG DE G QNP DERHK G AQN<BR> A24 preferred 1° Anchor P YFWP P 1° Anch<BR> YFWM or<BR> FLIW<BR> deleterious GDE QN RHK DE A QN DEA<BR> A3101 preferred RHK 1° Anchor YFW P YFW YFW AP 1° Anchor<BR> MVTALIS RK<BR> deleterious DEP DE ADE DE DE DE<BR> A3301 preferred 1° Anchor YFW AYFW 1° Anchor<BR> MVLFIST RK<BR> deleterious GP DE<BR> A6801 preferred YFWSTC 1° AnchorYFWLIVM YFW P 1° Anchor<BR> AVTMSLI RK<BR> deleterious GP DEG RHK A<BR> B0702 preferred RHKFWY 1° Anchor RHK RHK RHK RHK PA 1° Anchor<BR> P LMFWYAIV<BR> delterious DEQNP DEP DE DE GDE QN DE<BR> B3501 preferred FWYLIVM 1° Anchor FWY 1° Anchor<BR> P LMFWYIVA 1 2 3 45 6 7 8 9 C-ter-<BR> minus<BR> deleterious AGP G G<BR> B51 preferred LIVMFW 1° Anchor FWY STC FWY G FWY 1° Anchor<BR> P LIVFWYAM<BR> deleterious AGPDERIKS DE G DEQN GDE<BR> TC<BR> B5301 preferred LIVMFWY 1° Anchor FWY STC FWY LIVMEFWY FWY 1° Anchor<BR> P IMFWYALV<BR> deleterious AGPQN G RHKQN DE<BR> B5401 preferred PWY 1° Anchor FWYLIVM LIVM ALIVM FWYAP 1° Anchor<BR> P ATIVLMFWY<BR> deleterious GPQNDE GDESTC RHKDE DE QNDGE DE<BR> Italicized residues indicate less preferred or "tolerated" residures. The infromation in this Table is specific for 9-mers unles otherwise specified.

TABLE V (A) HLA Peptide Scoring Results-83P2H3-Al, 9-mers Score (Estiamte of Half Time of SEQ ID NO: Start Subsequence Residue Rank disassociation of a Moelcule Position Listing Containing this Subsequence) 1 401 LVEVPDIFR 45.000 1. 2 292 LLELIITTK 18.000 2. 3 248 GVEGNTVMF 18.000 3. 4 55 DNDVQALNK 12.500 4. 5 516 DEPEELGHFY 11.250 5. 6 448 SGVVPMSF 11.250 6. 7 182 LIEHGADIUR 9.000 7. 8 373 LQEAYMTPK 2.700 8. 9 59 QALNKLLKY 2.500 9. 10 331 MLGAIYLLY 2.500 10. 11 548 NVDLPFMYS 2.500 11. 12 513 QTEDPEELG 2.250 12. 13 658 DLDKDSVEK 2.000 13. 14 666 KLELGCPFS 1.800 14. 15 91 NLEAAMVLM 1.800 15. 16 174 NSEEIVRLL 1.350 16. 17 111 TSELYEGQT 1.350 17. 18 655 GSEDLDKDS 1.350 18. 19 514 TEDPEELGH 1.250 19. 20 715 LEDGESWEY 1.250 20. 21 214 QMYNLLLSY 1.250 21. 22 153 RRSPCNLIY 1.250 22. 23 81 TALHIAALY 1.000 23. 24 459 VLGWCNVMY 1.000 24. 25 501 ILGFASAFY 1.000 25. 26 539 TIIDGPANY 1.000 26. 27 632 RVEDRQDLN 0.900 27. 28 154 RSPCNLIYF 0.750 28. 29 615 RSGICRGREY 0.750 29.

TABLE V (A) HLA Peptide Scoring Results-83P2H3-Al, 9-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 30 404 VPDIFRMGV 0.625 30. 31 547 YNVDLPFMY 0. 625 31. 32 551 LPFMYSITY 0. 625 32. 33 487 FGDLMRFCW 0.625 33. 34 46 ESPLLLAAK 0. 600 34. 35 341 ICFTMCCIY 0. 500 35. 36 254 VMFQHLMQK 0.500 36. 37 523 DYDYPMALF 0.500 37. 38 485 MIFGDLMRF 0.500 38. 39 316 LVSLKWKRY 0.500 39. 40 504 FASAFYIIF 0.500 40. 41 146 RATGTAFRR 0. 500 41. 42 598 VATTVMLER 0. 500 42. 43 474 MLGPFTIMI 0. 500 43. 44 624 GLGDRWFLR 0. 500 44. 45 240 GLTPEKLAG 0.500 45. 46 377 YMTPKDDIR 0. 500 46. 47 450 EVVPMSFAL 0.500 47. 48 599 ATTVMLERK 0.500 48. 49 479 TIMIQKMIF 0.500 49. 50 462 WCNVMYFAR 0. 500 50.

TABLE V (B) HLA Pop tide Scoring Results-CaTrF2E1l-Al, 9-mers Score (Estimate of Half Time of SEQ ID : Start Subsequence Residue Rank Disassociation of a Molecule Containing Position Listing (This Subsequence) 1 63 FLEPPLAG 45.000 51. 2 944 RCDGHQQGY 52. 3 774 DLEMLSSTK 53. 4 209 SSDNKRWRK 15.000 54. 5 838 RSFPVFLRK 55.

TABLE V (B) HLA Peptide Scoring Results-CaTrF2Ell-A1, 9-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of of Molecule Containing Position Listing This Subsequence) 6 850 SGEMVTVGK 9. 000 56. 7 543 AVEPINELL 9.000 8 859 SSDGTPDRR 7.500 9 353 GADVHAQAR 5. 000 59. 10 93 MADSSEGPR 5. 000 60. 11 337 AIERRCKHY 4. 500 61. 12 818 SKESKHIWK 4.500 13 320 NSPFRDIYY 3. 750 63. 14 523 ASVLEILVY 3. 750 64. 15 597 TVDYLRLAG 2. 500 65. 16 231 APQPPPILK 2. 500 66. 17 762 LLDLFKLTI 2. 500 67. 18 387 TNQPHIVNY 2. 500 68. 19 692 GTYSIMIQK 2. 500 69. 20 741 QTNCTVPTY 2. 500 70. 21 759 STFLLDLFK 2.500 22 368 KDEGGYFYF 2. 250 72. 23 396 LTENPHKKA 2.250 24 727 LLNPCANMK 2.000 25 345 YVELLVAQG 1.800 26 525 VLEILVYNS 1.800 27 177 LLESTLYES 1. 800 77. 28 662 GIEAYLAMM 1.800 29 835 DIERSFPVF 1.800 30 754 DSETFSTFL 1. 350 80. 31 319 INSPFRDIY 1.250 32 488 DEDTRHLSR 1.250 33 300 DTIPVLLDI 1.250 34 486 VTDEDTRHL 1.250 35 830 ATTILDIER 1.250 36 119 GGEAFPLSS 1.25 TABLE V (B) HLA Peptide Scoring Results-CaTrF2Ell-A1, 9-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of of Molecule Containing Position Listing This Subsequence) 37 604 AGEVITLET 87. 38 575 VIFTLTAYY 88. 39 675 VLGWMNALY 89. 40 651 LVIVSAALY 1.000 90. 41 257 DLDGLLPFL 1.000 91. 42 85 SADGPGAGM 92. 43 534 KIENRHEML 0.900 93. 44 308 IAERTGNMR 0. 900 94. 45 921 VVELNKNSN 95. 46 107 VAELPGDES 0.900 96. 47 197 DSLFDYGTY 97. 48 77 LSFPCRLSS 98. 49 194 APMDSLFDY 99. 50 772 MGDLEMLSS 0.625 100.

TABLE VI (A) HLA Peptide Scoring Results-83P2H3-Al, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Disassociation of a Molecule Rank Position Listing Containing This Subsequence) 1 632 RVEDRQDLNR 45.000 101. 2 714 GLEDGESWEY 45.000 102. 3 106 VFEPMTSELY 22. 500 103. 4 98 LMEAAPELVF 2.500 104. 5 292 LLELIITTKK 18.000 105. 6 513 QTEDPEELGH 11.250 106. 7 174 NSEEIVRLLI 6.750 107. 8 248 GVEGNTVMFQ 4.500 108. 9 401 LVEVPDIFRM 4.500 109. 10 182 LIEHGADIRA 4. 500 110. 11 677 LSLPMPSVSR 3.000 111.

TABLE VI (A) HLA Peptide Scoring Results-83P2H3-Al, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Disassociation of a Molecule Rank Position Listing Containing This Subsequence) 12 514 TEDPEELGHF 2. 500 112. 13 538 LTIIDGPANY 2. 500 113. 14 550 DLPFMYSITY 2. 500 114. 15 80 ETALHIAALY 2. 500 115. 16 655 GSEDLDKDSV 1. 350 116. 17 111 TSELYEGQTA 1.350 117. 18 478 FTIMIQKMIF 1. 250 118. 19 330 CMLGAIYLLY 1. 250 119. 20 704 RRDLRGIINR 1. 250 120. 21 78 MGETALHIAA 1. 125 121. 22 387 VGELVTVIGA 1. 125 122. 23 162 FGEHPLSFAA 1. 125 123. 24 253 TVMFQHLMQK 1. 000 124. 25 458 LVLGWCNVMY 1.000 125. 26 57 DVQALNKLLK 1. 000 126. 27 540 IIDGPANYNV 1. 000 127. 28 548 NVDLPFMYSI 1.000 128. 29 500 VILGFASAFY 1. 000 129. 30 33 RDEQNLLQQK 0. 900 130. 31 313 VKELVSKLWK 0.900 131. 32 447 ASGEVVPMSF 0. 750 132. 33 286 SGDEQSLLEL 0. 625 133. 34 225 HGDHLQPLDL 0. 625 134. 35 423 GPFHVLIITY 0. 625 135. 36 585 VAHERDELWR 0. 500 136. 37 495 WLMAWILGF 0. 500 137. 38 400 LLVEVPDIFR 0. 500 138. 39 597 IVATTVMLER 0.500 139. 40 186 GADIRAQDSL 0.500 140. 41 171 ACVNSEEIVR 0. 500 141. 42 282 EIDSSGDEQS0.500 142.

TABLE VI (A) HLA Peptide Scoring Results - 83P2H3 - A1, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Disassociation of a Molecule Rank Position Listing Containing This Subsequence) 43 341 ICFTMCCIYR 0.500 143. 44 204 ILQPNKTFAC 0.500 144. 45 601 TVMLERKLPR 0.500 145. 46 307 ILDQTPVKEL 0.500 146. 47 12 ILCLWSKFCR 0.500 147. 48 340 IICFTMCCIY 0.500 148. 49 315 ELVSLKWKRY 0.500 149. 50 334 AIYLLYIICF 0.500 150.

TABLE VI (B) HLA Peptide Scoring Results - CaTrF2E11 - A1, 10-mers Score (Estimate of Half Time of SEQ ID NO : Start Subsequence Residue Disassociation of a Molecule Containing Rank Position Listing This Subsequence) 1 543 AVEPINELLR 450.000 151. 2 63 FLEPPPLAGE 180.000 152. 3 636 FIDGSFQLLY 125.000 153. 4 774 DLEMLSSTKY 45.000 154. 5 255 TADLDGLLPF 25.000 155. 6 525 VLFILVVYNSK 18. 000 156. 7 209 SSDNKRWRKK 15.000 157. 8 888 NEDPGKNETY 12.500 158. 9 423 IADNTRENTK 10.000 159. 10 585 PLEGTPPYPY 9.000 160. 11 810 MGETVGQVSK 9.000 161. 12 754 DSETFSTFLL 6.750 162. 13 319 INSPFRDIYY 6.250 163. 14 4 VVGPGANLCF 5.000 164. 15 403 KADMRRQDSR 5.000 165. 16 662 GIEAYLAMMV 4.500 166. 17 893 KNETYQYYGF 4.500 167. 18 534 KIENRHEMAL 4. 500 168.

TABLE VI (B) HLA Peptide Scoring Results-CaTrF2Ell-Al, 10-mers Score (Estiamte of Half time of SEQ ID NO: Start Subsequence Residue Disassociatin of a Molecule Containing Rank Position Listing this Subsequence) 19 462 GLSPLMMAAK 4.000 169. 20 639 GSFQLLYFIY 3.750 170. 21 744 CTVPTYPSCR 2.500 171. 22 498 SKDWAYGPVY 2.500 172. 23 193 KAPMDSLFDY 2.500 173. 24 174 PIDLLESTLY 2.500 174. 25 283 ILDIERSFPV 2.500 175. 26 618 FTNIKDLFMK 2.500 176. 27 386 CTNQPHIVNY 2.500 177. 28 711 LVYLLFMIGY 2.500 178. 29 257 DLDGLLPFLL 2.500 179. 30 522 EASVLEILVY 2.500 180. 31 487 TDEDTRHLSR 2.250 181. 32 547 INELLRDKWR 2.250 182. 33 260 GLLPFLLTHK 2.000 183. 34 73 GLTPLSFPCR 2.000 184. 35 219 IIEKQPQSPK 1.800 185. 36 427 TRENTKFVTK 1.800 186. 37 758 FSTFLLDLFK 1.500 187. 38 561 VSFYINVVSY 1.500 188. 39 96 SSEGPRAGPG 1.350 189. 40 486 VTDEDTRHLS 1.250 190. 41 272 LTDEEFREPS 1.250 191. 42 331 QTALHIAIER 1.250 192. 43 608 ITLFTGVLFF 1.250 193. 44 376 FGELPLSLAA 1.125 194. 45 604 AGEVITLFTG 1.125 195. 46 519 CGEEASVLEI 1.215 196. 47 353 GADVHAQARG 1.000 197. 48 695 SIMIQKILFK 1.000 198. 49 785 VVFILLVTY 1.000 199.

TABLE VI (B) HLA Peptide Scoring Results-CaTrF2Ell-Al, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Disassociation of a Molecule Containing Rank Position Listing This Subsequence) 50 93 MADSSEGPRA 1. 000 200.

TABLE VII (A) HLA Peptide Scoring Results-83P2H3-A2,9-mers Score (Estimate of Half Time of SEQ ID NO : Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 1 577 MMGDTHWRV 2625. 878 201. 2 336 YLLYIICFT 1604.438 202. 3 97 VLMEAAPEL 550.915 203. 4 291 SLLELIITT 260. 008 204. 5 135 ALLARRASV 257. 342 205. 6 419 TILGGPFHV 205. 231 206. 7 385 RLVGELVTV 159. 970 207. 8 337 LLYIICFTM 156. 750 208. 9 399 ILLVEVPDI 150. 931 209. 10 330 CMLGAIYLL 131. 296 210. 11 457 ALVLGWCNV 118.238 211. 12 50 LLAAKDNDV 118.238 212. 13 472 FQMLGPFTI 104.419 213. 14 43 RIWESPLLL 99. 957 214. 15 623 YGLGDRWFL 97.904 215. 16 371 KLLQEAYMT 96.503 216. 17 428 LIITYAFMV 94.295 217. 18 436 VLVTMVMRL 83.527 218. 19 87 ALYDNLEAA 73.458 219. 20 181 LLIEHGADI 72.717 220. 21 427 VLIITYAFM 69.676 221. 22 474 MLGPFTIMI 67.396 222. 23 553 FMYSITYAA 52.815 223. 24 569 LMLNLLIAM 51.908 224.

TABLE VIT (A) HLA Peptide Scoring Results - 83P2H3 - A2, 9-mers Score (Estimate of Half Time of SEQ ID NO : Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence( 25 12 ILCLWSKFC 46.451 225. 26 204 ILQPNKTFA 46. 451 226. 27 430 ITYAFMVLV 45. 227. 28 489 DLMRFCWLM 39. 291 228. 29 567 TLLMLNLLI 38.601 229. 30 77 AMGETALHI 30. 893 230. 31 568 LLMLNLLIA 29.468 231. 32 420 ILGGPFHVL 28. 290 232. 33 194 SLGNTVLHI 23. 995 233. 34 125 VVNQNMNLV 23.795 234. 35 396 AIIILLVEV 21. 996 235. 36 113 ELYEGQTAL 21. 236. 37 512 FQTEDPEEL 20. 016 237. 38 451 VVPMSFALV 19.657 238. 39 570 MLNLLIAMM 19.425 239. 40 159 LIYFGEHPL 15. 979 240. 41 129 NMNLVRALL 15.428 241. 42 502 LGFASAFYI 13.665 242. 43 329 FCMLGAIYL 13. 054 243. 44 202 ILILQPNKT 12.668 244. 45 339 YIICFTMCC 11.941 245. 46 393 VIGAIIILL 11.485 246. 47 528 MALFSTFEL 10.824 247. 48 473 QMLGPFTIM 10. 248. 49 443 RLISASGEV 9. 042 249. 50 556 SITYAAFAI 8. 320 250.

TABLE VII (B) HLA Peptide Scoring Results - CaTrF2F11 - A2, 9-mers Score estimate of Half Time of SEQ ID NO : Start Subsequence Residue Rank Disassociation of a Molecule Containing Position Listing This Subsequence) TABLE VII (B) HLA Peptide Scoring Results-CaTrF2Ell-A2, 9-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of of Molecule Containing Position Listing This Subsequence) 1 642 QLLYFIYSV 2249.173 251. 2 666 YLAMMVFAL 1310. 882 252. 3 709 FLLVYLLFM 1069.625 253. 4 761 FLLDLFKLT 988.029 254. 5 659 YLAGIEAYL 540. 469 255. 6 570 YLCAMVIFT 433. 632 256. 7 264 FLLTHKKRL 363. 588 257. 8 801 LLLNMLIAL 309.050 258. 9 710 LLVYLLFMI 236.595 259. 10 49 KQLAALLLV 210.038 260. 11 73 YLLFMIGYA 139.051 261. 12 440 LLLLKCARL 134.369 262. 13 646 FIYSVLVIV 132. 749 263. 14 163 FQGAFRKGV 123. 265 264. 15 777 MLSSTKYPV 118. 238 265. 16 348 LLVAQGADV 118.238 266. 17 809 LMGETVGQV 104.685 267. 18 304 VLLDIAERT 94.168 268. 19 668 AMMVFALVL 88.939 269. 20 787 FIILLVTYI 83. 474 270. 21 789 ILLVTYIIL 82. 637 271. 22 643 LLYFIYSVL 71.470 272. 23 716 FMIGYASAL 70.971 273. 24 34 WEWPPCAPV 51.635 274. 25 602 RLAGEVITL 49. 134 275. 26 286 CLPKALLNL 49. 134 276. 27 795 IILTSVLLL 42.494 277. 28 650 VLVIVSAAL 36.316 278. 29 578 TLTAYYQPL 32.044 279. 30 826 KLQWATTIL 30.655 280. 31 800 VLLLNMLIA 29.468 281.

FABLE VU (B) HLA Peptide Scoring Results-CaTrF2Ell-A2, 9-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Containing Position Listing This Subsequence) 32 790 LLVTYIILT 282. 33 611 FTGVLFFFT 283. 34 73 CLTPLSFPC 28. 814 284. 35 652 VIVSAALYL 285. 36 388 NQPHIVNYL 27.399 286. 37 674 LVLGWMNAL 287. 38 762 LLDLFKLTI 288. 39 819 KESKHIWKL 25.079 289. 40 613 GVLFFFTNI 24.386 290. 41 767 KLTIGMGDL 22.356 291. 42 897 YQYYGFSHT 21. 131 292. 43 802 LLNMLIALM 19.425 293. 44 726 SLLNPCANM 18. 382 294. 45 717 MIGYASALV 16.258 295. 46 657 ALYLAGIEA 15.898 296. 47 573 AMVIFTLTA 13.634 297. 48 794 YIILTSVLL 13.512 298. 49 792 VTYIILTSV 12.087 299. 50 667 LAMMVFALV 11.545 300.

ITABLE VIII (A) HLA Peptide Scoring Results-83P2H3-A2,10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 1 529 ALFSTFELFL 1651. 954 301. 2 576 AMMGDTHWRV 1393.938 302. 3 63 KLLKYEDCKV 900.698 303. 4 97 VLMEAAPELV 878.901 304. 5 427 VLIITYAFMV 685. 783 305. 6 501 ILGFASAFYI 565.771 306.

TABLE VIII(A) HLA Peptide Scoring Results - 83P2H3 - A2, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 7 624 GLGDRWFLRV 541. 810 307. 8 336 YLLYIICFTM 490. 421 308. 9 49 LLLAAKDNDV 437.482 309. 10 245 KLAGVEGNTV 243. 432 310. 11 331 MLGAIYLLYI 224.357 311. 12 602 VMLERKLPRC 212. 821 312. 13 240 GLTPFKLAGV 159. 970 313. 14 429 IITYAFMVLV 142. 093 314. 15 465 VMYFARGFQM 113. 209 315. 16 473 QMLGPFTIMI 105. 939 316. 17 568 LLMLNLLIAM 71. 872 317. 18 377 YMPTKDDIRL 70.971 318. 19 420 ILGGPFHVLI 67. 396 319. 20 87 ALYDNLEAAM 65.180 320. 21 43 RIWESPLLLA 53.466 321. 22 569 LMLNLLIAMM 51.908 322. 23 337 LLYIICFTMC 51. 349 323. 24 204 ILQPNKTFAC 48.984 324. 25 105 LVFEBPMTSEL 48.205 325. 26 180 RLLIEHGADI 38.601 326. 27 432 YAFMVLVTMV 37.815 327. 28 393 VIGAIIILLV 37. 393 328. 29 307 ILDQTPVKEL 33.411 329. 30 456 FALVLGWCNV 27.950 330. 31 435 MVLVTMVMRL 27. 042 331. 32 203 LILQPNKTFA 23. 632 332. 33 11 LILCLWSKFC 23. 632 333. 34 158 NLIYFGEHPL 21. 362 334. 35 2 GLSLPKEKGL 21. 362 335. 36 398 IILLVEVPDI 20. 753 336. 37 194 SLGNTVLHIL 20. 145 337.

TABLE VIII (A) HLA Peptide Scoring Results-83P2H3-A2, I Score (Estimate of Half Time of SEQ ID NO : Start Subsequence Residue Rank Disassociation of Molecule Position Listing Containing This Subsequence) 38 367 LLQQKLLQEA 19.425 338. 39 567 TLLMLNLLIA 17.334 339. 40 339 YIICFTMCCI 15. 177 340. 41 490 LMRFCWLMAV 14.927 341. 42 124 AVVNQNMNLV 13.997 342. 43 443 RLISASGEVV 13.973 343. 44 77 AMGETALHIA 13. 872 344. 45 96 MVLMEAAPEL 11.757 345. 46 564 IIATLLMLNL 11. 485 346. 47 485 MIFGDLMRFC 10.871 347. 48 591 ELWRAQIVAT 10. 669 348. 49 496 LMAVVILGFA 10.031 349. 50 385 RLVGELVTVI 9.838 350.

TABLE VIII (B) HLA Peptide Scoring Results-CaTrF2Ell-A2, 10-mers Score (Estimate of Half Time of SEQ ID NO : Start Subsequence Residue Rank Disassociation of a Molecule Containing Position Listing This Subsequence) 1 761 FLLDLFKLTI 2766.482 351. 2 709 FLLVYLLFMI 2368.734 352. 3 570 YLCAMVIFTL 1310.882 353. 4 641 FQLLYFIYSV 1048.989 354. 5 666 YLAMMVFALV 607. 884 355. 6 634 SLFIDGSFQL 458.437 356. 7 897 YQYYGFSHTV 394.449 357. 8 436 KMYDLLLLKC 378. 950 358. 9 643 LLYFIYSVLV 378.363 359. 10 800 VLLLNMLIAL 309.050 360. 11 701 ILFKDLFRFL 280.832 361. 12 833 ILDIERSFPV 274.313 362. 13 716 FMIGYASALV 231.067 363.

TABLE VIII (B) HLA Peptide Scoring Results - CaTfF2E11 - A2, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of of Molecule Containing Position Listing This Subsequence) 14 50 QLAALLLVHV 159. 970 364. 15 727 LLNPCANMKV 118. 238 365. 16 789 ILLVTYILLT 107.808 366. 17 509 SLYDLSSLDT 97. 770 367. 18 395 YLTENPHKKA 93. 696 368. 19 457 VLNNDGLSPL 83.527 369. 20 541 MLAVEPINEL 83. 527 370. 21 808 ALMGETVGQV 76.945 371. 22 705 DLFRFLLVYL 74.990 372. 23 777 MLSSTKYPVV 72.717 373. 24 801 LLLNMLIALM 71.872 374. 25 681 ALYFTRGLKL 68. 360 375. 26 805 MLIALMGETV 57. 937 376. 27 673 ALVLGWMNAL 49. 134 377. 28 642 QLLYFIYSVL 48.610 29 714 LLFMIGYASA 46. 873 379. 30 130 NLFEGEDGSL 42. 129 380. 31 689 KLTGTYSIMI 36.515 381. 32 827 LQWATTILDI 34. 328 382. 33 794 YIILTSVLLL 31. 077 383. 34 55 LLVHVGGGFL 25. 966 384. 35 264 FLLTHKKRLT 25.367 385. 36 791 LVTYIILTSV 23. 795 386. 37 659 YLAGIEAYLA 22.853 387. 38 609 TLFTGVLFFF 20.230 388. 39 796 ILTSVLLLNM 19.425 389. 40 623 DLFMKKCPGV 19. 301 390. 41 347 ELLVAQGADV 19. 301 391. 42 447 RLFPDSNLEA 18.382 392. 43 651 LVIVSAALYL 17. 477 393. 44 759 STFLLDLFKL 14. 645 394.

TABLE VIII (B) HLA Peptide Scoring Results-CaTrF2E11-A2, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Containing Position Listing This Subsequence) 45 776 EMLSSTKYPV 13.939 395. 46 592 YPYRTTVDYL 12.724 396. 47 558 FGAVSFYINV 11.904 397. 48 42 VITTVALKQL 11.485 398. 49 788 IILLVTYIIL 11.363 399. 50 697 MIQKILFKDL 9.488 400.

TABLE IX (A) HLA Peptide Scoring Results-83P2H3-A3,9-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 1 10 GLILCLWSK 405.000 401. 2 254 VMFQHLMQK 300.000 402. 3 63 KLLKYEDCK 270.000 403. 4 214 QMYNLLLSY 60. 000 404. 5 607 KLPRCLWPR 54.000 405. 6 292 LLELIITTK 45.000 406. 7 624 GLGDRWFLR 36.000 407. 8 484 KMIFGDLMR 36.000 408. 9 529 ALFSTFELF 30.000 409. 10 131 NLVRALLAR 18.000 410. 11 602 VMLERKLPR 18. 000 411. 12 476 GPFTIMIQK 13.500 412. 13 331 MLGAIYLLY 12.000 413. 14 318 SLKWKRYGR 12.000 414. 15 576 AMMGDTHWR 9.000 415. 16 496 LMAVVILGF 9.000 416. 17 697 RLRQGTLRR 8.000 417. 18 400 LLVEVPDIF 6.750 418. 19 330 CMLGAIYLL 6.075 419.

TABLE IX (A) HLA Peptide Scoring Results-83P2H3-A3,9-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 20 678 SLPMPSVSR 6.000 420. 21 377 YMTPKDDIR 6. 000 421. 22 658 DLDKDSVEK 6.000 422. 23 315 ELVSLKWKR 5.400 423. 24 474 MLGPFTIMI 5.400 424. 25 436 VLVTMVMRL 5.400 425. 26 553 FMYSITYAA 4.500 426. 27 485 MIFGDLMRF 4.500 427. 28 337 LLYIICFTM 4. 500 428. 29 420 ILGGPFHVL 4.050 429. 30 501 ILGFASAFY 4. 000 430. 31 459 VLGWCNVMY 4.000 431. 32 194 SLGNTVLHI 3.600 432. 33 306 QILDQTPVK 3.000 433. 34 201 HILILQPNK 3.000 434. 35 14 CLWSKFCRW 3. 000 435. 36 399 ILLVEVPDI 2.700 436. 37 373 LQEAYMTPK 2.700 437. 38 638 DLNRDRIQR 2.400 438. 39 347 CIYRPLKPR 2.250 439. 40 473 QMLGPFTIM 2.025 440. 41 567 TLLMLNLLI 1. 800 441. 42 77 AMGETALHI 1. 800 442. 43 87 ALYDNLEAA 1. 500 443. 44 599 ATTVMLERK 1. 500 444. 45 500 VILGFASAF 1.350 445. 46 97 VLMEAAPEL 1. 350 446. 47 113 ELYEGQTAL 1.350 447. 48 532 STFELFLTI 1.350 448. 49 181 LLIEHGADI 1.350 449. 50 426 HVLIITYAF 1.350 450.

TABLE IX (B) HLA Peptide Scoring Results - CaTrF2E11 - A3, 9-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Containing Position Listing This Subsequence) 1 436 KMYDLLLLK 900. 000 451. 2 614 VLFFFTNIK 300. 000 452. 3 696 IMIQKILFK 90. 000 453. 4 692 GTYSIMIQK 67.500 454. 5 198 SLFDYGTYR 60.000 455. 6 609 TLFTGVLFF 60.000 456. 7 705 DLFRFLLVY 54.000 458. 8 701 ILFKDLFRF 45.000 459. 9 466 LMMAAKTGK 30.000 459. 10 727 LLNPCANMK 30.000 460. 11 261 LLPFLLTHK 30. 000 461. 12 681 ALYFTRGLK 30.000 462. 13 395 YLTENPHKK 30. 000 463. 14 549 ELLRDKWRK 27. 000 464. 15 476 GIFQHIIRR 18. 000 465. 16 260 GLLPFLLTH 12.150 466. 17 620 NIKDLFMKK 12.000 467. 18 678 WMNALYFTR 12. 000 468. 19 759 STFLLDLFK 10. 000 469. 20 885 GIINEDPGK 9.000 470. 21 838 RSFPVFLRK 6. 750 471. 22 774 DLEMLSSTK 6. 000 472. 23 333 ALHIAIERR 6. 000 473. 24 239 KVFNRPILF 6.000 474. 25 290 ALLNLSNGR 6.000 475. 26 602 RLAGEVITL 5.400 476. 27 666 YLAMMVFAL 5.400 477. 28 668 AMMVFALVL 5.400 478. 29 6 LLYFIYSVL 4. 500 479. 30 716 FMIGYASAL 4.050 480.

TABLE IX (B) HLA Peptide Scoring Results-CaTrF2Ell-A3, 9-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of Molecule Molecule Containing Position Listing This Subsequence) 31 710 LLVYLLFMI 4.050 481. 32 642 QLLYFIYSV 482. 33 675 VLGWMNALY 4.000 483. 34 160 RMKFQGAFR 4.000 484. 35 762 LLDLFKLTI 3.600 485. 36 700 KILFKDLFR 3.600 486. 37 462 GLSPLMMAA 487. 38 709 FLLVYLLFM 488. 39 801 LLLNMLIAL 2.700 489. 40 613 GVLFFFTNI 2.430 490. 41 575 VIFTLTAYY 2.000 491. 42 281 STGKTCLPK 2.000 492. 43 657 ALYLAGIEA 2.000 493. 44 286 CLPKALLNL 1.800 494. 45 578 TLTAYYQPL 1.800 495. 46 826 KLQWATTIL 1.800 496. 47 439 DLLLLKCAR 497. 48 789 ILLVTYIIL 1.800 498. 49 573 AMVIFTLTA 1.800 499. 50 790 LLVTYIILT 1.350 500.

TABLE X (A) HLA Peptide Scoring Results-83P2H3-A3, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 1 372 LLQEAYMTPK 135. 000 501. 2 291 SLLELIITTK 101.250 502. 3 344 TMCCIYRPLK 60.000 503. 4 714 GLEDGESWEY 36. 000 504. 5 292 LLELIITTKK 30. 000 505.

TABLE X (A) HLA Peptide Scoring Results-83P2H3-A3, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 6 254 VMFQHLMQKR 30.000 506. 7 400 LLVEVPDIFR 27. 000 507. 8 330 CMLGAIYLLY 27. 000 508. 9 484 KMIFGDLMRF 27. 000 509. 10 529 ALFSTFELFL 18. 000 510. 11 495 WLMAWILGF 13. 500 511. 12 459 VLGWCNVMYF 12. 000 512. 13 12 ILCLWSKFCR 12. 000 513. 14 624 GLGDRWFLRV 10. 800 514. 15 553 FMYSITYAAF 10. 000 515. 16 253 TVMFQHLMQK 9. 000 516. 17 10 GLILCLWSKF 9.000 517. 18131 NLVRALLARR 9. 000 518. 19 334 AIYLLYIICF 9. 000 519. 20 181 LLIEHGADIR 9. 000 520. 21 434 FMVLVTMVMR 9.000 521. 22 473 QMLGPFTIMI 8. 100 522. 23 550 DLPFMYSITY 7.200 523. 24 98 LMEAAPELVF 6.000 524. 25 331 MLGAIYLLYI 5. 400 525. 26 399 ILLVEVPDIF 4. 500 526. 27 385 RLVGELVTVI 4. 050 527. 28 652 HTRGSEDLDK 3. 000 528. 29 337 LLYIICFTMC 3. 000 529. 30 465 VMYFARGFQM 3. 000 530. 31 14 CLWSKFCRWF 3.000 531. 32 420 ILGGPFHVLI 2. 700 532. 33 427 VLIITYAFMV 2. 700 533. 34 202 ILILQPNKTF 2. 250 534. 35 209 KTFACQMYNL 2.025 535. 36 501 ILGFASAFYI 1.800 536.

TABLE X (A) HLA Peptide Scoring Results-83P2H3-A3, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of Molecule Position Listing Containing This Subsequence) 37 490 LMRFCWLMAV 1.800 537. 38 423 GPFHVLIITY 1.800 538. 39 638 DLNRQRIQRY 1. 800 539. 40 597 IVATTVMLER 1.800 540. 41 377 YMTPKDDIRL 1.800 541. 42 336 YLLYIICFTM 1.350 542. 43 240 GLTPFKLAGV 1.350 543. 44 194 SLGNTVLHIL 1.350 544. 45 576 AMMGDTHWRV 1.350 545. 46 307 ILDQTPVKEL 1. 350 546. 47 601 TVMLERKLPR 1.200 547. 48 4 SLPKEKGLIL 1.200 548. 49 57 DVQALNKLLK 1.200 549. 50 532 STFELFLTII 1.012 550.

TABLE X (B) HLA Peptide Scoring Results - CaTrF2E11 - A3, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of Molecule Containing Position Listing This Subsequence) 1 260 GLLPFLLTHK 202.500 551. 2 462 GLSPLMMAAK 135.000 552. 3 609 TLFTGVLFFF 67.500 553. 4 160 RMKFQGAFRK 60.000 554. 5 657 ALYLAGIEAY 30.000 555. 6 525 VLEILVYNSK 30.000 556. 7 726 SLLNPCANMK 30.000 557. 8 613 GVLFFFTNIK 27.000 558. 9 261 LLPFLLTHKK 20.000 559. 10 73 CLTPLSFPCR 18.000 560. 11 711 LVYLLFMIGY 18.000 561. 12 689 KLTGTYSIMI 16.200 562.

TABLE X (B) HLA Peptide Scoring Results-CaTrF2Ell-A3, 10-mers Score (Estimate of Half Time of SEQ ID NO : Start Subsequence Residue Rank Disassociation of a Molecule Containing Position Listing This Subsequence) 13 634 SLFIDGSFQL 9. 000 563. 14 573 AMVIFTLTAY 9. 000 564. 15 695 SIMIQKILFK 9. 000 565. 16 436 KMYDLLLLKC 9.000 566. 17 602 RLAGEVITLF 6. 750 567. 188 63 FLEPPPLAGF 6.750 568. 19 681 ALYFTRGLKL 6. 000 569. 20 419 ALVALADNTR 6. 000 570. 21 650 VLVIVSAALY 6. 000 571. 22 917 VVPRVVELNK 6. 000 572. 23 761 FLLDLFKLTI 5. 400 573. 24 687 GLKLTGTYSI 5. 400 574. 25 314 NMREFINSPF 4. 500 575. 26 618 FTNIKDLFMK 4. 500 576. 27 570 YLCAMVIFTL 4.050 28 709 FLLVYLLFMI 4.050 29 673 ALVLGWMNAL 4. 050 30 700 KILFKDLFRF 4. 050 580. 31 675 VLGWMNALYF 4. 000 581. 32 92 GMADSSEGPR 3. 600 582. 33 636 FIDGSFQLLY 3. 600 583. 34 219 IIEKQPQSPK 3. 000 584. 35 643 LLYFIYSVLV 3.000 585. 36 529 LVYNSKIENR 3.000 586. 37 785 VVFIILLVTY 3.000 587. 38 447 RLFPDSNLEA 3.000 588. 39 465 PLMMAAKTGK 3. 000 589. 40 198 SLFDYGTYRH 3. 000 590. 41 800 VLLLNMLIAL 2. 700 591. 42 359 QARGRFFQPK 2. 700 592. 43 585 PLEGTPPYPY 2.700 593.

TABLE X (B) HLA Peptide Scoring Results-CaTrF2Ell-A3, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Containing Position Listing This Subsequence) 44 474 KIGIFQHIIR 2.400 594. 45 813 TVGQVSKESK 2.000 595. 46 158 NLRMKFQGAF 1.800 596. 47 669 MMVFALVLGW 1.800 597. 48 54 LLLVHVGGGF 1.350 598. 49 541 MLAVEPINEL 1.350 599. 50 789 ILLVTYIILT 1.350 600.

TABLE XI (A) HLA Peptide Scoring Results - 83P2H3 - A11, 9-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subseuqnce) 1 10 GLILCLWSK 3.600 601. 2 476 GPFTIMIQK 2.400 602. 3 63 KLLKYEDCK 1.800 603. 4 254 VMFQHLMQK 1.600 604. 5 58 VQALNKLLK 1.200 605. 6 599 ATTVMLERK 1.000 606. 7 172 CVNSEEIVR 0.800 607. 8 401 LVEVPDIFR 0.800 608. 9 624 GLGDRWFLR 0.720 609. 10 484 KMIFGDLMR 0.720 610. 11 306 QILDQTPVK 0.600 611. 12 201 HILILQPNK 0.600 612. 13 435 MVLVTMVMR 0. 600 613. 14 355 RTNNRTSPR 0.600 614. 15 373 LQEAYMTPK 0.600 615. 16 607 KLPRCLWPR 0.480 616. 17 697 RLRQGTLRR 0.480 617. 18 292 LLELIITTK 0.400 618.

TABLE XI (A) HLA Peptide Scoring Results - 83P2H3 - A11, 9-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Susequence) 19 132 LVRALLARR 0.400 619. 20 146 RATGTAFRR 0.360620. 21 256 FQHLMQKRK 0.300 621. 22 602 VMLERKLPR 0.240 622. 23 646 RYAQAFHTR 0.240 623. 24 131 NLVRALLAR 0.240 624. 25 345 MCCIYRPLK 0.200 625. 26 297 ITTKKREAR 0.200 626. 27 312 PVKELVSLK 0.200 627. 28 13 LCLWSKFCR 0.180 628. 29 576 AMMGDTHWR 0.160 629. 30 318 SLKWKRYGR 0.160 630. 31 314 KELVSLKWK 0.135 631. 32 658 DLDKDSVER 0.120 632. 33 462 WCNVMYFAR 0.120 633. 34 18 KFCRWFQRR 0.120 634. 35 293 LELIITTKK 0.090 635. 36 347 CIYRPLKPR 0.080 636. 37 598 VATTVMLER 0.080 637. 38 678 SLPMPSVSR 0.080 638. 39 342 CFTMCCIYR 0.080 639. 40 182 LIEHGADIR 0.080 640. 41 377 YMTPKDDIR 0.080 641. 42 315 ELVSLKWKR 0.072 642. 43 363 RDNTLLQQK 0.060 643. 44 426 HVLIITYAF 0.060 644. 45 392 TVIGAIIIL 0.060 645. 46 584 RVAHERDEL 0.060 646. 47 124 AVVNQNMNL 0.060 647. 48 248 GVEGNTVMF 0.060 648. 49 406 DIFRMGVTR 0.048 649.

TABLE XI (A) HLA Peptide Scoring Results-83P2H3-All, 9-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 50 638 DLNRQRIDR 0.048 650.

TABLE XI (B) HLA Peptide Scoring Results - CaTrF2E11 - A11, 9-mers Score (Estimate of Half Time of SEQ ID NO : Start Subsequence Residue Rank Disassociation of of Molecule Containing Position Listing This Subsequence) 1 692 GTYSIMIQK 12. 000 651. 2 436 KMYDLLLLK. 4. 800 652. 3 759 STFLLDLFK 4. 000 653. 4 281 STGKTCLPK 2. 000 654. 5 885 GIINEDPGK 1. 800 655. 6 696 IMIQKIFK 1. 200 656. 7 476 GIFQHIIRR 0.960 657. 8 620 NIKDLFMKK 0. 800 658. 9 681 ALYFTRGLK 0. 800 659. 10 614 VLFFFTNIK 0. 800 660. 11 466 LMMAAKTGK 0. 800 661. 12 700 KILFKDLFR 0. 720 662. 13 394 NYLTENPHK 0. 600 663. 14 727 LLNPCANMK 0. 400 664. 15 395 YLTENPHKK 0. 400 665. 16 420 LVAIADNTR 0. 400 666. 17 745 TVPTYPSCR 0. 400 667. 18 918 VPRWELNK 0. 400 668. 19 231 APQPPPILK 0. 400 669. 20 830 ATTILDIER 0. 670. 21 261 LLPFLLTHK 0. 400 671. 22 262 LPFLLTHKK 0. 400 672. 23 549 ELLRDKWRK 0.360673. 24 41 PVITTVALK 0. 300 674. 25 838 RSFPVFLRK 0.240 675.

TABLE XI (B) HLA Peptide Scoring Results - CaTrF2E11 - A11, 9-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Containing Position Listing This Subsequence) 26 160 RMKFQGAFR 0. 240 676. 27 678 WMNALYFTR 0. 677. 28 239 KVFNRPILF 0. 678. 29 74 LTPLSFPCR 0. 200 679. 30 619 TNIKDLFMK 0. 180 680. 31 428 RENTKFVTK 0. 180 681. 32 811 GETVGQVSK 0. 180 682. 33 321 SPFRDIYYR 0. 160 683. 34 198 SLFDYGTYR 0.160 684. 35 161 MKFQGAFRK 0. 120 685. 36 214 RWRKKIIEK 0. 120 686. 37 148 RPAGPGDGR 0. 687. 38 8 GANLCFQVR 0. 120 688. 39 871 RVDEVNWSH 0. 120 689. 40 774 DLEMLSSTK 0. 120 690. 41 332 TALHIAIER 0. 120 691. 42 290 ALLNLSNGR 0. 120 692. 43 353 GADVHAQAR 0. 120 693. 44 3 RWGPGANL 0. 090 694. 45 613 VLFFFTNI 0.090 695. 46 526 LEILVYNSK 0.090 696. 47 670 MVFALVLGW 0. 080 697. 48 333 ALHIAIERR 0. 080 698. 49 530 VYNSKIENR 0. 080 699. 50 841 PVFLRKAFR 0.080 700.

|TABLE XII (A) HLA Peptide Scoring Results-83P2H3-All, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) TABLE XII (A) HLA Peptide Scoring Results-83P2H3-All, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 1253TVMFQHLMQK8. 000701. 2 305 RQILDQTPVK 2.700 702. 3 632 RVEDRQDLNR 2.400 703. 4 652 HTRGSEDLDK 2.000 704. 5 601 TVMLERKLPR 1.600 705. 6 57 DVQALNKLLK 1. 200 706. 7 597 IVATTVMLER 0.800 707. 8 125 VVNQNMNLVR 0.800 708. 9 291 SLLELIITTK 0.600 709. 10 292 LLELIITTKK 0. 40. 11 344 TMCCIYRPLK 0. 400 711. 12 372 LLQEAYMTPK 0. 400 13 699 RQGTLRRDLR 0. 360 713. 14 311 TPVKELVSLK 0.300 714. 15 66 KYEDCKVHQR 0. 240 715. 16 12 ILCLWSKFCR 0. 240 716. 17 400 LLVEVPDIFR 0.240 18 598 VATTVMLERK 0. 200 718. 19 9 KGLILCLWSK 0.180 719. 20 350 RPLKPRTNNR 0. 180 720. 21 341 ICFTMCCIYR 0. 160 721. 22 215 MYNLLLSYDR 0. 160 722. 23 376 AYMTPKDDIR 0. 160 723. 24 254 VMFQHLMQKR 0. 160 724. 25 54 KDNDVQALNK 0. 120 725. 26 131 NLVRALLARR 0. 120 726. 27 181 LLIEHGADIR 0.120 28 434 FMVLVTMVMR 0. 120 728. 29 209 KTFACQMYNL 0.120 729. 30 171 ACVNSEEIVR 0.120 730. 31 314 KELVSLKWKR 0.108 731.

TABLE XII (A) HLA Peptide Scoring Results-83P2H3-All, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 32 255 MFQHLMQKRK 732. 33 575 IAMMGDTHWR 0.080 733. 34 296 IITTKKREAR 0.080 734. 35 585 VAHERDELWR 0.080 735. 36 33 RDEQNLLQQK 0.060 736. 37 392 TVIGAIIILL 0.060 38 580 DTHWRVAHER 738. 39 584 RVAHERDELW 0.060 739. 40 411 GVTRFFGQTI 0.060 740. 41 401 LVEVPDIFRM 741. 42 45 WESPLLLAAK 0. 060 742. 43 435 MVLVTMVMRL 0.060 743. 44 43 RIWESPLLLA 0.048 744. 45 418 QTILGGPFHV 0.045 745. 46 681 MPSVSRSTSR 0.040 746. 47 137 LARRASVSAR 747. 48 144 SARATGTAFR 748. 49 390 LVTVIGAIII 749. 50 451 VVPMSFALVL 0. 040 750.

TABLE XII (B) HLA Peptide Scoring Results - CaTrF2E11 - A11, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Containing Position Listing This Subsequence) 1 613 GVLFFFTNIK 9.000 751. 2 917 VVPRVVELNK 4.000 752. 3 160 RMKFQGAFRK 3.600 753. 4 618 FTNIKDLFMK 3.000 754. 5 813 TVGQVSKESK 2.000 755. 6 260 GLLPFLLTHK 1.800 756. 7 695 SIMIQKILFK 757.

TABLE XII (B) HLA Peptide Scoring Results-CaTrF2Ell-All, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of of Molecule Containing Position Listing This Subsequence) 8 462 GLSPLMMAAK 1.200 9 529 LVYNSKIENR 0. 800 759. 10 543 AVEPINELLR 0. 800 760. 11 726 SLLNPCANMK 0.600 761. 12 862 GTPDRRWCFR 0.600 13 394 NYLTENPHKK 0. 600 763. 14 474 KIGIFQHIIR 0.480 764. 15 331 QTALHIAIER 0. 400 765. 16 204 TYRHHSSDNK 0.400 766. 17 525 VLEILVYNSK 0.400 767. 18 219 IIEKQPQSPK 0.400 768. 19 261 LLPFLLTHKK 0. 400 769. 20 40 APVITTVALK 0. 300 770. 21 744 CTVPTYPSCR 0.300 22 680 NALYFTRGLK 0.300 23 213 KRWRKKIIEK 0. 240 773. 24 92 GMADSSEGPR 0.240 25 359 QARGRFFQPK 0. 200 775. 26 423 IADNTRENTK 0.200 27 289 KALLNLSNGR 0.180 28 243 RPILFDIVSR 0. 180 778. 29 548 NELLRDKWRK 0.180 30 490 DTRHLSRKSK 0.150 31 619 TNIKDLFMKK 0.120 32 403 KADMRRQDSR 0.120 33 419 ALVAIADNTR 0.120 34 151 GPGDGRPNLR 0.120 35 773 GDLEMLSSTK 0.090 36 116 GTPGGEAFPL 0.090 37 471 KTGKIGIFQH 0.090 38 899 YYGFSHTVGR 0.080 TABLE XII (B) HLA Peptide Scoring Results - CaTrF2E11 - A11, 10-mers Score (Estimate of Half Time of SEQ @Q ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Containing Position Listing This Subsequence) 39 399 NPHKKADMRR 0.080 789 40 465 PLMMAAKTGK 0.080 790. 41 829 WATTILDIER 0.080 791. 42 435 TKMYDLLLLK 0.080 792. 43 393 VNYLTENPHK 0. 080 793. 44 73 CLTPLSFPCR 0.080 794 45 711 LVYLLFMIGY 0.080 795 46 677 GWMNALYFTR 0.072 796. 47 849 RSGEMVTVGK 797. 48 759 STFLLDLFKL 798. 49 840 FPVFLRKAFR 799. 50 692 GTYSIMIQKI 0. 060 800.

TABLE III (A) HLA Peptide Scoring Results-83P2H3-A24, 9-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 1 269 TYGPLTSTL 288.000 801. 2 221 SYDRHGDHL 200.000 802. 3 335 IYLLYIICF 15.000 803. 4 554 MYSITYAAF 100.000 804. 5 622 GYGLGDRWF 100.000 805. 6 523 FYDYPMALF 100.000 806. 7 376 AYMTPKDDI 807. 8 323 RYGRPYFCM 50.000 808. 9 106 VFEPMTSEL 39.600 809. 10 546 NYNVDLPFM 37.500 810. 11 88 LYDNLEAAM 30. 000 811. 12 467 YFARGFQML 28. 800 812. 13 561 AFAIIATLL 28.000 813.

TABLE XIII (A) HLA Peptide Scoring Results-83P2H3-A24,9-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of Molecule Position Listing Containing This Subsequence) 14 466 MYFARGFQM 25.000 814. 15 522 HFYDYPMAL 24.000 815. 16 530 LFSTFELFL 20.000 816. 17 151 AFRRSPCNL 20.000 817. 18 210 TFACQMYNL 20.000 19 161 YFGEHPLSF 12. 000 819. 20 359 RTSPRDNTL 11.520 820. 21 174 NSEEIVRLL 10.080 821. 22 407 IFRMGVTRF 10.000 822. 23 699 RQGTLRRDL 9. 600 823. 24 43 RIWESPLLL 9. 600 824. 25 338 LYIICFTMC 9.000 825. 26 584 RVAHERDEL 8.800 826. 27 233 DLVPNHQGL 8.640 827. 28 195 LGNTVLHIL 8.400 828. 29 129 NMNLVRALL 8.400 829. 30 75 RGAMGETAL 8.000 830. 31 690 RSSANWERL 8.000 831. 32 97 VLMEAAPEL 7.920 832. 33 600 TTVMLERKL 7. 920 833. 34 525 DYPMALFST 7. 500 834. 35 533 TFELFLTII 7. 500 835. 36 128 QNMNLVRAL 7.200 836. 37 3 LSLPKEKGL 7.200 38 450 EVVPMSFAL 7.200 39 90 DNLEAAMVL 7.200 40 643 RIQRYAQAF 7. 200 840. 41 566 ATLLMLNLL 7.200 841. 42 348 IYRPLKPRT 7. 200 842. 43 57 DVQALNKLL 7.200 843. 44 482 IQKMIFGDL 6.720 844.

TABLEXIII (A) HLA Peptide Scoring Results-83P2H3-A24,9-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 45 528 MALFSTFEL @ 6.600 845. 46 364 DNTLLQQKL 6.336 846. 47 563 AIIATLLML 847. 48 197 NTVLHILIL 848. 49 329 FCMLGAIYL 6.000 849. 50 596 QIVATTVML 6. 000 850.

TABLE XIII (B) HLA Peptide Scoring Results-CaTrF2Ell-A24, 9-mers Score (Estimate of Half Time of SEQ ID NO : Start Subsequence Residue Rank Disassociation of Molecule Containing Position Listing This Subsequence) 1 782 KYPVVFIIL 1008.000 851. 2 563 FYINVVSKYL 420.000 852. 3 793 TYIILTSVL 360.000 853. 4 502 AYGPVYSSL 336.000 854. 4 682 LYFTRGLKL 220.000 855. 6 719 GYASALVSL 200.000 856. 7 326 IYYRGQTAL 200.000 857. 8 569 SYLCAMVIF 150.000 858. 9 693 TYSIMIQKI 66.000 859. 10 432 KFVTKMYDL 60.000 860. 11 708 RELLVYLIF 42.000 861. 12 635 LFIDGSFQL 36.000 862. 13 702 LFKDLFREL 34. 560 863. 14 760 TFLLDLFKL 33.000 864. 15 131 LFEGEDGSL 30.000 865. 16 375 YFGELPLSL 28.800 866. 17 706 LFRFLLVYL 867. 18 757 TESTFLLDL 20.000 868. 19 373 YFYFGELPL 20.000 869. 20 593 PYRTTVDYL 870.

TABLE XIII (B) HLA Peptide Scoring Results - CaTrF2E11 - A24, 9-mers Score (Estimate of Half Time of SEQ ID NO : Start Subsequence Residue Rank Disassociation of a Molecule Containing Position Listing This Subsequence) 21 901 GFSHTVGRL 20.000 22 616 FFFTNIKDL 20.000 23 412 RGNTVLHAL 16.800 24 169 KGVPNPIDL 874. 25 617 FFTNIKDLF 14.000 875. 26 610 LFTGVLFFF 14.000 876. 27 557 KFGAVSFYI 14.000 877. 28826KLQWATTIL12. 000878. 29 3 RVVGPGANL 12.000 30 534 KIENRHEML 12. 000 31 298 RNDTIPVLL 11.200 881. 32 543 AVEPINELL 10.080 882. 33 388 NQPHIVNYL 10.080 883. 34 71 GFCLTPLSF 10.000 884. 35 599 DYLRLAGEV 9.900 885. 36 542 LAVEPINEL 9.504 886. 37 798 TSVILLNML 8.640 887. 38 694 YSIMIQKIL 8. 400 888. 39 650 VLVIVSAAL 8.400 40 628 KCPGVNSLF 8. 400 890. 41 284 KTCLPKALL 8.000 891. 42 767 KLTIGMGDL 8.000 892. 43 341 RCKHYVELL 8.000 893. 44 955 KWRTDDAPL 8.000 894. 45 602 RLAGEVITL 8.000 895. 46 18 RGSCCSSRL 8.000 896. 47 595 RTTVDYLRL 8.000 897. 48 916 SVVPRVVEL 7.920 898. 49 645 YFIYSVLVI 7.500 899. 50 665 AYLAMMVFA 7.500 900.

TABLE XIV (A) HLA Peptide Scoring Results-83P2H3-A24,10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 1 323 RYGRPYFCML 480.000 901. 2 466 MYFARGFQML 288.000 902. 3 525 DYPMALFSTF 216.000 903. 4 622 EYGLGDRWFL 200. 000 904. 5 160 IYFGEHPLSF 100. 000 905. 6 114 LYEGQTALHI 75. 000 906. 7 431 TYAFMVLVTM 35. 000 907. 8 511 IFQTEDPEEL 33. 000 908. 9 210 TFACQMYNLL 24.000 909. 10 650 AFHTRGSEDL 20.000 910. 11 328 YFCMLGAIYL 20.000 911. 12 407 IFRMGVTRFF 14.000 912. 13 522 HFYDYPMALF 12.000 913. 14 335 IYLLYIICFT 10. 500 914. 15 481MIQKMIFGDL 10.080 915. 16 492 RFCWLMAVVI 10.000 916. 17 503 GFASAFYIIF 10. 000 917. 18 359 RTSPRDNTLL 9.600 918. 19 546 NYNVDLPFMY 9.000 919. 20 250 EGNTVMFQHL 8. 640 920. 21 392 TVIGAIIILL 8. 400 921. 22 343 FTMCCIYRPL 8. 400 922. 23 128 QNMNLVRALL 8.400 923. 24 209 KTFACQMYNL 8.000 924. 25 338 LYIICFTMCC 7. 500 925. 26 471 GFQMLGPFTI 7. 500 926. 27 603 MLERKLPRCL 7.200 927. 28 419 TILGGPFHVL 7.200 928. 29 29 WAQSRDEQNL 7.200 929. 30 428 LIITYAFMVL 7. 200 930.

TABLE XIV (A) HLA Peptide Scoring Results-83P2H3-A24,10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 31 127 NQNMNLVRAL 7.200 931. 32 173 VNSEEIVRLL 6.720 932. 33 96 MVLMEAAPEL 6. 600 933. 34 348 IYRPLKPRTN 6.000 934. 35 391 VTVIGAIIIL 6.000 935. 36 4 SLPKEKGLIL 6.000 936. 37 542 DGPANYNVDL 6.000 937. 38 329 FCMLGAIYLL 6. 000 938. 39 670 GCPFSPHLSL 6.000 939. 40 562 FAIIATLLML 6.000 940. 41 271 GPLTSTLYDL 6.000 941. 42 694 NWERLRQGTL 6.000 942. 43 616 SGICGREYGL 6.000 943. 44 484 KMIFGDLMRF 6.000 944. 45 435 MVLVTMVMRL 6.000 945. 46 151 AFRRSPCNLI 6.000 946. 47 310 QTPVKELVSL 6.000 947. 48 158 NLIYFGEHPL 6. 000 948. 49 595 AQIVATTVML 6.000 949. 50 123 IAVVNQNMNL 6.000 950.

TABLE XIV (B) HLA Peptide Scoring Results-CaTrF2Ell-A24, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 1 782 KYPWFIILL 600. 000 1 2 658 LYLAGIEAYL 420. 000 2 3 665 AYLAMMVFAL 300. 000 3 4 793 TYIILTSVLL 300.000 4 5 693 TYSIMIQKIL 280. 000 5 6 719 GYASALVSLL 240.000 TABLE XIV (B) HLA Peptide Scoring Results - CaTrF2E11 - A24, 10-mers Score (Estimate of Half Time of SEQ ID NO : Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 7 374 FYFGELPLSL 240. 000 7 372 GYFYFGELPL 200. 000 8 9 599 DYLRLAGEVI 75. 000 9 10 432 KFVTKMYDLL 60. 000 10 11 635 LFIDGSFQLL 51. 840 11 12 644 LYFIYSVLVI 50. 000 12 13 327 YYRGQTALHI 50. 000 13 14 122 AFPLSSLANL 30. 000 14 15 715 LFMIGYASAL 30. 000 15 16 562 SFYINVVSYL 28. 000 16 17 702 LFKDLFRFLL 24. 000 17 18 706 LFRFLLVYLL 24. 000 18 19 615 LFFFTNIKDL 20. 000 19 20 839 SFPVFLRKAF 18. 000 20 21 169 KGVPNPIDLL 14. 400 21 22 616 FFFTNIKDLF 14. 000 22 23 387 TNQPHIVNYL 12. 096 23 24 757 TFSTFLLDLF 12. 000 24 25 101 RAGPGEVAEL 10.560 25 26 240 VFNRPILFDI 10. 500 26 25 563 FYINVVSYLC 10.50 27 28 542 LAVEPINELL 10'. 080 28 29 928 SNPDEWVPL 10. 080 29 30 786 VFIILLVTYI 9. 000 30 31 896 TYQYYGFSHT 9. 000 31 32 317 EFINSPFRDI 9. 000 32 33 173 NPIDLLESTL 8. 640 33 34 697 MIQKILFKDL 8. 640 34 35 166 AFRKGVPNPI 8. 400 35 36 649 SVLVIVSAAL 8. 400 36 37 642 QLLYFIYSVL 8. 400 37 TABLE XIV (B) HLA Peptide Scoring Results-CaTrF2Ell-A24, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 38 748 TYPSCRDSET 8. 250 38 39 408 RQDSRGNTVL 8. 000 39 40 252 RGSTADLDGL 8. 000 40 41 712 VYLLFMIGYA 7. 500 41 42 569 SYLCAMVIFT 7. 500 42 43 708 RFLLVYLLFM 7. 500 43 44 285 TCLPKALLNL 7. 200 44 45 673 ALVLGWMNAL 7. 200 45 46 577 FTLTAYYQPL 7. 200 46 47 46 VALKQLAALL 7. 200 47 48 65 EPPPLAGFCL 7. 200 48 49 647 IYSVLVIVSA 7. 000 49 50 915 SSVVPRVVEL 6. 600 50 TABLE XV (A) HLA Peptide Scoring Results-83P2H3-B7,9-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 1 452 VPMSFALVL 240.000 951. 2 671 CPFSPHLSL 120.000 952. 3 5 LPKEKGLIL 80.000 953. 4 311 TPVKELVSL 80.000 954. 5 543 GPANYNVDL 80.000 955. 124 AVVNQNMNL 60. 000 956. 7 324 YGRPYFCML 40.000 957. 8 31 QSRDEQNLL 40.000 958. 9 560 AAFAIIATL 36.000 959. 10 584 RVAHERDEL 30.000 960. 11 450 EWPMSFAL 20. 000 961. 12 57 DVQALNKLL 20.000 962.

TABLE XV (A) HLA Peptide Scoring Results-83P2H3-B7,9-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence 13 392 TVIGAIIIL 20.000 963. 14 102 APELVFEPM 18.000 964. 15 151 AFRRSPCNL 12.000 965. 16 528 MALFSTFEL 12.000 966. 17 128 QNMNLVRAL 12.000 967. 18 566 ATLLMINLL 12.000 968. 19 211 FACQMYNLL 12.000 969. 20 565 IATLLMLNL 12.000 970. 21 212 ACQMYNLLL 12.000 971. 22 329 FCMLGAIYL 12.000 972. 23 97 VLMEAAPEL 12.000 973. 24 30 AQSRDEQNL 12.000 974. 25 563 AIIATLLML 12.000 975. 26 699 RQGTLRRDL 6.000 976. 27 623 YGLGDRWFL 6.000 977. 28 420 ILGGPFHVL 6.000 978. 29 300 KKREARQIL 6.000 979. 30 129 NMNLVRALL 6.000 980. 31 458 LVLGWCNVM 5.000 981. 32 690 RSSANWERL 4.000 98.2 33 702 TLRRDLRGI 4.000 983. 34 364 DNTLLQQKL 4.000 984. 35 284 DSSGDEQSL 4.000 985. 36 165 HPLSFAACV 4.000 986. 37 353 KPRTNNRTS 4.000 987. 38 3 LSLPKEKGL 4.000 988. 39 379 TPKDDIRLV 4.000 989. 40 330 CMLGAIYLL 4.000 990. 41 197 NTVLHILIL 4.000 991. 42 436 VLVTMVMRL 4.000 992. 43 378 MTPKDDIRL 4.000 993.

TABLE XV (A) HLA Peptide Scoring Results-83P2H3-B7, 9-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 44 285 SSGDEQSLL 4. 000 994. 45 75 RGAMGETAL 4.000 995. 46 195 LGNTVLHIL 4.000 996. 47 80 ETALHIAAL 997. 48 617 GICGREYGL 998. 49 113 ELYEGQTAL 4.000 999. 50 596 QIVATTVML 4.000 1000.

TABLE XV (B) HLA Peptide Scoring Results - CaTrF2E11 - B7, 9-mers Score (Estimate of Half Time of SEQ ID NO : Start Subsequence Residue Rank Disassociation of of Molecule Containing Position Listing This Subsequence) 1 40 APVITTVAL 240.000 1001. 2 151 GPGDGRPNL 120.000 1002. 3 123 FPLSSLANL 80.000 1003. 4 783 YPVVFIILL 80.000 1004. 5 117 TPGGEAFPL 80.000 1005. 6 75 TPLSFPCRL 80.000 1006. 7 279 EPSTGKTCL 80. 000 1007. 8 99 GPRAGPGEV 40.000 1008. 9 250 VSRGSTADL 40.000 1009. 10 668 AMMVFALVL 36.000 1010. 11 170 GVPNPIDLL 30.000 1011. 12 3 RVVGPGANL 30.000 1012. 13 229 APAPQPPPI 24.000 1013. 14 929 NPDEVVVPL 24.000 1014. 15 674 LVLGWMNAL 20.000 1015. 16 916 SWPRWEL 20.000 1016. 17 433 FVTKMYDLL 20.000 1017. 18 188 VPGPKKAPM 20.000 1018. 19 1 MPRVVGPGA 20.000 1019.

TABLE XV (B) HLA Peptide Scoring Results - CaTrF2E11 - B7, 9-mers Score (Estimate o Halft Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Containing Positon Listing This Subsequence) 20 56 LVHVGGGFL 20.000 1020. 21 542 LAVEPINEL 18.000 1021. 22 543 AVEPINELL 18.000 1022. 23 455 EAVLNNDGL 12.000 1023. 24 46 VALKQLAAL 12.000 1024. 25 770 IGMGDLEML 12.000 1025. 26 680 NALYFTRGL 12.000 1026. 27 69 LAGFCLTPL 12.000 1027. 28 47 ALKQLAALL 12.000 1028. 29 102 AGPGEVAEL 12.000 1029. 30 720 YASALVSLL 12.000 1030. 31 629 CPGVNSLFI 8.000 1031. 32 66 PPPLAGFCL 8.000 1032. 33 932 EVVVPLDSM 7.500 1033. 34 284 KTCLPKALL 6.000 1034. 35 20 SCCSSRLRL 6.000 1035. 36 14 QVRERGSCC 5.000 1036. 37 566 NVVSYLCAM 5.000 1037. 38 706 LFRFLLVYL 4.000 1038. 39 694 YSIMIQKIL 4.000 1039. 40 600 YLRLAGEVI 4.000 1040. 41 371 GGYFYFGEL 4.000 1041. 42 440 LLLLKCARL 4.000 1042. 43 795 IILTSVLLL 4.000 1043. 44 716 FMIGYASAL 4.000 1044. 45 650 VLVIVSAAL 4.000 1045. 46 607 VITLFTGVL 4.000 1046. 47 43 ITTVALKQL 4.000 1047. 48 434 VTKMYDLLL 4.000 1048. 49 578 TLTAYYQPL 4.000 1049. 50 61 GGFLEPPPL 4.000 1050.

TABLE XVI (A) HLA Peptide Scoring Results-83P2H3-B7,10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 271 GPLTSTLYDL 80. 000 1051. 2 630 FLRVEDRQDL 60. 000 1052. 3 706 DLRGIINRGL 40.000 1053. 4 412 VTRFFGQTIL 40.000 1054. 5 52 AAKDNDVQAL 36.000 1055. 6 560 AAFAIIATLL 36. 000 1056. 7 451 WPMSFALVL 20. 000 1057. 476 GPFTIMIQKM 20.000 1058. 9 96 MVLMEAAPEL 20. 000 1059. 10 172 CVNSEEIVRL 20. 000 1060. 11 392 TVIGAIIILL 20. 000 1061. 12 105 LVFEPMTSEL 20. 000 1062. 13 206 QPNKTFACQM 20. 000 1063. 14 435 MVLVTMVMRL 20. 000 1064. 15 128 QNMNLVRALL 18. 000 1065. 16 559 YAAFAIIATL 12. 000 1066. 17 329 FCMLGAIYLL 12. 000 1067. 18 29 WAQSRDEQNL 12. 000 1068. 19 562 FAIIATLLML 12. 000 1069. 20 150 TAFRRSPCNL 12. 000 1070. 21 599 ATTVMLERKL 12. 000 1071. 22 30 AQSRDEQNLL 12.000 1072. 23 123 IAVVNQNMNL 12. 000 1073. 24 595 AQIVATTVML 12. 000 1074. 25 343 FTMCCIYRPL 12. 000 1075. 26 565 IATLLMLNLL 12.000 1076. 27 211 FACQMYNLLL 12. 000 1077. 28 529 ALFSTFELFL 12. 000 1078. 29 101 AAPELVFEPM 9.000 1079.

TABLE XVI (A) HLA Peptide Scoring Results-83P2H3-B7,10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of Molecule Position Listing Containing This Subsequence) 30 326 RPYFCMLGAI 8.000 1080. 31 670 GCPFSPHLSL 6. 000 1081. 32 702 TLRRDLRGII 6.000 1082. 33 679 LPMPSVSRST 6.000 1083. 34 419 TILGGPFHVL 6. 000 1084. 35 132 LVRALLARRA 5.000 1085. 36 426 HVLIITYAFM 5.000 1086. 37 178 IVRLLIEHGA 5. 000 1087. 38 472 FQMLGPFTIM 4. 500 1088. 39 564 IIATLLMLNL 4. 000 1089. 40 493 FCWLMAWIL 4. 000 1090. 41 4 SLPKEKGLIL 4. 000 1091. 42 127 NQNMNLVRAL 4. 000 1092. 43 616 SGICGREYGL 4. 000 1093. 44 220 LSYDRHGDHL 4. 000 1094. 45 310 QTPVKELVSL 4. 000 1095. 46 359 RTSPRDNTLL 4. 000 1096. 47 2 GLSLPKEKGL 4. 000 1097. 48 364 DNTLLQQKLL 4. 000 1098. 49 542 DGPANYNVDL 4. 000 1099. 50 40 QQKRIWESPL 4. 000 1100.

TABLE XVI (B) HLA Peptide Scoring Results-CaTrF2Ell-B7, 10-mers Score (estimate ot llalt'l'ime of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Containing Position Listing This Subsequence) 1 229 APAPQPPPIL 360. 000 1101. 2 445 CARLFPDSNL 180.000 1102. 3 190 GPKKAPMDSL 120.000 1103. 4 504 GPVYSSLYDL 80.000 1104. 5 592 YPYRTTVDYL 80.000 1105.

TABLE XVI (B) HLA Peptide Scoring Results - CaTrF2E11 - B7, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of Molecule Containing Position Listing This Subsequence) 6 173 NPIDLLESTL 80. 000 1106. 7 65 EPPPLAGFCL 80. 000 1107. 8 296 NGRNDTIPVL 40. 000 1108. 9 667 LAMMVFALVL 36. 000 1109. 10 99 GPRAGPGEVA 30. 000 1110. 11 449 FPDSNLEAVL 24.000 1111. 12 485 EVTDEDTRHL 20.000 1112. 13 433 FVTKMYDLLL 20. 000 1113. 14 249 IVSRGSTADL 20.000 1114. 15 651 LVIVSAALYL 20.000 1115. 16 649 SVLVIVSAAL 20. 000 1116. 17 45 TVALKQLAAL 20.000 1117. 18 952 YPRKWRTDDA 20. 000 1118. 19 606 EVITLFTGVL 20. 000 1119. 20 150 AGPGDGRPNL 18. 000 1120. 21 231 APQPPPILKV 18. 000 1121. 22 542 LAVEPINELL 12. 000 1122. 23 39 CAPVITTVAL 12. 000 1123. 24 46 VALKQLAALL 12. 000 1124. 25 47 ALKQLAALLL 12. 000 1125. 26 673 ALVLGWMNAL 12. 000 1126. 27 101 RAGPGEVAEL 12. 000 1127. 28 501 WAYGPVYSSL 12. 000 1128. 29 681 ALYFTRGLKL 12. 000 1129. 30 589 TPPYPYRTTV 6. 000 1130. 31 541 MLAVEPINEL 6. 000 1131. 32 19 GSCCSSRLRL 6. 000 1132. 33 169 KGVPNPIDLL 6.000 1133. 34 237 ILKVFNRPIL 6.000 1134. 35 670 MVFALVLGWM 5. 000 1135. 36 187 VVPGPKKAPM 5.000 1136.

TABLE XVI (B) HLA Peptide Scoring Results - CaTrF2E11 - B7, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of Molecule Containing Position Listing This Subsequence) 37 718 IGYASALVSL 4.000 1137. 38 434 VTKMYDLLLL 4.000 1138. 39 1 MPRVVGPGAN 4.000 1139. 40 577 FTLTAYYQPL 4.000 1140. 41 252 RGSTADLDGL 4. 000 1141. 42 900 YGFSHTVGRL 4.000 1142. 43 794 YIILTSVLLL 4.000 1143. 44 325 DIYYRGQTAL 4.000 1144. 45 759 STFLLDLFKL 4.000 1145. 46 570 YLCAMVIFTL 4.000 1146. 47 339 ERRCKHYVEL 4.000 1147. 48 55 LLVHVGGGFL 4.000 1148. 49 42 VITTVALKQL 4.000 1149. 50 253 GSTADLDGLL 4.000 1150.

TABLE XVII (A) HLA Peptide Scoring Results-83P2H3-B35,9-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 1 5 LPKEKGLIL 120. 000 1151. 2 31 QSRDEQNLL 45. 000 1152. 3 551 LPFMYSITY 40.000 1153. 4 379 TPKDDIRLV 36.000 1154. 5 311 TPVKELVSL 30.000 1155. 6 516 DPEELGHFY 24.000 1156. 7 452 VPMSFALVL 20.000 1157. 8 526 YPMALFSTF 20.000 1158. 9 615 RSGICGREY 20.000 1159. 10 671 CPFSPHLSL 20.000 1160. 11 543 GPANYNVDL 20. 000 1161.

TABLE XVII (A) HLA Peptide Scoring Results-83P2H3-B35,9-mers Score (Estimate of Half Time of SEQ ID NO : Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 12 285 SSGDEQSLL 15.000 1162. 13 353 KPRTNNRTS 12. 000 1163. 14 102 APELVFEPM 12. 000 1164. 15 154 RSPCNLIYF 10. 000 1165. 16 690 RSSANWERL 10. 000 1166. 17 673 FSPHLSLPM 10. 000 1167. 18 446 SASGEWPM 9. 000 1168. 19 144 SARATGTAF 9. 000 1169. 20 284 DSSGDEQSL 7. 500 1170. 21 360 TSPRDNTLL 7. 500 1171. 22 608 LPRCLWPRS 6. 000 1172. 23 369 QQKLLQEAY 6. 000 1173. 24 81 TALHIAALY 6.000 1174. 25 639 LNRQRIQRY 6. 000 1175. 26 59 QALNKLLKY 6. 000 1176. 27 432 YAFMVLVTM 6.000 1177. 28562FAIIATLLM6. 000"7S. 29 3 LSLPKEKGL 5. 000 1179. 30 547 YNVDLPFMY 4. 000 1180. 31 326 RPYFCMLGA 4. 000 1181. 32 165 HPLSFAACV 4. 000 1182. 33 247 AGVEGNTVM 4. 000 1183. 34 539 TIIDGPANY 4. 000 1184. 35 43 RIWESPLLL 4.000 1185. 36 350 RPLKPRTNN 4. 000 1186. 37 324 YGRPYFCML 3. 000 1187. 38 173 VNSEEIVRL 3. 000 1188. 39 713 RGLEDGESW 3. 000 1189. 40 565 IATLLMLNL 3.000 1190. 41 585 VAHERDELW 3. 000 1191. 42 174 NSEEIVRLL 3. 000 1192.

TABLE XVII (A) HLA Peptide Scoring Results-83P2H3-B35, 9-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 43 528 MALFSTFEL 3.000 1193. 44 211 FACQMYNLL 3.000 1194. 45 482 IQKMIFGDL 3.000 1195. 46 512 FQTEDPEEL 3. 000 1196. 47 560 AAFAIIATL 3.000 1197. 48 504 FASAFYIIF 3.000 1198. 49 584 RVAHERDEL 3.000 1199. 50 454 MSFALVLGW 2.500 1200.

TABLE XVII (B) HLA Peptide Scoring Results - CaTrF2E11 - B35, 9-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Containing Position Listing This Subsequence) 1 366 QPKDEGGYF 180.000 1201. 2 194 APMDSLFDY 80.000 1202. 3 584 QPLEGTPPY 80.000 1203. 4 188 VPGPKKAPM 40.000 1204. 5 151 GPGDGRPNL 40.000 1205. 6 592 YPYRTTVDY 40.000 1206. 7 117 TPGGEAFPL 30.000 1207. 8 783 YPVVFIILL 20.000 1208. 9 40 APVITTVAL 20.000 1209. 10 840 FPVFLRKAF 20.000 1210. 11 279 EPSTGKTCL 20.000 1211. 12 233 QPPPILKVF 20.000 1212. 13 123 FPLSSLANL 20.000 1213. 14 75 TPLSFPCRL 20.000 1214. 15 523 ASVLEILVY 15.000 1215. 16 197 SLFDYGTY 15.000 1216. 17 817 VSKESKHIW 15.000 1217. 18 250 VSRGSTADL 15.000 1218.

TABLE XVII (B) HLA Peptide Scoring Results - CaTrF2E11 - B35, 9-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of of Molecule Containing Position Listing This Subsequence) 19 929 NPDEVVVPL 12.000 1219. 20 99 GPRAGPGEV 12. 000 1220. 21 320 NSPFRDIYY 10. 000 1221. 22 229 APAPQPPPI 8.000 1222. 23 629 CPGVNSLFI 8.000 1223. 24 508 SSLYDLSSL 7. 500 1224. 25 253 GSTADLDGL 7. 500 1225. 26 341 RCKHYVELL 6. 000 1226. 27 430 NTKFVTKMY 6. 000 1227. 28 287 LPKALLNLS 6. 000 1228. 29 1 MPRVVGPGA 6.000 1229. 30 542 LAVEPINEL 6.000 1230. 31 550 LLRDKWRKF 6. 000 1231. 32 603 LAGEVITLF 6.000 1232. 33 190 GPKKAPMDS 6.000 1233. 34 694 YSIMIQKIL 5. 000 1234. 35 633 NSLFIDGSF 5. 000 1235. 36 758 FSTFLLDLF 5. 000 1236. 37 798 TSVLLLNML 5. 000 1237. 38 779 SSTKYPVVF 5.000 1238. 39 173 NPIDLLEST 4.000 1239. 40 307 DIAERTGNM 4.000 1240. 41 689 KLTGTYSIM 4. 000 1241. 42 142 SPADASRPA 4.000 1242. 43 661 AGIEAYLAM 4. 000 1243. 44 686 RGLKLTGTY 4.000 1244. 45 243 RPILFDIVS 4. 000 1245. 46 469 AAKTGKIGI 3. 600 1246. 47 595 RTTVDYLRL 3. 000 1247. 48 602 RLAGEVITL 3.000 1248. 49 69 LAGFCLTPL 3.000 1249.

FABLE XVII (B) HLA Peptide Scoring Results - CaTrF2E11 - B35, 9-mers Score Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Containing Position Listing This Subsequence) 50 664 EAYLAMMVF 3.000 1250.

ITABLE XVIII (A) HLA Peptide Scoring Results-83P2H3-B35, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Itank Disassociation of u Molecule Position Listing Containing This Subsequence) 1 423 GPFHVLIITY 40. 000 1251. 2 206 QPNKTFACQM 40. 000 1252. 3 476 GPFTIMIQKM 40. 000 1253. 4 52 AAKDNDVQAL 27. 000 1254. 5 271 GPLTSTLYDL 20. 000 1255. 6 235 VPNHQGLTPF 20.000 1256. 7 326 RPYFCMLGAI 16. 000 1257. 8 445 ISASGEVVPM 15.000 1258. 9 5 LPKEKGLILC 12.000 1259. 10 101 AAPELVFEPM 12.000 1260. 11 594 RAQIVATTVM 12. 000 1261. 12 220 LSYDRHGDHL 10. 000 1262. 13 447 ASGEWPMSF 10. 000 1263. 14 284 DSSGDEQSLL 7. 500 1264. 15 482 IQKMIFGDLM 6. 000 1265. 16 369 QQKLLQEAYM 6.000 1266. 17 246 LAGVEGNTVM 6. 000 1267. 18 69 DCKVHQRGAM 6. 000 1268. 19 686 RSTSRSSANW 5. 000 1269. 20 143 VSARATGTAF 5. 000 1270. 21 29 WAQSRDEQNL 4.500 1271. 22 630 FLRVEDRQDL 4. 500 1272. 23 87 ALYDNLEAAM 4.000 1273. 24. 90 DNLEAAMVLM 4. 000 1274.

TABLE XVIII (A) HLA Peptide Scoring Results-83P2H3-B35,10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Position Listing Containing This Subsequence) 25 123 IAVVNQNMNL 3.000 1275. 26 562 FAIIATLLML 3.000 1276. 27 150 TAFRRSPCNL 3.000 1277. 28 560 AAFAIIATLL 3.000 1278. 29 528 MALFSTFELF 3.000 1279. 30 3 LSLPKEKGLI 3.000 1280. 31 359 RTSPRDNTLL 3.000 1281. 32 211 FACQMYNLLL 3.000 1282. 33 412 VTRFFGQTIL 3.000 1283. 34 545 ANYNVDLPFM 3.000 1284. 35 40 QQKRIWESPL 3.000 1285. 36 565 IATLLMLNLL 3.000 1286. 37 559 YAAFAIIATL 3.000 1287. 38 706 DLRGIINRGL 3.000 1288. 39 274 TSTLYDLTEI 3.000 1289. 40 484 KMIFGDLMRF 3.000 1290. 41 190 RAQDSLGNTV 2.400 1291. 42 340 IICFTMCCIY 2.000 1292. 43 330 CMLGAIYLLY 2.000 1293. 44 472 FQMLGPFTIM 2.000 1294. 45 679 LPMPSVSRST 2.000 1295. 46 519 ELGHFYDYPM 2.000 1296. 47 251 GNTVMFQHLM 2.000 1297. 48 213 CQMYNLLLSY 2.000 1298. 49 58 VQALNKLLKY 2.000 1299. 50 568 LLMLNLLIAM 2.000 1300.

TABLE XVIII (B) HLA Peptide Scoring Results-CaTrF2Ell-B35, 10-mers Start Score (Estimate of Half Time of SEQ ID NO : Start Subsequence Residue Rank Disassociation of a Molecule Containing Position Listing This Subsequence) TABLE XVIII (B) HLA Peptide Scoring Results - CaTrF2E11 - B35, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Containing Position Listing This Subsequence) 1 366 QPKDEGGYFY 240.000 1301. 2 190 GPKKAPMDSL 60.000 1302. 3 890 DPGKNETYQY 60.000 1303. 4 173 NPIDLLESTL 40.000 1304. 5 494 LSRKSKDWAY 30.000 1305. 6 532 NSKIENRHEM 30.000 1306. 7 749 YPSCRDSETF 30.000 1307. 8 592 YPYRTTVDYL 20.000 1308. 9 65 EPPPLAGFCL 20.000 1309. 10 84 SSADGPGAGM 20.000 1310. 11 504 GPVYSSLYDL 20.000 1311. 12 123 FPLSSLANLF 20.000 1312. 13 229 APAPQPPPIL 20.000 1313. 14 193 KAPMDSLFDY 12.000 1314. 15 336 IAIERRCKHY 12.000 1315. 16 497 KSKDWAYGPV 12.000 1316. 17 561 VSFYINVVSY 10.000 1317. 18 639 GSFQLLYFIY 10.000 1318. 19 725 VSLLNPCANM 10.000 1319. 20 522 EASVLEILVY 9.000 1320. 21 101 RAGPGEVAEL 9.000 1321. 22 445 CARLFPDSNL 9.000 1322. 23 469 AAKTGKIGIF 9.000 1323. 24 918 VPRVVELNKN 9.000 1324. 25 507 YSSLYDLSSL 7.500 1325. 26 820 ESKHIWKLQW 7.500 1326. 27 449 FPDSNLEAVL 6.000 1327. 28 542 LAVEPINELL 6.000 1328. 29 952 YPRKWRTDDA 6.000 1329. 30 314 NMREFINSPF 6.000 1330. 31 1 MPRVVGPGAN 6.000 1331.

TABLE XVIII (B) HLA Peptide Scoring Results-CaTrF2EIl-B35, 10-mers Score (Estimate of Half Time of SEQ ID NO: Start Subsequence Residue Rank Disassociation of a Molecule Containing Position Listing This Subsequence) 32 287 LPKALLNLSN 6.000 1332. 33 99 GPRAGPGEVA 6.000 1333. 34 660 LAGIEAYLAM 6.000 1334. 35 915 SSWPRWEL 5.000 1335. 36 114 ESGTPGGEAF 5.000 1336. 37 694 YSIMIQKILF 5.000 1337. 38 19 GSCCSSRLRL 5.000 1338. 39 778 LSSTKYPWF 5.000 1339. 40 253 GSTADLDGLL 5.000 1340. 41 568 VSYLCAMVIF 5.000 1341. 42 434 VTKMYDLLLL 4.500 1342. 43 231 APQPPPILKV 4.000 1343. 44 458 LNNDGLSPLM 4.000 1344. 45 661 AGIEAYLAMM 4.000 1345.. 46 589 TPPYPYRTTV 4.000 1346. 47 783 YPVVFIILLV 4.000 1347. 48 6 GPGANLCFQV 4.000 1348. 49 700 KILFKDLFRF 3.000 1349. 50 501 WAYGPVYSSL 3.000 1350.

Table XIX (A): Motif-bearing Subsequences of the 83P2H3 Protein

Post translational modifications N-glycosylation site 1 208-211 NKTF 2 358-361 NRTS cAMP-and cGMP-dependent protein kinase phosphorylation site 1 25-28 RRES 2 139-142 RRAS 3 263-266 RKHT Protein kinase C phosphorylation site

1 144-146 SAR 2 298-300 TTK 3 299-301 TKK 4 318-320 SLK 5 361-363 SPR 6 379-381 TPK 7 688-690 TSR 8 702-704 TLR Casein kinase II phosphorylation site 1 32-35 SRDE 2 276-279 TLYD 3 281-284 TEID 4 285-288 SSGD 5 286-289 SGDE 6 291-294 SLLE 7 361-364 SPRD 8 379-382 TPKD 9 532-535 STFE 10 539-542 TIID N-myristoylation site 1 10-15 GLILCL 2 248-253 GVEGNT 3 714-719 GLEDGE Motifs and Domains: Ank repeat aa 44 76 aa 78.. 108 aa 116.. 148 aa 162.. 194 Ion transport aa 409.. 578 Table XIX (B): Motif-bearing Subsequences of the CaTrF2Ell Protein Post translational modifications N-glycosylation site Number of matches: 5 1 233-236 NLSN 2 239-242 NDTI 3 683-686 NCTV 4 816-819 NWSH 5 834-837 NETY cAMP-and cGMP-dependent protein kinase phosphorylation site 210-213 KRLT Protein kinase C phosphorylation site Number of matches: 8 1 144-146 TYR 2 166-168 SPK 3 207-209 THK 4 222-224 TGK

5 412-414 TGK 6 222-224 TGK 7 412-414 TGK 8 435-437 SRK Casein kinase II phosphorylation site Number of matches : 17 1 24-27 SSAD 2 82-85 SPAD 3 121-124 TLYE 4 138-141 SLFD 5 194-197 STAD 6 213-216 TDEE 7 392-395 SNLE 8 427-430 TDED 9 449-452 SLYD 10 454-457 SSLD 11 458-461 TCGE 12 464-467 SVLE 13 473-476 SKIE 14 536-539 TTVD 15 691-694 SCRD 16 772-775 TILD 17 868-871 SNPD Tyrosine kinase phosphorylation site 436-443 RKSKDWAY N-myristoylation site Number of matches: 5 1 30-35 GAGMAD 2 32-37 GMADSS 3 56-61 GTPGGE 4 627-632 GLKLTG 5 881-886 GNPRCD Motifs and Domains Ankyrin binding domain aa 329-361 aa 376-408 aa 461-493 Transmembrane domain aa 561-583 aa 605-622 aa 638-660 aa 672-697 aa 707-725 aa 783-811 Table XX : Frequently Occurring Motifs arg. _ Name Description Potential Function identity Nucleic acid-binding protein functions as transcription factor, nuclear location zf-C2H2 34% Zinc finger, C2H2 type probable Cytochrome b (N- membrane bound oxidase, generate c ochrome b N 68% terminal)/b6/petB superoxide domains are one hundred amino acids long and include a conserved intradomain g 19% Immunoglobulin domain disulfide bond. tandem repeats of about 40 residues, each containing a Trp-Asp motif. Function in WD40 18% WD domain, G-beta repeat signal transduction and protein interaction may function in targeting signaling PDZ 23% DZ domain molecules to sub-membranous sites short sequence motifs involved in protein- LRR 28% Leucine Rich Repeat protein interactions conserved catalytic core common to both serine/threonine and tyrosine protein kinases containing an ATP binding site and pkinase 23% Protein kinase domain a catalytic site leckstrin homologyinvolvedin intracellular signaling or as constituents of PH 16% H domain the cytoskeleton 30-40 amino-acid long found in the extracellular domain of membrane-bound EGF 34% EGF-like domain roteins or'in secreted proteins Reverse transcriptase (RNA-dependent DNA rvt 49% polymerase) Cytoplasmic protein, associates integral ank 25% Ank repeat membrane proteins to the cytoskeleton NADH- Ubiquinone/plastoquinone membrane associated. Involved in proton oxidored q 32% (complex I), various chains. translocation across the membrane calcium-binding domain, consists of al2 residue loop flanked on both sides by a 12 efhand 24% EF hand residue alpha-helical domain Aspartyl or acid proteases, centered on a rvp 79% Retroviral aspartyl protease catalytic aspartyl residue extracellular structural proteins involved in formation of connective tissue. The Collagen triple helix repeat sequence consists of the G-X-Y and the Collagen 42% (20 copies) polypeptide chains forms a triple helix. Located in the extracellular ligand-binding region of receptors and is about 200 amino acid residues long with two pairs of fn3 20% Fibronectin type III domain cysteines involved in disulfide bonds seven hydrophobic transmembrane regions, with the N-terminus located extracellularly 7 transmembrane receptor while the C-terminus is cytoplasmic. 7tm 1 19% (rhodopsin family) Signal through G proteins

Table XXIA. Nucleotide sequence of splice variant A for PCaT.

1 GGTTCTGCAA GCCACACATG GCCTCACTGC ATGTTTTTCT TCTTTTTTAA CAATCCTTTT 61 AAAAAATGTA GAAACCCTTT TCAGTTCAAA GGCCACACCA AAGCAGGTCA GGTAGATCTG 121 GTCCACAGGC CATAGATAGC CAATCCCTGT CCCAGAGGTG GAGCTGTGAG ACTTGTCGGG 181 GTGAGACCTG TTAGAGGCTG GATGGGGCAA TTGCTTGGGG AATNTGTGCA GATGTTCTCT 241 GCCTCCTGCT CCTTCTAGAT GATTTTTGGG CGACCTGATG CGATTCTGCT GGCTGATGGC 301 TGTGGTCATC CTGGGGCTTT GCTTCAGGTA ATCATCTGTC CAGGGACCAG GGGCCATGGC 361 AGGGGAAGAG ATGAGGAAGT TTAGGGGGCA CTGGCNCTGG CTAAACTTGG GGAGGAGGAG.

421 TAATGCAGAG ATNCAGAGGA GACCTAT Table XXIIA. Nucleotide sequence alignment of Variant A with PCaT.

Score = 106 bits (55), Expect = le-l9 Identities = 69/71 (97%), Gaps = 2/71 (2%) Strand = Plus/Plus PCaT : 1651 agatgatttttgg- agatgatttttgggcgacctgatgcgattctgctggctgatggctgtggtcatcctgggg 316

Table XXIIA. Longest amino acid sequence alignment of Variant A and PCaT.

Score = 42. 8 bits (87), Expect = 0. 16 Identities = 16/16 (100%) Frame = +3/+2 PCaT : 1662 GDLMRFCWLMAWILG 1709 GDLMRFCWLMAWILG Vrnt A: 269 GDLMRFCWLMAWILG 316 Table XXIVA. Peptide sequences from the translation of the nucleotide sequence of variant A. Open reading frame Amino acid sequences Frame GSASHTWPHCMFFFFFNNPFKKCRNPFQFKGHTKAGQVDLVHRP*IANPCPRGG AVRLVGVRPVRGWMGQLLGE*VQMFSASCSF*MIFGRPDAILLADGCGHPGALL QVIICPGTRGHGRGRDEEV*GALALAKLGEEE*CRD*EETY Frame2 VLQATHGLTACFSSFLTILLKNVETLFSSKATPKQVR*IWSTGHR*PIPVPEVE L*DLSG*DLLEAGWGNCLGN*CRCSLPPAPSR*FLGDLMRFCWLMAVVILGLCF R*SSVQGPGAMAGEEMRKFRGHW*WLNLGRRSNAE*QRRP Frame3 FCKPHMASLHVFLLF*QSF*KM*KPFSVQRPHQSRSGRSGPQAIDSQSLSQRWS CETCRGETC*RLDGAIAWG*CADVLCLLLLLDDFWAT*CDSAG*WLWSSWGFAS GNHLSRDQGPWQGKR*GSLGGTG*G*TWGGGVMQR*RGDL

Note: Frame 2 gives the longest subsequence that is identical with PCaT amino acid sequence. In this Table'each (*) indicates a single unknown amino acid.

Table XXIB. Nucleotide sequence of splice Variant B for PCaT.

1 ATTCTGCTGG CTGATGGCTG TGGTCATCCT GGGCTTGCTT CAGCCTTCTA TATCATCTTC 61 CAGACAGAGG ACCCCGAGAG CTAGGCCACT TCTACGACTA CCCCACGCCC CTGTCCGGCA 121 CCTTCGAGCT GTTCCTTACC ATCATCGATG GCCCAGCCAA CTACAACGTG GACCTGCCCT 181 TCGTGTACAG CATCACCTAT GCTGCCTTTG CCATCATCGC CACACTGCTC ATGCTCAACC 241 TCCTCATTGC CATGATGGGC GACACTCACT GGCGAGTGGC CCATGAGCGG GATGAGCTGT 301 GGAGGGCCCA GATTGTGGCC ACCACGGTGA TGCTGGAGCG GAAGCTGCCT CGCTGCCTGT 361 GGCCTCGCTC CGGGATCTGC GGANNCGGGA GTATGGCCTG GGAGACCGCT GGTCCCTCGG 421 CGCGCTGGAA GAACAGGCAA CGATCTCAAC CGGCAGCGGA TCCAACGCCA CCGCACAGGC 481 CTTCCACACC CGGGGCTCCT GAGGATTCGG CCCCCAGACT CAGTGCAAAC AACTAGAGCT 541 GGCGCTGTCC CTTTCAGCCC CAGCGTGTCC CCTTCCTAAT TGCGCTCAAG GTCCCGAAAG 601 TACCTTCCCG TAGACGTGCC AATGGGCGCA AGCGCTCCGG GCAAGGGGGC CCCTGCCGGA 661 GAAGACCTGC GTGGCGACCA CTCCACCAGG GGCTCCGGAC GCACCGCGAA GCTGGGATAT 721 CCAGAACCGA CGCGTGTCCC ACCTGGCCCG GACCTGGCCC CCATTACCGG GGGGCCAACG 781 ACACAAACCG AAACCCAGGA GCCATCCCGG CCAGGGGAAA CAGCGGCCCC ACGCCGAACA 841 TCCTCG

Table XXIIB. Nucleotide sequence alignment of Variant B with PCaT.

Score = 798 bits (415), Expect = 0.0 Identities = 542/573 (94%), Gaps = 15/573 (2%) Strand = Plus/Plus

Table XXIIIB. Longest amino acid sequence alignment of Variant B and PCaT.

Score = 243 bits (525), Expect (6) = Se-77 Identities = 98/104 (94%) Frame = +3/+3 PCaT : 1749 PEELGHFYDYPMALFSTFELFLTIIDGPANYNVDLPFMYSITYAAFAIIATLLMLNLLIA 1928 P ELGHFYDYP L TFELFLTIIDGPANYNVDLPF+YSITYAAFAIIATLLMLNLLIA Vrnt B: 72 PRELGHFYDYPTPLSGTFELFLTIIDGPANYNVDLPFVYSITYAAFAIIATLLMLNLLIA 251 PCaT : 1929 MMGDTHWRVAHERDELWRAQIVATTVMLERKLPRCLWPRSGICG 2060 MMGDTHWRVAHERDELWRAQIVATTVMLERKLPRCLWPRSGICG Vrnt B: 252 MMGDTHWRVAHERDELWRAQIVATTVMLERKLPRCLWPRSGICG 383 Table XXIVB. Peptide sequences from the translation of the nucleotide sequence of variant B. Open reading frame Amino acid sequence Frame 1 ILLADGCGHPGLASAFYI I FQTEDPES *ATSTTTPRPCPAPSSCSLPSSMAQPTTT WTCPSCTASPMLPLPSSPHCSCSTSSLP*WATLTGEWPMSGMSCGGPRLWPPR*CW SGSCLAACGLAPGSA**GVWPGRPLVPRRAGRTGNDLNRQRIQRHRTGLPHPGLLR IRPPDSVQTTRAGAVPFSPSVSPS*LRSRSRKYLPVDVPMGASAPGKGAPAGEDLR GDHSTRGSGRTAKLGYPEPTRVPPGPDLAPITGGPTTQTETQEPSRPGETAAPRRTSS Frame2 FCWLMAW ILGLLQPSISSSRQRTPRARPLLRLPHAPVRHLRAVPYHHRWPSQLQR GPALRVQHHLCCLCHHRHTAHAQPPHCHDGRHSLASGP*AG*AVEGPDCGHHGDAG AEAASLPVASLRDLR*REYGLGDRWSLGALEEQATISTGSGSNATAQAFHTRGS*G FGPQTQCKQLELALSLSAPACPLPNCAQGPESTFP*TCQWAQALRARGPLPEKTCV ATTPPGAPDAPRSWDIQNRRVSHLARTWPPLPGGQRHKPKPRSHPGQGKQRPHAEHP Frame3 SAG*WLWSSWACFSLLYHLPDRGPRELGHFYDYPTPLSGTFELFLTIIDGPANYNV DLPFVYS I TYAAFAI IATLLMLNLL IAMMGDTHWRVAHERDELWRAQItATTVMLE RKLPRCLWPRSGICG*GSMAWETAGPSARWKNRQRSQPAADPTPPHRPSTPGAPED SAPRLSANN*SWRCPFQPQRVPFLIALKVPKVPSRRRANGRKRSGQGGPCRRRPAW RPLHQGLRTHREAGISRTDACPTWPGPGPHYRGANDTNRNPGAIPARGNSGPTPNIL

Note: Frame 3 gives the longest subsequence that is identical with PCaT amino acid sequence. In this Table each (*) indicates a single unknown amino acid.

Table XXIC. Nucleotide sequence of splice Variant C for PCaT.".

1 TTTATTTTCT CCAGGAATAT ATATTGATAT TCTAAGTGGG ATGTTTATAT TTATAAGTGG 61 CCTTTATGTC TGTAGGGTCA AAATATCTGG GAGCCCTTAA AAGCCCTTTC TATTTGCTTT 121 CTCTGGTGCC TGTGCTCCTG GGAATGGGGC TTCTGCTTCC TGTCTTTCTC CTGCCTCTGG 181 CCTGGCTGCG TCATGGATGT TGGGTCATTG GGTAAAGAAT TGTTGGTCTC AAGCTCTATC 241 AACTCTCTCC CACTGAAGAA GGTCAACAAA GGCTGCCCTA CCCCTACCTC TGTCTGCGCC 301 CAGCCTCATC TCTGACTTCT CCTTTTGTTC CCATACGCAG ATTGTGGCCA CCACGGTGAT 361 GCTGGAGCGG AAGCTGCCTC GCTGCCTGTG GCCTCGCTCC GGGATCTGCG GACGGGAGTA 421 TGGCCTGGGA GACCGCTGGT TCCTGCGGTG AGTGATATGC GGGGGTAGGT GTCCCCTGAG 481 AAGCCTCATC GGCAGGGTAT CCCCCTGCTC AGACAGCTTC CGGCTCCTGG GTTCCCTGTG 541 GAGGCCTGTG TGCTCCCTAG GCTCTATGCT TGTTGATTGA GCTGGTGAGG AAGGGGTCCC 601 GTTTGGAGCT CAGACTTCCC AAAGCATCCA GGGAGTCTGT GGCAGAGCCT GCTGCTTTCT 661 GAGGCCTAGC TGCCAAGGGG CCAGTTACCC AGGCATNCAC CATGGGNTNC AGAAAAGNGG 721 AAAAGGCCAG CAATGGCGGT GGAT Table XXIIC. Nucleotide sequence alignment of Variant C with PCaT.

Score = 214 bits (111), Expect = 4e-52 Identities = 111/111 (100%) Strand = Plus/Plus

Table XXIIIC. Longest amino acid sequence alignment of Variant C and PCaT.

Score = 97. 3 bits (206), Expect = 6e-18 Identities = 37/37 (100%) Frame = +31+2 PCaT : 1986 QIVATTVMLERKLPRCLWPRSGICGREYGLGDRWFLR 2096 QIVATTVMLERKLPRCLWPRSGICGREYGLGDRWFLR Vrnt C: 338 QIVATTVMLERKLPRCLWPRSGICGREYGLGDRWFLR 448 Table XXIVC. Peptide sequences from the translation of the nucleotide sequence of variant C. Open reading frame Amino acid sequences Frame FIFSRNIY*YSKWDVYIYKWPLCL*GQNIWEPLKALSICFLWCLCSWEWGFCFLSFS CLWPGCVMDVGSLGKELLVSSSINSLPLKKVNKGCPTPTSVCAQPHL*LLLLFPYAD CGHHGDAGAEAASLPVASLRDLRTGVWPGRPLVPAVSDMRG*VSPEKPHRQGIPLLR QLPAPGFPVEACVLPRLYAC*LSW*GRGPVWSSDFPKHPGSLWQSLLLSEA*LPRGQ LPRH*PW**EK*KRPAMAVD Frame2 LFSPGIYIDILSGMFIFISGLYVCRVKISGSP*KPFLFAFSGACAPGNGASASCLSP ASGLAASWMLGHWVKNCWSQALSTLSH*RRSTKAALPLPLSAPSLISDFSFCSHTQI VATTVMLERKLPRCLWPRSGICGREYGLGDRWFLR*VICGGRCPLRSLIGRVSPCSD SFRLLGSLWRPVCSLGSMLVD*AGEEGVPFGAQTSQSIQGVCGRACCFLRPSCQGAS YPG*HHG*QK*GKGQQWRW Frame3 YFLQEYILIF*VGCLYL*VAFMSVGSKYLGALKSPFYLLSLVPVLLGMGLLLPVFLL PLAWLRHGCWVIG*RIVGLKLYQLSPTEEGQQRLPYPYLCLRPASSLTSPFVPIRRL WPPR*CWSGSCLAACGLAPGSADGSMAWETAGSCGE*YAGVGVP*EASSAGYPPAQT ASGSWVPCGGLCAP*ALCLLIELVRKGSRLELRLPKASRESVAEPAAF*GLAAKGPV TQA*TMG*RK*EKASNGGG

Note: Frame 2 gives the longest subsequence that is identical with PCaT amino acid sequence. In this Table each (*) indicates a single unknown amino acid.