WO2002014501A2

WO2002014501A2 - Nucleic acids and corresponding proteins entitled phor1-a11 and phor1-f5d6 useful in treatment and detection of cancer

Info

Publication number: WO2002014501A2
Application number: PCT/US2001/025862
Authority: WO
Inventors: Rene S. Hubert; Arthur B. Raitano; Mary Faris; Pia M. Challita-Eid; Wangmao Ge; Aya Jakobovits
Original assignee: Agensys Inc
Current assignee: Agensys Inc
Priority date: 2000-08-17
Filing date: 2001-08-17
Publication date: 2002-02-21
Anticipated expiration: 2003-02-17
Also published as: WO2002014501A3; AU2001286541A1

Abstract

A novel gene (designated PHOR1-A11 or PHOR1-F5D6) and its encoded protein are described. While PHOR1-A11 or PHOR1-F5D6 exhibits tissue specific expression in normal adult tissue, it is aberrantly expressed in prostate cancer. Consequently, PHOR1-A11 or PHOR-F5D6 provides a diagnostic and/or therapeutic target for cancer. The PHOR1-A11 or PHOR1-F5D6 gene or fragment thereof, or its encoded protein or a fragment thereof, can be used to elicit an immune response.

Description

NUCLEIC ACIDS AND CORRESPONDING PROTEINS ENTITLED PHORl-All AND PHOR1-F5D6 USEFUL IN TREATMENT AND DETECTION OF CANCER

This application claims the benefit of United States provisional application serial number 60/226,241 filed August 17, 2000, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention described herein relates to novel genes and their encoded protein, termed PHORl- All and PHOR1-F5D6, and to diagnostic and therapeutic methods and compositions useful in the management of various cancers that express PHORl-Al 1 or PHOR1-F5D6. 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 (SOD) 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.

la-509107 Most bladder cancers recur in the bladder. Bladder cancer is managed with a combination of transurethral resection of the bladder (TUR) and intravesical chemotherapy or immunotherapy. The multifocal and recurrent nature of bladder cancer points out the limitations of TUR. Most muscle-invasive cancers are not cured by TUR alone. Radical cystectomy and urinary diversion is the most effective means to eliminate the cancer but carry an undeniable impact on urinary and sexual function. There continues to be a significant need for treatment modalities that are beneficial for bladder cancer patients.

An estimated 130,200 cases of colorectal cancer occurred in 2000 in the United States, including 93,800 cases of colon cancer and 36,400 of rectal cancer. Colorectal cancers are the third most common cancers in men and women. Incidence rates declined significantly during 1992-1996 (-2.1% per year). Research suggests that these declines have been due to increased screening and polyp removal, preventing progression of polyps to invasive cancers. There were an estimated 56,300 deaths (47,700 from colon cancer, 8,600 from rectal cancer) in 2000, accounting for about 11% of all U.S. cancer deaths.

At present, surgery is the most common form of therapy for colorectal cancer, and for cancers that have not spread, it is frequently curative. Chemotherapy, or chemotherapy plus radiation is given before or after surgery to most patients whose cancer has deeply perforated the bowel wall or has spread to the lymph nodes. A permanent colostomy (creation of an abdominal opening for elimination of body wastes) is occasionally needed for colon cancer and is infrequently required for rectal cancer. There continues to be a need for effective diagnostic and treatment modalities for colorectal cancer.

There were an estimated 164,100 new cases of lung and bronchial cancer in 2000, accounting for 14% of all U.S. cancer diagnoses. The incidence rate of lung and bronchial cancer is declining significantly in men, from a high of 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s, the rate of increase among women began to slow. In 1996, the incidence rate in women was 42.3 per 100,000.

Lung and bronchial cancer caused an estimated 156,900 deaths in 2000, accounting for 28% of all cancer deaths. During 1992-1996, mortality from lung cancer declined significantly among men (-1.7% per year) while rates for women were still significantly increasing (0.9% per year). Since 1987, more women have died each year of lung cancer than breast cancer, which, for over 40 years, was the major cause of cancer death in women. Decreasing lung cancer incidence and mortality rates most likely resulted from decreased smoking rates over the previous 30 years; however, decreasing smoking patterns among women lag behind those of men. Of concern, although the declines in adult tobacco use have slowed, tobacco use in youth is increasing again.

Treatment options for lung and bronchial cancer are determined by the type and stage of the cancer and include surgery, radiation therapy, and chemotherapy. For many localized cancers, surgery is usually the treatment of choice. Because the disease has usually spread by the time it is discovered, radiation therapy and chemotherapy are often needed in combination with surgery. Chemotherapy alone or

3 la-509107 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-

4 la-509107 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 novel genes, designated PHORl-Al 1 and PHOR1-F5D6, that are over-expressed in cancers listed in Table I. Northern blot expression analysis of PHORl-Al 1 and PHOR1- F5D6 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 PHORl-Al 1 and PHOR1- F5D6 are provided. The tissue-related profile of PHORl-All and PHOR1-F5D6 in normal adult tissues, combined with the over-expression observed in prostate and other tumors, shows that PHORl-Al 1 and PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 genes, mRNAs, and/or coding sequences, preferably in isolated form, including polynucleotides encoding PHORl-Al 1- or PHORl-F5D6-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 PHORl-All- or PHORl-F5D6-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 PHORl-Al 1 or PHOR1-F5D6 genes or mRNA sequences or parts thereof, and polynucleotides or oligonucleotides that hybridize to the PHORl-Al 1 or PHOR1-F5D6 genes, mRNAs, or to PHORl-Al 1- or PHORl-F5D6-encoding polynucleotides. Also provided are means for isolating cDNAs and the genes encoding PHORl-Al 1 or PHOR1-F5D6. Recombinant DNA molecules containing PHORl-Al 1 or PHOR1-F5D6 polynucleotides, cells transformed or transduced with such molecules, and host-vector systems for the expression of PHORl-Al 1 or PHOR1-F5D6

5 la-509107 gene products are also provided. The invention further provides antibodies that bind to PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or

PHOR1-F5D6 polynucleotides and proteins in various biological samples, as well as methods for identifying cells that express PHORl-Al 1 or PHOR1-F5D6. A typical embodiment of this invention provides methods for monitoring PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 such as prostate cancers, including therapies aimed at inhibiting the transcription, translation, processing or function of PHORl-Al 1 or PHOR1-F5D6 as well as cancer vaccines.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. PHORl-Al 1 and PHOR1-F5D6 fragment sequences. Figure 2. The cDNA and amino acid sequences of PHORl-Al 1 and PHOR1-F5D6. Figure 3. Amino acid sequence of PHORl-All and PHOR1-F5D6. Figure 4. Sequence alignment of PHORl-All with a Marmota olfactory receptor (GenBank accession AF044033). The sequences are 83% identical.

Figure 5. Sequence alignment of PHOR1-F5D6 with human olfactory receptor, family 2, subfamily A, member 4 (GenBank accession NP_112170.1). The sequences are 100% identical.

Figure 6. Restricted normal tissue expression of PHORl-All in Testis, Placenta, Prostate, and Prostate Cancer. First strand cDNA was prepared from normal tissues, and from prostate cancer pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to PHORl-Al 1, was performed at 30 cycles of amplification. Expression of PHORl-Al 1 is observed in normal testis and placenta kidney and prostate, and in prostate cancer pool.

Figure 7. Expression of PHORl-All in Ovarian Cancer Patient Specimens 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), and ovarian cancer pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to PHORl-All, was performed at 30 cycles of amplification. Expression of PHORl-Al 1 is observed in the ovarian cancer pool but not in VP1 and VP2.

la-509107 Figure 8. Expression of PHORl-All in normal human tissues. Two multiple tissue northern blots, with 2 μg of mRNA/lane, were probed with the PHORl-Al 1 fragment. Size standards in kilobases (kb) are indicated on the side. The results show restricted expression of PHORl-Al 1 in placenta and prostate. Figure 9. Expression of PHORl-All in prostate xenograft tissues and cancer cell lines. RNA was extracted from prostate xenografts LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC-9AI (A), and from the prostate cancer cell line PC3 and the bladder cancer cell line J82 (B). Northern blots with 10 μg of total RNA/lane were probed with the PHORl-Al 1 fragment. Size standards in kilobases (kb) are indicated on the side. The results show expression of PHORl-Al 1 in all prostate xenografts an in the cancer cell lines tested.

Figure 10. Expression of PHOR1-F5D6 in normal and cancer tissues by RT-PCR. First strand cDNA was prepared from normal tissues, kidney cancer pool, bladder cancer pool, prostate cancer pool, and from prostate xenograft tissues (LAPC-4AD, LAPC-4AI, LAPC-9AD and LAPC-9AI). Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to PHOR1-F5D6, was performed at 30 cycles of amplification. Expression of PHOR1-F5D6 is observed in normal ovary and prostate, in prostate cancer pool and kidney cancer pool as well as all 4 xenograft tissues tested.

Figure 11. Expression of PHOR1-F5D6 in kidney and ovarian cancer patient specimens 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), kidney cancer pool and ovarian cancer pool. Normalization was performed by PCR using primers to actin and GAPDH. Semi-quantitative PCR, using primers to PHOR1- F5D6, was performed at 30 cycles of amplification. Expression of PHOR1-F5D6 is observed in the kidney cancer pool, and to lower levels in VP1, VP2 and ovarian cancer pool.

Figure 12. Expression of PHOR1-F5D6 in normal human tissues. Two multiple tissue northern blots, with 2 μg of mRNA/lane, were probed with the PHOR1-F5D6 fragment. Size standards in kilobases (kb) are indicated on the side. The results show absence of expression of PHOR1-F5D6 in all 16 normal tissues tested.

Figure 13. Expression of PHOR1-F5D6 in kidney cancer patient specimens. RNA was extracted from normal kidney (N), kidney tumors (T) and their matched normal adjacent tissue (NAT) isolated from kidney cancer patients. Northern blots with 10 μg of total RNA/lane were probed with the PHOR1-F5D6 fragment. Size standards in kilobases (kb) are indicated on the side. The results show expression of PHOR1-F5D6 in kidney tumor tissues tested. The expression detected in normal adjacent

la-509107 tissues (isolated from patients) but not in normal tissues, isolated from healthy donors, may indicate that this tissue is not fully normal and that PHOR1-F5D6 may be expressed in early stage tumors.

Figure 14A. Hydrophilicity amino acid profile of PHORl-Al 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 14B. Hydrophilicity amino acid profile of PHOR1-F5D6 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 PHORl-Al 1 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 PHOR1-F5D6 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 PHORl-All 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 of PHOR1-F5D6 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 PHORl-All 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 PHOR1-F5D6 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.

8 la-509107 Figure 18A. Beta-turn amino acid profile of PHORl-Al 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 18B. Beta-turn amino acid profile of PHOR1-F5D6 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.

DETAILED DESCRIPTION OF THE INVENTION Outline of Sections I.) Definitions

II.) PHORl-All or PHOR1-F5D6 Polynucleotides II.A.) Uses of PHORl-All or PHOR1-F5D6 Polynucleotides

II.A.1.) Monitoring of Genetic Abnormalities II.A.2.) Antisense Embodiments II.A.3.) Primers and Primer Pairs

II.A.4.) Isolation of PHORl-All or PHORl-F5D6-Encoding Nucleic Acid Molecules

II.A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems III.) PHORl-All- or PHORl-F5D6-related Proteins III.A.) Motif-bearing Protein Embodiments

III.B.) Expression of PHORl-All- or PHORl-F5D6-reIated Proteins III.C.) Modifications of PHORl-All- or PHORl-F5D6-related Proteins

III.D.) Uses of PHORl-All- or PHORl-F5D6-related Proteins rV.) PHORl-All or PHOR1-F5D6 Antibodies V.) PHORl-All or PHOR1-F5D6 Cellular Immune Responses VI.) PHORl-All or PHOR1-F5D6 Transgenic Animals VII.) Methods for the Detection of PHORl-All or PHOR1-F5D6

VIII.) Methods for Monitoring the Status of PHORl-All- or PHORl-F5D6-related Genes and Their Products

IX.) Identification of Molecules That Interact With PHORl-All or PHOR1-F5D6 X.) Therapeutic Methods and Compositions

9 la-509107 X.A.) Anti-Cancer Vaccines

X.B.) PHORl-All or PHOR1-F5D6 as a Target for Antibody-Based Therapy X.C.) PHORl-All or PHOR1-F5D6 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 Tmmunotherapy X.E.) Administration of Vaccines for Therapeutic or Prophylactic Purposes

XL) Diagnostic and Prognostic Embodiments of PHORl-All or PHOR1-F5D6. XII.) Inhibition of PHORl-All or PHOR1-F5D6 Protein Function

XII.A.) Inhibition of PHORl-All or PHOR1-F5D6 With Intracellular Antibodies XII.B.) Inhibition of PHORl-All or PHOR1-F5D6 with Recombinant Proteins XII.C.) Inhibition of PHORl-All or PHOR1-F5D6 Transcription or Translation

XII.D.) General Considerations for Therapeutic Strategies XIII.) KITS

L) Definitions: Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.

The terms "advanced prostate cancer", "locally advanced prostate cancer", "advanced disease" and ' "locally advanced disease" mean prostate cancers that have extended through the prostate capsule, and are meant to include stage C disease under the American Urological Association (AUA) system, stage Cl - C2 disease under the Whitmore-Jewett system, and stage T3 - T4 and N+ disease under the TNM (tumor, node,

10 la-509107 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 PHORl-Al 1 or PHOR1-F5D6 (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 PHORl-Al 1 or

PHOR1-F5D6. 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 PHORl-Al 1- or PHORl-F5D6-related protein). For example an analog of the PHORl-Al 1- or PHOR1-F5D6 protein can be specifically bound by an antibody or T cell that specifically binds to PHORl-Al 1- or PHOR1-F5D6.

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-PHORl- Al 1- or PHOR1-F5D6 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-PHORl-Al 1 or PHOR1-F5D6 antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-PHORl-Al 1 or PHOR1-F5D6 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."

11 la-509107 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 At²¹¹, 1¹³¹, 1¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes of Lu. Antibodies may also be conjugated to an anti-cancer pro-drug activating enzyme capable of converting the pro-drug to its active form.

The term "homolog" refers to a molecule which exhibits homology to another molecule, by for example, having sequences of chemical residues that are the same or similar at corresponding positions. "Human Leukocyte Antigen" or "HLA" is a human class I or class II Major Histocompatibility

Complex (MHC) protein (see, e.g., Stites, et al., IMMUNOLOGY, 8™ 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 μg/ml ssDNA, in which temperatures for hybridization are above 37 degrees C and temperatures for washing in O.lXSSC/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 PHORl-Al 1 or PHOR1-F5D6 gene or that encode polypeptides other tha PHORl-Al 1 or PHOR1-F5D6 gene product or fragments thereof, respectively. A skilled artisan can readily employ nucleic acid isolation procedures to obtain an isolated PHORl-Al 1 or PHOR1-F5D6 polynucleotide. A protein is said to be "isolated," for example, when physical, mechanical or chemical methods are employed to remove the PHORl-All or PHOR1-F5D6 protein from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated PHORl-Al 1 or PHOR1-F5D6 protein. Alternatively, an isolated protein can be prepared by chemical means.

12 la-509107 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 a PHORl-All- or PHORl-F5D6-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.

13 la-509107 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 FIGURE 2) can also be uracil (U); this definition pertains to the differences between the chemical structures of DNA and RNA, in particular the observation that one of the four major bases in RNA is uracil (U) instead of thymidine (T).

The term "polypeptide" means a polymer of at least about 4, 5, 6, 7, or 8 amino acids. Throughout the specification, standard three letter or single letter designations for amino acids are used. In the art, this term is often used interchangeably with "peptide" or "protein".

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

14 la-509107 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 NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42 °C, with washes at 42°C in 0.2 x SSC (sodium chloride/sodium, citrate) and 50% formamide at 55 °C, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 55 °C. "Moderately stringent conditions" are described by, but not limited to, those in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 xDenhardt'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

15 la-509107 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 PHORl-Al 1 or PHOR1-F5D6 protein shown in Figure 2 or Figure 3). An analog is an example of a variant protein.

The PHORl-Al 1- or PHORl-F5D6-related proteins of the invention include those specifically identified herein, as well as allelic variants, conservative substitution variants, analogs and homologs that can be isolated/generated and characterized without undue experimentation following the methods outlined herein or readily available in the art. Fusion proteins that combine parts of different PHORl-Al 1 or PHOR1-F5D6 proteins or fragments thereof, as well as fusion proteins of a PHORl-Al 1 or PHOR1-F5D6 protein and a heterologous polypeptide are also included. Such PHORl-Al 1 or PHOR1-F5D6 proteins are collectively referred to as the PHORl-Al 1- or PHORl-F5D6-related proteins, the proteins of the invention, or PHORl-Al 1 or PHOR1-F5D6, respectively. The term 'PHORl-Al 1- or PHORl-F5D6-related protein" refers to a polypeptide fragment or a PHORl-All or PHOR1-F5D6 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, respectively. II.) PHORl-All or PHOR1-FSD6 Polynucleotides One aspect of the invention provides polynucleotides corresponding or complementary to all or part of a PHORl-Al 1 or PHOR1-F5D6 gene, mRNA, and/or coding sequence, preferably in isolated form, including polynucleotides encoding a PHORl-Al 1- or PHORl-F5D6-related protein and fragments thereof, DNA, RNA, DNA/RNA hybrid, and related molecules, polynucleotides or oligonucleotides complementary to a PHORl-Al 1 or PHOR1-F5D6 gene or mRNA sequence or a part thereof, and polynucleotides or oligonucleotides that hybridize to a PHORl-Al 1 or PHOR1-F5D6 gene, mRNA, or to a PHORl-All or PHOR1-F5D6 encoding polynucleotide (collectively, "PHORl-All orPHORl-F5D6 polynucleotides" respectively). In all instances when referred to in this section, T can also be U in Figure 2.

16 la-509107 Embodiments of a PHORl-Al 1 polynucleotides include: a PHORl-Al 1 polynucleotides having the sequence shown in Figure 2A, the nucleotide sequence of PHORl-All as shown in Figure 2A, wherein T is U; at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in Figure 2A; or, at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in Figure 2A where T is U. For example, embodiments of PHORl-Al 1 nucleotidespr polypeptide comprise, without limitation:

(a) a polynucleotide comprising or consisting of the sequence as shown in Figure 2A (SEQ ID NO.: ), wherein T can also be U;

(b) a polynucleotide comprising or consisting of the sequence as shown in Figure 2A (SEQ ID NO.: ), from nucleotide residue number 15 through nucleotide residue number 959, wherein T can also be U;

(c) a polynucleotide that encodes a PHORl-Al 1-related protein that is at least 90% homologous to the entire amino acid sequence shown in SEQ ID NO.: ;

(d) a polynucleotide that encodes a PHORl-Al 1-related protein that is at least 90% identical to the entire amino acid sequence shown in SEQ ID NO: ;

(e) a polynucleotide that encodes at least one peptide set forth in part (A) of Tables V-XVIII;

(f) a polynucleotide that encodes a peptide region of at least 5 amino acids of Figure 3 A in any whole number increment up to 314 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of Figure 14A;

(g) a polynucleotide that encodes a peptide region of at least 5 amino acids of Figure 3 A in any whole number increment up to 314 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of Figure 15 A;

(h) a polynucleotide that encodes a peptide region of at least 5 amino acids of Figure 3 A in any whole number increment up to 314 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 16A;

(i) a polynucleotide that encodes a peptide region of at least 5 amino acids of Figure 3 A in any whole number increment up to 314 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile on Figure 17A;

17 la-509107 (j) a polynucleotide that encodes a peptide region of at least 5 amino acids of Figure 3 A in any whole number increment up to 314 that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of Figure 18 A;

(k) a polynucleotide that is fully complementary to a polynucleotide of any one of (a)-(j); (1) a polynucleotide that selectively hybridizes under stringent conditions to a polynucleotide of (a)-(k); and

(n) a peptide that is encoded by any of (a)-(k).

(o) a polynucleotide of any of (a)-(l) or peptide of (n) together with a pharmaceutical excipient and/or in a human unit dose form. As used herein, a range is understood to specifically disclose all whole unit positions thereof.

Embodiments of a PHOR1-F5D6 polynucleotide include: a PHOR1-F5D6 polynucleotide having the sequence shown in Figure 2B, the nucleotide sequence of PHOR1-F5D6 as shown in Figure 2B, wherein T is U; at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in Figure 2B; 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 PHOR1-F5D6 nucleotides comprise, without limitation:

(a) a polynucleotide comprising or consisting of the sequence as shown in Figure 2B (SEQ

ID NO.: ), wherein T can also be U;

(b) a polynucleotide comprising or consisting of the sequence as shown in Figure 2B (SEQ

ID NO.: ), from nucleotide residue number 1 through nucleotide residue number 933, wherein T can also be U;

(c) a polynucleotide that encodes a PHORl-F5D6-related protein that is at least 90% homologous to the entire amino acid sequence shown in SEQ ID NO.: ;

(d) a polynucleotide that encodes a PHORl-F5D6-related protein that is at least 90% identical to the entire amino acid sequence shown in SEQ ED NO: ;

(e) a polynucleotide that encodes at least one peptide set forth in parts (B) of Tables V-

XVIII;

18 la-509107 (f) a polynucleotide that encodes a peptide region of at least 5 amino acids of Figure 3B in any whole number increment up to 310 that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of Figure 14B;

(g) a polynucleotide that encodes a peptide region of at least 5 amino acids of Figure 3B in any whole number increment up to 310 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of Figure 15B;

(h) a polynucleotide that encodes a peptide region of at least 5 amino acids of Figure 3B in any whole number increment up to 310 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 16B;

(i) a polynucleotide that encodes a peptide region of at least 5 amino acids of Figure 3B in any whole number increment up to 310 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile on Figure 17B;

(j) a polynucleotide that encodes a peptide region of at least 5 amino acids of Figure 3B in any whole number increment up to 310 that includes an amino acid position having a value greater than 0.5 in the Beta-turn profile of Figure 18B ;

(k) a polynucleotide that is fully complementary to a polynucleotide of any one of (a)-(j);

(1) a polynucleotide that selectively hybridizes under stringent conditions to a polynucleotide of (a)-(k); and

(n) a peptide that is encoded by. any of (a)-(k). (o) a polynucleotide of any of (a)-(l) or peptide of (n) together with a pharmaceutical excipient and/or in a human unit dose form.

As used herein, a range is understood to specifically disclose all whole unit positions thereof.

Typical embodiments of the invention disclosed herein include PHORl-All or PHOR1-F5D6 polynucleotides that encode specific portions of the PHORl-All or PHOR1-F5D6 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, or 300 contiguous amino acids, up to the full length of the proteins (314 contiguous amino acids for PHORl-Al 1 ; 310 contiguous amino acids for PHOR1-F5D6).

19 la-509107 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 PHORl-Al 1 or PHOR1-F5D6 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 10 to about amino acid 20 of the PHORl-Al 1 or PHOR1-F5D6 protein shown in Figure 2, or Figure 3, polynucleotides encoding about amino acid 20 to about amino acid 30 of the PHORl-Al 1 or PHOR1-F5D6 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 30 to about amino acid 40 of the PHORl-Al 1 or PHOR1-F5D6 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 40 to about amino acid 50 of the PHORl-Al 1 or PHOR1-F5D6 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 50 to about amino acid 60 of the PHORl-Al 1 or PHOR1-F5D6 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 60 to about amino acid 70 of the PHORl-Al 1 or PHOR1-F5D6 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 70 to about amino acid 80 of the PHORl-Al 1 or PHOR1-F5D6 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 80 to about amino acid 90 of the PHORl-Al 1 or PHOR1-F5D6 protein shown in Figure 2 or Figure 3, polynucleotides encoding about amino acid 90 to about amino acid 100 of the PHORl-Al 1 or PHOR1- F5D6 protein shown in Figure 2 or Figure 3, and polynucleotides encoding portions of the amino acid sequence 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 PHORl-Al 1 or PHOR1-F5D6 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 PHORl-All or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 sequence as shown in Figure 2 or Figure 3.

Additional illustrative embodiments of the invention disclosed herein include PHORl-All or PHOR1-F5D6 polynucleotide fragments encoding one or more of the biological motifs contained within the PHORl-Al 1 or PHOR1-F5D6 protein. sequence, including one or more of the motif-bearing subsequences of the PHORl-Al 1 or PHOR1-F5D6 protein set forth in Tables V-XVIII. In another embodiment, typical polynucleotide fragments of the invention encode one or more of the regions of PHORl-Al 1 or PHOR1-F5D6 that exhibit homology to a known molecule. In another embodiment of the invention, typical polynucleotide fragments can encode one or more of the PHORl-All or PHOR1-F5D6

20 . la-509107 N-glycosylation sites, cAMP and cGMP-dependent protein kinase phosphorylation sites, casein kinase II phosphorylation sites or N-myristoylation site and amidation sites.

II.A.) Uses of PHORl-All or PHOR1-F5D6 Polynucleotides II.A.1.) Monitoring of Genetic Abnormalities

The polynucleotides of the preceding paragraphs have a number of different specific uses. The huma PHORl-Al 1 or PHOR1-F5D6 gene maps to the chromosomal location set forth in Example 3. For example, because the PHORl-Al 1 or PHOR1-F5D6 gene maps to this chromosome, polynucleotides that encode different regions of the PHORl-Al 1 or PHOR1-F5D6 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 PHOR1- Al 1 or PHOR1-F5D6 protein provide new tools that can be used to delineate, with greater precision than previously possible, cytogenetic abnormalities in the chromosomal region that encodes PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 was shown to be highly expressed in prostate and other cancers, PHORl-Al 1 or PHOR1-F5D6 polynucleotides are used in methods assessing the status of PHORl-Al 1 or PHOR1-F5D6 gene products in normal versus cancerous tissues. Typically, polynucleotides that encode specific regions of the PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 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

21 la-509107 include molecules capable of inhibiting the RNA or protein expression of PHORl-Al 1 or PHOR1-F5D6. 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 PHORl-Al 1 or PHOR1-F5D6 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., PHORl-Al 1 or PHOR1-F5D6. See for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1:1-5 (1988). The PHORl-Al 1 or PHOR1-F5D6 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-l,2-benzodithiol-3-one-l,l-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 PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 antisense oligonucleotides of the present invention typically can be RNA or DNA that is complementary to and stably hybridizes with the first 1005' codons or last 100 3' codons of the PHORl-Al 1 or PHOR1-F5D6 genomic sequence or the corresponding mRNA, respectively. 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 PHORl-Al 1 or PHOR1-F5D6 mRNA and not to mRNA specifying other regulatory subunits of protein kinase. In one embodiment, PHORl-Al 1 or PHOR1-F5D6 antisense oligonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to PHORl-Al 1 or PHOR1-F5D6 mRNA. Optionally, PHORl-Al 1 or PHOR1-F5D6 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 PHORl-All or PHOR1-F5D6. Alternatively, the antisense molecules are modified to employ ribozymes in the inhibition of PHORl-Al 1 or PHOR1-F5D6 expression, see, e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996). II.A.3.) Primers and Primer Pairs

22 la-509107 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 PHORl-Al 1 or PHOR1-F5D6 polynucleotide in a sample and as a means for detecting a cell expressing a PHORl-Al 1 or PHOR1-F5D6 protein.

Examples of such probes include polypeptides comprising all or part of the huma PHORl-Al 1 or PHOR1-F5D6 cDNA sequence shown in Figure 2. Examples of primer pairs capable of specifically amplifying PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 mRNA.

The PHORl-Al 1 or PHOR1-F5D6 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 PHORl-All or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 polypeptides; as tools for modulating or inhibiting the expression of the PHORl-Al 1 or PHOR1-F5D6 gene(s) and/or translation of the PHORl-Al 1 or PHOR1- F5D6 transcript(s); and as therapeutic agents.

II.A.4.) Isolation of PHORl-All or PHOR1-F5D6-Encoding Nucleic Acid Molecules The PHORl-Al 1 or PHOR1-F5D6 cDNA sequences described herein enable the isolation of other polynucleotides encoding PHORl-Al 1 or PHOR1-F5D6 gene product(s), as well as the isolation of polynucleotides encoding PHORl-Al 1 or PHOR1-F5D6 gene product homologs, alternatively spliced isoforms, allelic variants, and mutant forms of the PHORl-Al 1 or PHOR1-F5D6 gene product as well as polynucleotides that encode analogs of PHORl-Al 1- or PHORl-F5D6-related proteins. Various molecular cloning methods that can be employed to isolate full length cDNAs encoding a PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 gene cDNAs can be identified by probing with a labeled PHORl-Al 1 or PHOR1-F5D6 cDNA or a fragment thereof. For example, in one embodiment, the PHORl- Al 1 or PHOR1-F5D6 cDNA (Figure 2) or a portion thereof can be synthesized and used as a probe to retrieve

23 la-509107 overlapping and full-length cDNAs corresponding to a PHORl-Al 1 or PHOR1-F5D6 gene. The PHORl-Al 1 or PHOR1-F5D6 gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with PHORl-Al 1 or PHOR1-F5D6 DNA probes or primers, respectively. II.A.5.) Recombinant Nucleic Acid Molecules and Host- Vector Systems

The invention also provides recombinant DNA or RNA molecules containing a PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 or a fragment, analog or homolog thereof can be used to generate PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 proteins or fragments thereof are available, see for example, Sambrook et al., 1989, supra; Current Protocols in Molecular Biology, 1995, supra). Preferred vectors for mammalian expression include but are not limited to pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vector pSRαtkneo (Muller et al., 1991, MCB 11 : 1785). Using these expression vectors, PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 protein or fragment thereof. Such host- vector systems can be employed to study the functional properties of PHORl-Al 1 or PHOR1-F5D6 and PHORl-Al 1 or PHOR1-F5D6 mutations or analogs.

Recombinant huma PHORl-Al 1 or PHOR1-F5D6 protein or an analog or homolog or fragment thereof can be produced by mammalian cells transfected with a construct encoding a PHORl-Al 1- or PHORl-F5D6-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding PHORl-All or PHOR1-F5D6 or fragment, analog or homolog thereof, the PHORl-All or

24 la-509107 PHOR1-F5D6 or related protein is expressed in the 293T cells, and the recombinant PHORl-Al 1 or PHOR1-F5D6 protein is isolated using standard purification methods (e.g., affinity purification using anti- PHORl-Al 1 or PHOR1-F5D6 antibodies). In another embodiment, a PHORl-Al 1 or PHOR1-F5D6 coding sequence is subcloned into the retroviral vector pSRoMSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPrl, 293 and rat-1 in order to establish PHORl-Al 1 or

PHOR1-F5D6 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 PHORl-Al 1 or PHOR1- F5D6 coding sequence can be used for the generation of a secreted form of recombinant PHORl-Al 1 or PHOR1-F5D6 protein. As discussed herein, redundancy in the genetic code permits variation in PHORl-Al 1 or PHOR1-

F5D6 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.°o.ip/~nakamura/codon.html.

Additional sequence modifications are known to enhance protein expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon/intron splice site signals, transposon-like repeats, and/or other such well-characterized sequences that are deleterious to gene expression. The GC content of the sequence is adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. Where possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures. Other useful modifications include the addition of a translational initiation consensus sequence at the start of the open reading frame, as described in Kozak, Mol. Cell Biol, 9:5073-5080 (1989). Skilled artisans understand that the general rule that eukaryotic ribosomes initiate translation exclusively at the 5 ' proximal AUG codon is abrogated only under rare conditions (see, e.g., Kozak PNAS 92(7): 2662-2666, (1995) and Kozak NAR 15(20): 8125-8148 (1987)).

III.) PHORl-All- or PHORl-F5D6-related Proteins Another aspect of the present invention provides PHORl-Al 1- or PHORl-F5D6-related proteins.

Specific embodiments of PHORl-Al 1 or PHOR1-F5D6 proteins comprise a polypeptide having all or part of the amino acid sequence of huma PHORl-Al 1 or PHOR1-F5D6 as shown in Figure 2 or Figure 3. Alternatively, embodiments of PHORl-All or PHOR1-F5D6 proteins comprise variant, homolog or

25 la-509107 analog polypeptides that have alterations in the amino acid sequence of PHORl-Al 1 or PHOR1-F5D6 shown in Figure 2 or Figure 3.

In general, naturally occurring allelic variants of huma PHORl-Al 1 or PHOR1-F5D6 share a high degree of structural identity and homology (e.g., 90% or more homology). Typically, allelic variants of the PHORl-Al 1 or PHOR1-F5D6 protein contain conservative amino acid substitutions within the PHORl-Al 1 or PHOR1-F5D6 sequences described herein or contain a substitution of an amino acid from a corresponding position in a homologue of PHORl-Al 1 or PHOR1-F5D6. One class of PHORl-Al 1 or PHOR1-F5D6 allelic variants are proteins that share a high degree of homology with at least a small region of a particular PHORl- Al 1 or PHOR1-F5D6 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" 2^nd 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 PHORl-Al 1 or PHOR1-F5D6 proteins such as polypeptides having amino acid insertions, deletions and substitutions. PHORl-All or PHOR1-F5D6 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., 73/4331 (1986); Zoller et al., Nucl. Acids Res., 70:6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selection mutagenesis (Wells

26 la-509107 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 PHORl-Al 1 or PHOR1-F5D6 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, PHORl-Al 1 or PHOR1-F5D6 variants, analogs or homologs, have the distinguishing attribute of having at least one epitope that is "cross reactive" with a PHORl-Al 1 or PHOR1-F5D6 protein having the amino acid sequence of FIGURE 2, respectively. As used in this sentence, "cross reactive" means that an antibody or T cell that specifically binds to a PHORl-Al 1 or PHOR1-F5D6 variant also specifically binds to the PHORl-Al 1 or PHOR1-F5D6 protein having the amino acid sequence of FIGURE 2, respectively. A polypeptide ceases to be a variant of the PHORl-Al 1 or PHOR1-F5D6 protein when it no longer contains any epitope capable of being recognized by an antibody or T cell that specifically binds to the PHORl-Al 1 or PHOR1-F5D6 protein, respectively. 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 PHORl-All- or PHORl-F5D6-related protein variants share 70%, 75%, 80%, 85% or 90% or more similarity with the amino acid sequence of FIGURE 2 or a fragment thereof. Another specific class of PHORl-Al 1 or PHOR1-F5D6 protein variants or analogs comprise one or more of the PHORl-All or PHOR1-F5D6 biological motifs described herein or presently known in the art. Thus, encompassed by the present invention are analogs of PHORl-All or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 protein shown in Figure 2 or Figure 3. For example, representative embodiments of the invention comprise peptides/proteins having any 4, 5,

27 la-509107 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of the PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 10 to about amino acid 20 of the PHORl-Al 1 or PHOR1-F5D6 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 20 to about amino acid 30 of the PHORl-Al 1 or PHOR1-F5D6 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 30 to about amino acid 40 of the PHORl-All or PHOR1-F5D6 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 40 to about amino acid 50 of the PHORl-Al 1 or PHOR1-F5D6 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 50 to about amino acid 60 of the PHORl-Al 1 or PHOR1- F5D6 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 60 to about amino acid 70 of the PHORl-Al 1 or PHOR1-F5D6 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 70 to about amino acid 80 of the PHORl-Al 1 or PHOR1-F5D6 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 80 to about amino acid 90 of the PHORl-Al 1 or PHOR1-F5D6 protein shown in Figure 2 or Figure 3, polypeptides consisting of about amino acid 90 to about amino acid 100 of the PHORl-Al 1 or PHOR1-F5D6 protein shown in Figure 2 or Figure 3, etc. throughout the entirety of the PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 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.

PHORl-Al 1- or PHORl-F5D6-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 PHORl-Al 1- or PHORl-F5D6-related protein. In one embodiment, nucleic acid molecules provide a means to generate defined fragments of the PHORl-Al 1 or PHOR1-F5D6 protein (or variants, homologs or analogs thereof). HLA.) Motif-bearing Protein Embodiments Additional illustrative embodiments of the invention disclosed herein include PHORl-All or PHOR1-F5D6 polypeptides comprising the amino acid residues of one or more of the biological motifs contained within the PHORl-Al 1 or PHOR1-F5D6 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/struc-predict.html psort.ims.u-tokvo.ac.ip/; www.cbs.dtu.dk/:

28 la-509107 www.ebi.ac.ukinteφro/scan.html: www.expasv.ch/tools/scnpsitl.html; Epimatrix™ and Epimer™, Brown University, www.brown.edu/Researcli/TB-HIV Lab/epimatrix/epimatrix.html; and BIMAS, bimas.dcrt.nih.gov/.).

Motif bearing subsequences of the PHORl-All and PHOR1-F5D6 proteins 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 PHORl-Al 1 or PHOR1-F5D6 motifs discussed above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the PHORl-Al 1 or PHOR1-F5D6 motifs discussed above are associated with growth dysregulation and because PHORl-Al 1 or PHOR1-F5D6 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 OΗrian, 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(l):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 a PHORl-Al 1 or PHOR1-F5D6 protein that are capable of optimally binding to specified HLA alleles (e.g., Table IV; Epimatrix™ and Epimer™, Brown University, URL www.brown.edu/Research/TB- HIV Lab/epimatrix epimatrix.html; and BBVIAS, URL bimas.dcrt.nih.gov/.l 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 ■

29 la-509107 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 199950(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.

PHORl-All- or PHORl-F5D6-related proteins are embodied in many forms, preferably in isolated form. A purified PHORl-Al 1 or PHOR1-F5D6 protein molecule will be substantially free of other proteins or molecules that impair the binding of PHORl-All or PHOR1-F5D6 to antibody, T cell or other ligand. The nature and degree of isolation and purification will depend on the intended use. Embodiments of a PHORl-Al 1- or PHORl-F5D6-related proteins include purified PHORl-Al 1- or PHORl-F5D6-related proteins and functional, soluble PHORl-Al 1- or PHORl-F5D6-related proteins. In one embodiment, a functional, soluble PHORl-All or PHOR1-F5D6 protein or fragment thereof retains the ability to be bound by antibody, T cell or other ligand.

30 la-509107 The invention also provides PHORl-Al 1 or PHOR1-F5D6 proteins comprising biologically active fragments of the PHORl-Al 1 or PHOR1-F5D6 amino acid sequence shown in Figure 2 or Figure 3. Such proteins exhibit properties of the PHORl-Al 1 or PHOR1-F5D6 protein, respectively, such as the ability to elicit the generation of antibodies that specifically bind an epitope associated with the PHOR1- Al 1 or PHOR1-F5D6 protein; to be bound by such antibodies; to elicit the activation of HTL or CTL; and/or, to be recognized by HTL or CTL.

PHORl-Al 1- or PHORl-F5D6-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-PHORl-Al 1 or PHOR1-F5D6 antibodies, or T cells or in identifying cellular factors that bind to PHORl-All or PHOR1-F5D6.

CTL epitopes can be determined using specific algorithms to identify peptides within a PHORl-Al 1 or PHOR1-F5D6 protein that are capable of optimally binding to specified HLA alleles (e.g., by using the SYFPEITHI site at World Wide Web URL syfipeithi.bmi-heidelberg.com/; the listings in Table IV(A)-(E); Epimatrix™ and Epimer™, Brown University, URL (www.brown.edu/Research/TB- HIV Lab/epimatrix/epimatrix.html); and BIMAS, URL bimas.dcrt.nih.goW). Illustrating this, peptide epitopes from PHORl-All orPHORl-F5D6 that are presented in the context of human MHC class I molecules HLA-A1, A2, A3, All, A24, B7 and B35 were predicted (Tables V-XVIII). Specifically, the complete amino acid sequence of the PHORl-Al 1 and PHOR1-F5D6 proteins weres 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 PHORl-Al 1 and PHOR1-F5D6 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

31 la-509107 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, Epimer™ and Epimatrix™ 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 PHORl-Al 1 and PHOR1-F5D6 proteins. As used in this context "applied" means that the PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 protein of 8, 9, 10, or 11 amino acid residues that bears an HLA Class I motif, or a subsequence of 9 or more amino acid residues that bear an HLA Class II motif are within the scope of the invention.

III.B.) Expression of PHORl-All- or PHORl-F5D6-related Proteins In an embodiment described in the examples that follow, PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 protein in transfected cells. The secreted HIS-tagged PHORl-Al 1 or PHOR1-F5D6 in the culture media can be purified, e.g., using a nickel column using standard techniques.

III.C.) Modifications of PHORl-All- or PHORl-F5D6-related Proteins Modifications of PHORl-Al 1- or PHORl-F5D6-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 PHORl-All or PHOR1-F5D6 polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the PHORl- Al 1 or PHOR1-F5D6 protein. Another type of covalent modification of the PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 comprises linking the PHORl-All or PHOR1-F5D6 polypeptide to one of a variety of nonproteinaceous

32 la-509107 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 PHORl-Al 1- or PHORl-F5D6-related proteins of the present invention can also be modified to form a chimeric molecule comprising PHORl-All- or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1- or PHOR1-F5D6. A chimeric molecule can comprise a fusion of a PHORl- Al 1- or PHORl-F5D6-related protein with a polyhistidine epitope tag, which provides an epitope to which immobilized nickel can selectively bind, with cytokines or with growth factors. The epitope tag is generally placed at the amino- or carboxyl- terminus of the PHORl-Al 1 or PHOR1-F5D6 protein. In an alternative embodiment, the chimeric molecule can comprise a fusion of a PHORl-Al 1 or PHOR1-F5D6- 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 PHORl-Al 1 or PHOR1-F5D6 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 PHORl-All- or PHORl-F5D6-related Proteins The proteins of the invention have a number of different specific uses. As PHORl-Al 1 or PHOR1-F5D6 is highly expressed in prostate and other cancers, PHORl-Al 1- or PHORl-F5D6-related proteins are used in methods that assess the status of PHORl-Al 1 or PHOR1-F5D6 gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of the PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1- or PHORl-F5D6-related proteins comprising the amino acid residues of one or more of the biological motifs contained within the PHORl-Al 1 or PHOR1-F5D6 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, PHORl-Al 1- or PHORl-F5D6-related proteins that contain the amino acid

33 la-509107 residues of one or more of the biological motifs in the PHORl-Al 1 or PHOR1-F5D6 protein are used to screen for factors that interact with that region of PHORl-Al 1 or PHOR1-F5D6, respectively.

PHORl-Al 1 or PHOR1-F5D6 protein fragments/subsequences are particularly useful in generating and characterizing domain-specific antibodies (e.g., antibodies recognizing an extracellular or intracellular epitope of a PHORl-Al 1 or PHOR1-F5D6 protein), for identifying agents or cellular factors that bind to PHORl-Al 1 or PHOR1-F5D6 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 PHORl-All or PHOR1-F5D6 genes, or by analogs, homologs or fragments thereof, have a variety of uses, including but not limited to generating antibodies and in methods for identifying ligands and other agents and cellular constituents that bind to a PHORl-Al 1 or PHOR1-F5D6 gene product. Antibodies raised against a PHORl-Al 1 or PHOR1-F5D6 protein or fragment thereof are useful in diagnostic and prognostic assays, and imaging methodologies in the management of human cancers characterized by expression of PHORl-Al 1 or PHOR1-F5D6 protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers. PHORl-Al 1- or PHORl-F5D6-related nucleic acids or proteins are also used in generating HTL or CTL responses.

Various immunological assays useful for the detection of PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHORl-F5D6-expressing cells (e.g., in radioscintigraphic imaging methods). PHORl-All or PHOR1-F5D6 proteins are also particularly useful in generating cancer vaccines, as further described herein. IV.) PHORl-All or PHOR1-F5D6 Antibodies Another aspect of the invention provides antibodies that bind to PHORl-Al 1- or PHOR1-F5D6- related proteins. Preferred antibodies specifically .bind to a PHORl-Al 1- or PHORl-F5D6-related protein and do not bind (or bind weakly) to peptides or proteins that are not PHORl-Al 1- or PHORl-F5D6-related proteins. For example, antibodies bind PHORl-Al 1 or PHOR1-F5D6 can bind PHORl-Al 1- or PHOR1- F5D6-related proteins such as the homologs or analogs thereof. PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 is also expressed or overexpressed in these other cancers. Moreover, intracellularly expressed antibodies

34 la-509107 ι (e.g., single chain antibodies) are therapeutically useful in treating cancers in which the expression of PHORl-All or PHOR1-F5D6 is involved, such as advanced or metastatic prostate cancers.

The invention also provides various immunological assays useful for the detection and quantification of PHORl-All or PHOR1-F5D6 and mutant PHORl-Al 1- or PHORl-F5D6-related proteins. Such assays can comprise one or more PHORl-Al 1 or PHOR1-F5D6 antibodies capable of recognizing and binding a PHORl-Al 1- or PHORl-F5D6-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 PHORl-Al 1 or PHOR1-F5D6 are also provided by the invention, including but not limited to radioscintigraphic imaging methods using labeled PHORl-Al 1 or PHOR1-F5D6 antibodies. Such assays are clinically useful in the detection, monitoring, and prognosis of PHORl-Al 1 or PHOR1-F5D6 expressing cancers such as prostate cancer.

PHORl-Al 1 or PHOR1-F5D6 antibodies are also used in methods for purifying a PHORl-Al 1- or PHORl-F5D6-related protein and for isolating PHORl-Al 1 or PHOR1-F5D6 homologues and related molecules. For example, a method of purifying a PHORl-Al 1- or PHORl-F5D6-related protein comprises incubating a PHORl-Al 1 or PHOR1-F5D6 antibody, which has been coupled to a solid matrix, with a lysate or other solution containing a PHORl-Al 1- or PHORl-F5D6-related protein under conditions that permit the PHORl-Al 1 or PHOR1-F5D6 antibody to bind to the PHORl-Al 1- or PHORl-F5D6-related protein; washing the solid matrix to eliminate impurities; and eluting the PHORl-Al 1- or PHORl-F5D6-related protein from the coupled antibody. Other uses of the PHORl-Al 1 or PHOR1-F5D6 antibodies of the invention include generating anti-idiotypic antibodies that mimic the PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1- or PHORl-F5D6-related protein, peptide, or fragment, in isolated or immunocoηjugated 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 PHORl-Al 1 or PHOR1-F5D6 can also be used, such as a PHORl-Al 1 or PHOR1-F5D6

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 PHORl-Al 1- or PHORl-F5D6-related protein is synthesized and used as an immunogen.

35 la-509107 In addition, naked DNA immunization techniques known in the art are used (with or without purified PHORl-Al 1- or PHORl-F5D6-related protein or PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 as shown in Figure 2 or Figure 3 can be analyzed to select specific regions of the PHORl-Al 1 or PHOR1-F5D6 protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyses of the PHORl-All or PHOR1-F5D6 amino acid sequences are used to identify hydrophilic regions in the PHORl-Al 1 or PHOR1-F5D6 structure. Regions of the PHORl-Al 1 or PHOR1-F5D6 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-Dool e, 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 PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 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.

PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1- or PHORl-F5D6-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 PHORl-Al 1 or PHOR1-F5D6 protein can also be produced in the context of chimeric or complementarity determining region (CDR) grafted antibodies of multiple species origin. Humanized or huma PHORl-Al 1 or PHOR1-F5D6 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

36 la-509107 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 huma PHORl-Al 1 or PHOR1-F5D6 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 huma PHORl-Al 1 or PHOR1-F5D6 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 PHORl-All or PHOR1-F5D6 antibodies with a PHORl-All- or PHOR1-F5D6- related protein can be established by a number of well known means, including Western blot, immunoprecipitation, ELISA, and FACS analyses using, as appropriate, PHORl-Al 1- or PHOR1-F5D6- related proteins, PHORl-Al 1- or PHORl-F5D6-expressing cells or extracts thereof. A PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 epitopes are generated using methods generally known in the art. Homodimeric antibodies can also be generated by cross-linking techniques known in the art (e.g., Wolff et al., Cancer Res. 53: 2560-2565).

V.) PHORl-All or PHOR1-FSD6 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.

37 la-509107 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 etal, 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,

38 la-509107 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 ->lCr-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 ^Cr-release assay involving peptide sensitized target cells and target cells expressing endogenously generated antigen.

3) Demonstration of recall T cell responses from immune individuals who have been either effectively vaccinated and/or from chronically ill patients (see, e.g., Rehermann, B. et al, J. Exp. Med. 181:1047, 1995; Doolan, D. L. et al, Immunity 7:97, 1997; Bertoni, R. et al, J. Clin. Invest. 100:503, 1997; Threlkeld, S. C. et al, J. Immunol. 159:1648, 1997; Diepolder, H. M. et al, J. Virol. 71:6011, 1997). Accordingly, recall responses are detected by culturing PBL from subjects that have been exposed to the antigen due to disease and thus have generated an immune response "naturally", or from patients who were vaccinated against the antigen. PBL from subjects are cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APC) to allow activation of "memory" T cells, as compared to

"naive" T cells. At the end of the culture period, T cell activity is detected using assays including ->lCr release involving peptide-sensitized targets, T cell proliferation, or lymphokine release.

VI.) PHORl-All or PHOR1-F5D6 Transgenic Animals Nucleic acids that encode a PHORl-Al 1- or PHORl-F5D6-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 PHORl-Al 1 or PHOR1-F5D6 can be used to clone genomic DNA that encodes PHORl-Al 1 or PHOR1- F5D6. The cloned genomic sequences can then be used to generate transgenic animals containing cells that express DNA that encode PHORl-Al 1 or PHOR1-F5D6. 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.

39 la-509107 Typically, particular cells would be targeted for PHORl-Al 1 or PHOR1-F5D6 transgene incorporation with tissue-specific enhancers.

Transgenic animals that include a copy of a transgene encoding PHORl-Al 1 or PHOR1-F5D6 can be used to examine the effect of increased expression of DNA that encodes PHORl-Al 1 or PHOR1- F5D6. 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 PHORl-All or PHOR1-F5D6 can be used to construct a PHORl-Al 1 or PHOR1-F5D6 "knock out" animal that has a defective or altered gene encoding PHORl- Al 1 or PHOR1-F5D6 as a result of homologous recombination between the endogenous gene encoding PHORl-All or PHOR1-F5D6 and altered genomic DNA encoding PHORl-Al 1 or PHOR1-F5D6 introduced into an embryonic cell of the animal. For example, cDNA that encodes PHORl-Al 1 or PHOR1-F5D6 can be used to clone genomic DNA encoding PHORl-Al 1 or PHOR1-F5D6 in accordance with established techniques. A portion of the genomic DNA encoding PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 polypeptide.

VII.) Methods for the Detection of PHORl-All or PHOR1-F5D6

40 la-509107 Another aspect of the present invention relates to methods for detecting PHORl-Al 1 or PHOR1- F5D6 polynucleotides and PHORl-Al 1- or PHORl-F5D6-related proteins, as well as methods for identifying a cell that expresses PHORl-Al 1 or PHOR1-F5D6. The expression profile of PHORl-Al 1 or PHOR1- F5D6 makes it a diagnostic marker for metastasized disease. Accordingly, the status of PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 polynucleotides in a biological sample, such as serum, bone, prostate, and other tissues, urine, semen, cell preparations, and the like. Detectable PHORl-Al 1 or PHOR1-F5D6 polynucleotides include, for example, a PHORl-Al 1 or PHOR1-F5D6 gene or fragment thereof, PHORl-Al 1 or PHOR1-F5D6 mRNA, alternative splice variant PHORl-Al 1 or PHOR1-F5D6 mRNAs, and recombinant DNA or RNA molecules that contain a PHORl-Al 1 or PHOR1-F5D6 polynucleotide, respectively. A number of methods for amplifying and/or detecting the presence of PHORl-Al 1 or PHOR1-F5D6 polynucleotides are well known in the art and can be employed in the practice of this aspect of the invention.

In one embodiment, a method for detecting a PHORl-Al 1 or PHOR1-F5D6 mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using a PHORl-Al 1 or PHOR1-F5D6 polynucleotides as sense and antisense primers to amplify PHORl-Al 1 or PHOR1-F5D6 cDNAs therein; and detecting the presence of the amplified PHORl-Al 1 or PHOR1-F5D6 cDNA. Optionally, the sequence of the amplified PHORl- Al 1 or PHOR1-F5D6 cDNA can be determined. In another embodiment, a method of detecting a PHORl-Al 1 or PHOR1-F5D6 gene in a biological sample comprises first isolating genomic DNA from the sample; amplifying the isolated genomic DNA using PHORl-Al 1 or PHOR1-F5D6 polynucleotides as sense and antisense primers; and detecting the presence of the amplified PHORl-Al 1 or PHOR1-F5D6 gene. Any number of appropriate sense and antisense probe combinations can be designed from the nucleotide sequence provided for PHORl-Al 1 or PHOR1-F5D6 (Figure 2) and used for this purpose.

The invention also provides assays for detecting the presence of a PHORl-Al 1 or PHOR1-F5D6 protein in a tissue or other biological sample such as serum, semen, bone, prostate, urine, cell preparations, and the like. Methods for detecting a PHORl-Al 1- or PHORl-F5D6-related protein are also well known and include, for example, immunoprecipitation, immunohistochemical analysis, Western blot analysis, molecular

41 la-509107 binding assays, ELISA, ELIFA and the like. For example, a method of detecting the presence of a PHORl- Al 1- or PHORl-F5D6-related protein in a biological sample comprises first contacting the sample with a PHORl-Al 1 or PHOR1-F5D6 antibody, a PHORl-Al 1- or PHORl-F5D6-reactive fragment thereof, or a recombinant protein containing an antigen binding region of a PHORl-Al 1 or PHOR1-F5D6 antibody; and then detecting the binding of PHORl-Al 1- or PHORl-F5D6-related protein in the sample.

Methods for identifying a cell that expresses PHORl-Al 1 or PHOR1-F5D6 are also within the scope of the invention. In one embodiment, an assay for identifying a cell that expresses a PHORl-Al 1 or PHOR1- F5D6 gene comprises detecting the presence of PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT- PCR using complementary primers specific for PHORl-Al 1 or PHOR1-F5D6, 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 PHORl-Al 1 or PHOR1-F5D6 gene comprises detecting the presence of PHORl-Al 1- or PHORl-F5D6-related proteins 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 PHORl-Al 1- or PHOR1- F5D6-related proteins and cells that express PHORl-Al 1- or PHORl-F5D6-related proteins.

PHORl-Al 1 or PHOR1-F5D6 expression analysis is also useful as a tool for identifying and evaluating agents that modulate PHORl-Al 1 or PHOR1-F5D6 gene expression. For example, PHORl-Al 1 - or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 expression by RT- PCR, nucleic acid hybridization or antibody binding.

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 PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-

42 la-509107 F5D6 in a biological sample of interest can be compared, for example, to the status of PHORl-All or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 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. Neural. 1996 Dec 9;376(2):306-14 and U.S. Patent No. 5,837,501) to compare PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 expressing cells) as well as the level, and biological activity of expressed gene products (such as PHORl-Al 1 or PHOR1-F5D6 mRNA, polynucleotides and polypeptides). Typically, an alteration in the status of PHORl-Al 1 or PHOR1-F5D6 comprises a change in the location of PHORl-Al 1 or PHOR1-F5D6 and/or PHORl-Al 1- or PHOR1-F5D6 -expressing cells and or an increase in PHORl-Al 1 or PHOR1-F5D6 mRNA and/or protein expression.

PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 gene), Northern analysis and/or PCR analysis of PHORl-Al 1 or PHOR1-F5D6 mRNA (to examine, for example alterations in the polynucleotide sequences or expression levels of PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 proteins and/or associations of PHORl-Al 1 or PHOR1-F5D6 proteins with polypeptide binding partners). Detectable PHORl-Al 1 or PHOR1-F5D6 polynucleotides include, for example, a PHORl-All or PHOR1-F5D6 gene or fragment thereof, PHORl-Al 1 or PHOR1- F5D6 mRNA, alternative splice variants, PHORl-Al 1 or PHOR1-F5D6 mRNAs, and recombinant DNA or RNA molecules containing a PHORl-All or PHOR1-F5D6 polynucleotide.

43 la-509107 The expression profile of PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 provides information useful for predicting susceptibility to particular disease stages, progression, and/or tumor aggressiveness. The invention provides methods and assays for determining PHORl-Al 1 or PHOR1-F5D6 status and diagnosing cancers that express PHORl-Al 1 or PHOR1-F5D6, such as cancers of the tissues listed in Table I. For example, because PHORl- Al 1 or PHOR1-F5D6 mRNA is so highly expressed in prostate and other cancers relative to normal prostate tissue, assays that evaluate the levels of PHORl-Al 1 or PHOR1-F5D6 mRNA transcripts or proteins in a biological sample can be used to diagnose a disease associated with PHORl-Al 1 or PHOR1-F5D6 dysregulation, and can provide prognostic information useful in defining appropriate therapeutic options.

The expression status of PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 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 PHORl-All or PHOR1-F5D6 in a biological sample can be examined by a number of well-known procedures in the art. For example, the status of PHORl-All or PHOR1-F5D6 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 PHORl-All or PHOR1-F5D6 expressing cells (e.g. those that express PHORl-Al 1 or PHOR1-F5D6 mRNAs or proteins). This examination can provide evidence of dysregulated cellular growth, for example, when PHORl-All- or PHORl-F5D6-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 PHORl-Al 1 or PHOR1-F5D6 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 l):474-8).

In one aspect, the invention provides methods for monitoring PHORl-Al 1 or PHOR1-F5D6 gene products by determining the status of PHORl-Al 1 or PHOR1-F5D6 gene products expressed by cells from

44 la-509107 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 PHORl-All or PHOR1-F5D6 gene products in a corresponding normal sample. The presence of aberrant PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 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 PHORl-All or PHOR1-F5D6 mRNA can, for example, be evaluated in tissue samples including but not limited to those listed in Table I. The presence of significant PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 mRNA or express it at lower levels.

In a related embodiment, PHORl-Al 1 or PHOR1-F5D6 status is determined at the protein level rather than at the nucleic acid level. For example, such a method comprises determining the level of PHORl- Al 1 or PHOR1-F5D6 protein expressed by cells in a test tissue sample and comparing the level so determined to the level of PHORl-Al 1 or PHOR1-F5D6 expressed in a corresponding normal sample. In one embodiment, the presence of PHORl-Al 1 or PHOR1-F5D6 protein is evaluated, for example, using immunohistochemical methods. PHORl-Al 1 or PHOR1-F5D6 antibodies or binding partners capable of detecting PHORl-Al 1 or PHOR1-F5D6 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 PHORl-All or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 may be indicative of the presence or promotion of a tumor. Such assays therefore have diagnostic and predictive value where a mutation in PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 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

45 la-509107 sequences such as single strand conformation polymorphism analysis are well known in the art (see, e.g., U.S.¹ 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 PHORl-Al 1 or PHOR1-F5D6 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 PHORl-All orPHORl- F5D6. ■ Gene amplification is measured in a sample directly, for example, by conventional Southern blotting or Northern blotting to quantitate the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad. Sci. USA, 77:5201-5205), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies are employed that recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn are labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.

Biopsied tissue or peripheral blood can be conveniently assayed for the presence of cancer cells using for example, Northern, dot blot or RT-PCR analysis to detect PHORl-Al 1 or PHOR1-F5D6 expression. The presence of RT-PCR amplifiable PHORl-Al 1 or PHOR1-F5D6 mRNA provides an indication of the presence

46 la-509107 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 PHORl-Al 1 or PHOR1-F5D6 mRNA or PHORl-Al 1 or PHOR1-F5D6 protein in a tissue sample, its presence indicating susceptibility to cancer, wherein the degree of PHORl-All or PHOR1-F5D6 mRNA expression correlates to the degree of susceptibility. In a specific embodiment, the presence of PHORl-Al 1 or PHOR1-F5D6 in prostate or other tissue is examined, with the presence of PHORl-Al 1 or PHOR1-F5D6 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 of PHORl-All or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1- F5D6 mRNA or PHORl-Al 1 or PHOR1-F5D6 protein expressed by tumor cells, comparing the level so determined to the level of PHORl-Al 1 or PHOR1-F5D6 mRNA or PHORl-Al 1 or PHOR1-F5D6 protein expressed in a corresponding normal tissue taken from the same individual or a normal tissue reference sample, wherein the degree of PHORl-Al 1 or PHOR1-F5D6 mRNA or PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHORlrF5D6 is expressed in the tumor cells, with higher expression levels indicating more aggressive < tumors. Another embodiment is the evaluation of the integrity of PHORl-Al 1 or PHOR1-F5D6 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 PHORl-All orPHORl-F5D6 mRNA or PHORl-Al 1 or PHOR1-F5D6 protein expressed by cells in a sample of the tumor, comparing the

47 la-509107 level so determined to the level of PHORl-Al 1 or PHOR1-F5D6 mRNA or PHORl-Al 1 or PHOR1-F5D6 protein expressed in an equivalent tissue sample taken from the same individual at a different time, wherein the degree of PHORl-Al 1 or PHOR1-F5D6 mRNA or PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer. Also, one can evaluate the integrity PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 gene and PHORl-All orPHORl-F5D6 gene products (or perturbations in PHORl-All or PHOR1-F5D6 gene and PHORl-Al 1 or PHOR1-F5D6 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. ll(6):543-51; Baisden et al., 1999, Am. J. Surg. Pathol. 23(8):918-24). Methods for observing a coincidence between the expression of PHORl-Al 1 or PHOR1-F5D6 gene and PHORl-Al 1 or PHOR1-F5D6 gene products (or perturbations in PHORl-Al 1 or PHOR1-F5D6 gene and PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or

PHOR1-F5D6 gene and PHORl-All or PHOR1-F5D6 gene products (or perturbations in PHORl-All or PHOR1-F5D6 gene and PHORl-Al 1 or PHOR1-F5D6 gene products) and another factor associated with malignancy entails detecting the overexpression of PHORl-Al 1 or PHOR1-F5D6 mRNA or protein in a tissue sample, detecting the overexpression of PSA mRNA or protein in a tissue sample (or PSCA or PSM expression), and observing a coincidence of PHORl-Al 1 or PHOR1-F5D6 mRNA or protein and PSA mRNA or protein overexpression (or PSCA or PSM expression). In a specific embodiment, the expression of PHORl- Al 1 or PHOR1-F5D6 and PSA mRNA in prostate tissue is examined, where the coincidence of PHORl-Al 1 or PHOR1-F5D6 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.

48 la-509107 Methods for detecting and quantifying the expression of PHORl-Al 1 or PHOR1-F5D6 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 PHORl-All orPHORl-F5D6 mRNA include in situ hybridization using labeled PHORl-Al 1 or PHOR1-F5D6 riboprobes, Northern blot and related techniques using PHORl-Al 1 or PHOR1-F5D6 polynucleotide probes, RT-PCR analysis using primers specific for PHORl-Al 1 or PHOR1-F5D6, and other amplification type detection methods, such as, for example, branched DNA, SISB A, TMA and the like. In a specific embodiment, semi-quantitative RT-PCR is used to detect and quantify PHORl-Al 1 or PHOR1-F5D6 mRNA expression. Any number of primers capable of amplifying PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 protein can be used in an immunohistochemical assay of biopsied tissue.

IX.) Identification of Molecules That Interact With PHORl-All or PHOR1-F5D6 . The PHORl-All or PHOR1-F5D6 protein and nucleic acid sequences disclosed herein allow a skilled artisan to identify proteins, small molecules and other agents that interact with PHORl-All or PHOR1-F5D6, as well as pathways activated by PHORl-Al 1 or PHOR1-F5D6 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 PHORl-All or PHOR1-F5D6 protein sequences. In such methods, peptides that bind to a molecule such as PHORl-Al 1 or PHOR1-F5D6 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

49 la-509107 that interact with PHORl-Al 1 or PHOR1-F5D6 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 PHORl-All or PHOR1-F5D6 are used to identify protein- protein interactions mediated by PHORl-Al 1 or PHOR1-F5D6. Such interactions can be examined using immunoprecipitation techniques (see, e.g., Hamilton BJ, et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51). PHORl-All or PHOR1-F5D6 protein can be immunoprecipitated from PHORl-Al l- or PHORl-F5D6-expressing cell lines using anti-PHORl-Al 1 or PHOR1-F5D6 antibodies. Alternatively, antibodies against His-tag can be used in a cell line engineered to express PHORl-Al 1 or PHOR1-F5D6 (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 PHORl-All or PHOR1-F5D6 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 PHORl-All's or PHORl-F5D6'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 PHORl-Al 1 or PHOR1-F5D6 are identified and used to treat patients that have a cancer that expresses the PHORl-Al 1 or PHOR1-F5D6 antigen (see, e.g., Hille, B., Ionic Channels of Excitable Membranes 2^nd Ed., Sinauer Assoc, Sunderland, MA, 1992). Moreover, ligands that regulate PHORl-Al 1 or PHOR1-F5D6 function can be identified based on their ability to bind PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6.

An embodiment of this invention comprises a method of screening for a molecule that interacts with a PHORl-Al 1 or PHOR1-F5D6 amino acid sequence shown in Figure 2 or Figure 3, comprising the steps of contacting a population of molecules with the PHORl-Al 1 or PHOR1-F5D6 amino acid sequence, allowing the population of molecules and the PHORl-Al 1 or PHOR1-F5D6 amino acid sequence to

50 la-509107 interact under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the PHORl-Al 1 or PHOR1-F5D6 amino acid sequence, and then separating molecules that do not interact with the PHORl-Al 1 or PHOR1-F5D6 amino acid sequence from molecules that do. In a specific embodiment, the method further comprises purifying a molecule that interacts with the PHORl-Al 1 or PHOR1-F5D6 amino acid sequence. The identified molecule can be used to modulate a function performed by PHORl-Al 1 or PHOR1-F5D6. In a preferred embodiment, the PHORl-Al 1 or PHOR1- F5D6 amino acid sequence is contacted with a library of peptides.

X.) Therapeutic Methods and Compositions The identification of PHORl-Al 1 or PHOR1-F5D6 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 PHORl- Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 protein are useful for patients suffering from a cancer that expresses PHORl-Al 1 or PHOR1-F5D6. These therapeutic approaches generally fall into two classes. One class comprises various methods for inhibiting the binding or association of the PHORl-Al 1 or PHOR1-F5D6 protein with its binding partner or with other proteins. Another class comprises a variety of methods for inhibiting the transcription of the PHOR1- Al 1 or PHOR1-F5D6 gene or translation of PHORl-Al 1 or PHOR1-F5D6 mRNA. X.A.) Anti-Cancer Vaccines

The invention further provides cancer vaccines comprising a PHORl-Al 1- or PHORl-F5D6-related protein or PHORl-Al 1- or PHORl-F5D6-related nucleic acid. In view of the expression pattern of PHORl- Al 1 or PHOR1-F5D6, cancer vaccines prevent and/or treat PHORl-All- or PHORl-F5D6-exρressing 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 PHORl-Al 1- or PHORl-F5D6-related protein, or a PHORl-Al 1- or PHORl-F5D6-encoding nucleic acid molecule and recombinant vectors capable of expressing and presenting the PHORl-Al 1 or PHOR1-F5D6 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(l):66-78; Maruyama et al., Cancer Immunol Immunother 2000 Jun 49(3):123-32)

51 la-509107 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 PHORl-Al 1 or PHOR1-F5D6 protein shown in FIGURE 2 or analog or homolog thereof) so that the mammal generates an immune response that is specific for that epitope (e.g. generates antibodies that specifically recognize that epitope). In a preferred method, the PHORl-Al 1 or PHOR1- F5D6 immunogen contains a biological motif, see e.g., Tables V-XIX, or a peptide of a size range from PHORl-All or PHOR1-F5D6 indicated in Figure 14, Figure 15, Figure 16, Figure 17, or Figure 18.

The entire PHORl-All or PHOR1-F5D6 protein, immunogenic regions or epitopes thereof can be combined and delivered by various means. Such vaccine compositions can include, for example, lipopeptides (e.g., Vitiello, A. et al, J. Clin. Invest. 95:341, 1995), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) ("PLG") microspheres (see, e.g., Eldridge, et al., Molec. Immunol. 28:287- 294, 1991: Alonso et al, Vaccine 12:299-306, 1994; Jones et al, Vaccine 13:675-681, 1995), peptide compositions contained in immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al, Nature. 344:873-875, 1990; Hu et al. , Clin Exp Immunol. 113:235-243, 1998), multiple antigen peptide systems (MAPs) (see e.g., Tam, J. P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J.P., J. Immunol Methods 196:17-32, 1996), peptides formulated as multivalent peptides; peptides for use in ballistic delivery systems, typically crystallized peptides, viral delivery vectors (Perkus, M. E. et al, In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p. 379, 1996; Chakrabarti, S. et al, Nature 320:535, 1986; Hu, S. L. et al, Nature 320:537, 1986; Kieny, M.-P. et al, A!DS Bio Technology 4:790, 1986; Top, F. H. et al, J. Infect. Dis. 124:148, 1971; Chanda, P. K. et al, Virology 175:535, 1990), particles of viral or synthetic origin (e.g., Kofler, N. et al, J. Immunol. Methods. 192:25, 1996; Eldridge, J. H. et al, Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. et al, Nature Med. 7:649, 1995), adjuvants (Warren, H. S., Vogel, F. R., and Chedid, L. A. Annu. Rev. Immunol. 4:369, 1986; Gupta, R. K. et al, Vaccine 11:293, 1993), liposomes (Reddy, R. et al, J. Immunol 148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or, naked or particle absorbed cDNA (Ulmer, J. B. etal, 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 PHORl-All- or PHORl-F5D6-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.

52 la-509107 . Cellular Vaccines:

CTL epitopes can be determined using specific algorithms to identify peptides within PHORl-Al 1 or PHOR1-F5D6 protein that bind corresponding HLA alleles (see e.g., Table IV; Epimer™ and Epimatrix™, Brown University (URL www.brown.edvj/Research TB-HIV_Lab/epimatiix epimatrix.html); and, BDVLAS, (URL bimas.dcrt.nih. gov/; SYFPEITHI at URL syφeithi.bmi-heidelberg.com/). In a preferred embodiment, the PHORl-Al 1 or PHOR1-F5D6 immunogen contains one or more amino acid sequences identified using techniques well known in the art, such as the sequences shown in Tables V-XVIII or a peptide of 8, 9, 10 or 11 amino acids specified by an HLA Class I motif/supermotif (e.g., Table IV (A), Table IV (D), or Table IV (E)) and/or a peptide of at least 9 amino acids that comprises an HLA Class II motif/supermotif (e.g., Table IV (B) or Table IV (C)). As is appreciated in the art, the HLA Class I binding groove is essentially closed ended so that peptides of only a particular size range can fit into the groove and be bound, generally HLA Class I epitopes are 8, 9, 10, or 11 amino acids long. In contrast, the HLA Class II binding groove is essentially open ended; therefore a peptide of about 9 or more amino acids can be bound by an HLA Class II molecule. Due to the binding groove differences between HLA Class I and II, HLA Class I motifs are length specific, i.e., position two of a Class I motif is the second amino acid in an amino to carboxyl direction of the peptide. The amino acid positions in a Class II motif are relative only to each other, not the overall peptide, i.e., additional amino acids can be attached to the amino and/or carboxyl termini of a motif- bearing sequence. HLA Class II epitopes are often 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids long, or longer than 25 amino acids. Antibody-based Vaccines

A wide variety of methods for generating an immune response in a mammal are known in the art (for example as the first step in the generation of hybridomas). Methods of generating an immune response in a mammal comprise exposing the mammal's immune system to an immunogenic epitope on a protein (e.g. the PHORl-Al 1 or PHOR1-F5D6 protein) so that an immune response is generated. A typical embodiment consists of a method for generating an immune response to PHORl-Al 1 or PHOR1-F5D6 in a host, by contacting the host with a sufficient amount of at least one PHORl-Al 1 or PHOR1-F5D6 B cell or cytotoxic T-cell epitope or analog thereof; and at least one periodic interval thereafter re-contacting the host with the PHORl-Al 1 or PHOR1-F5D6 B cell or cytotoxic T-cell epitope or analog thereof. A specific embodiment consists of a method of generating an immune response against a PHORl-Al 1- or PHOR1- F5D6-related protein or a man-made multiepitopic peptide comprising: administering PHORl-Al 1 or

PHOR1-F5D6 immunogen (e.g. the PHORl-Al 1 or PHOR1-F5D6 protein or a peptide fragment thereof, a PHORl-Al 1 or PHOR1-F5D6 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 PADRE™ peptide (Epimmune Inc., San Diego, CA;

53 la-509107 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 PHORl-Al 1 or PHOR1-F5D6 immunogen by: administering in vivo to muscle or skin of the individual's body a DNA molecule that comprises a DNA sequence that encodes a PHORl-Al 1 or PHOR1-F5D6 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 Vaccines:

Vaccine compositions of the invention include nucleic acid-mediated modalities. DNA or RNA that encode ρrotein(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 PHORl-Al 1 or PHOR1-F5D6. Constructs comprising DNA encoding a PHORl-Al 1- or PHORl-F5D6-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 PHORl-Al 1 or PHOR1-F5D6 protein/immunogen. Alternatively, a vaccine comprises a PHORl-Al 1- or PHORl-F5D6-related protein. Expression of the PHORl-Al 1- or PHORl-F5D6-related protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells that bear PHORl-Al 1 or PHOR1-F5D6 protein, respectively. 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 e 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

54 la-509107 introducing naked DNA encoding a PHORl-Al 1- or PHORl-F5D6-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 etal, Nature 351:456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g. adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled in the art from the description herein.

Thus, gene delivery systems are used to deliver a PHORl-Al 1- or PHORl-F5D6-related nucleic acid molecule. In one embodiment, the full-length huma PHORl-Al 1 or PHOR1-F5D6 cDNA is employed. In another embodiment, PHORl-All or PHOR1-F5D6 nucleic acid molecules encoding specific cytotoxic T i lymphocyte (CTL) and/or antibody epitopes are employed. Ex Vivo Vaccines

Various ex vivo strategies can also be employed to generate an immune response. One approach involves the use of antigen presenting cells (APCs) such as dendritic cells (DC) to present PHORl-All or' PHOR1-F5D6 antigen to a patient's immune system. Dendritic cells express MHC class I and II molecules, B7 co-stimulator, and JL-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 PHORl-Al 1 or PHOR1-F5D6 peptides to T cells in the context of MHC class I or II molecules. In one embodiment, autologous dendritic cells are pulsed with PHORl-Al 1 or PHOR1-F5D6 peptides capable of binding to MHC class I and/or class II molecules. In another embodiment, dendritic cells are pulsed with the complete PHORl-Al 1 or PHOR1-F5D6 protein. Yet another embodiment involves engineering the overexpression of the PHORl-All or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 can also be engineered to express immune modulators, such as GM-CSF, and used as immunizing agents. X.B.) PHORl-All or PHOR1-F5D6 as a Target for Antibody-based Therapy

55 la-509107 PHORl-All and PHOR1-F5D6 are attractive targets 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 PHORl-All or PHOR1-F5D6 is expressed by cancer cells of various lineages relative to corresponding normal cells, systemic administration of PHORl-Al 1- or PHORl-F5D6-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 PHORl-All orPHORl-F5D6 are useful to treat PHORl-Al l- or PHORl-F5D6- expressing cancers systemically, either as conjugates with a toxin or therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.

PHORl-Al 1 or PHOR1-F5D6 antibodies can be introduced into a patient such that the antibody binds to PHORl-Al 1 or PHOR1-F5D6 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 PHORl- Al 1 or PHOR1-F5D6, inhibition of ligand binding or signal tiansduction 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 PHORl-All or PHORl -F5D6 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. PHORl-All or PHOR1-F5D6), 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-PHORl-Al 1 or PHOR1-F5D6 antibody) that binds to a marker (e.g. PHORl-All or PHOR1-F5D6) 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 PHORl-Al 1 or PHOR1-F5D6, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a PHORl-Al 1 or PHOR1-F5D6 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

56 la-509107 pharmaceutical composition comprising a therapeutically effective amount of an antibody conjugated to a cytotoxic and/or therapeutic agent.

Cancer immunotherapy using anti-PHORl-Al 1 or PHOR1-F5D6 antibodies can be done in accordance with various approaches that have been successfully employed in the treatment of other types of cancer, including but not limited to colon cancer (Arlen et al., 1998, Crit. Rev. Immunol. 18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari et al., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992, Cancer Res. 52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J. Immunother. Emphasis Tumor Immunol. 19:93-101), leukemia (Zhong et al., 1996, Leuk. Res. 20:581-589), colorectal cancer (Moun et al., 1994, Cancer Res. 54:6160-6166; Velders et al., 1995, Cancer Res. 55:4398-4403), and breast cancer (Shepard et al., 1991, J. Clin. Immunol. 11:117-127). Some therapeutic approaches involve conjugation of naked antibody to a toxin, such as the conjugation of Y⁹¹ or I¹³¹ to anti-CD20 antibodies (e.g., Zevalin™, IDEC Pharmaceuticals Corp. or Bexxar™, Coulter Pharmaceuticals), while others involve co-administration of antibodies and other therapeutic agents, such as Herceptin™ (trastuzumab) with paclitaxel (Genentech, Inc.). To treat prostate cancer, for example, PHORl-Al 1 or PHOR1-F5D6 antibodies can be administered in conjunction with radiation, chemotherapy or hormone ablation.

Although PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 expression, preferably using immunohistochemical assessments of tumor tissue, quantitative PHORl-Al 1 or PHOR1-F5D6 imaging, or other techniques that reliably indicate the presence and degree of PHORl- Al 1 or PHOR1-F5D6 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. Aήti-PHORl-Al 1 or PHOR1-F5D6 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-PHORl-Al 1 or PHOR1-F5D6 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

57 la-509107 receptor sites on complement proteins. In addition, anti-PHORl-Al 1 or PHOR1-F5D6 mAbs that exert a direct biological effect on tumor growth are useful to treat cancers that express PHORl-Al 1 or PHOR1- F5D6. 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-PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 antigen with high affinity but exhibit low or no antigenicity in the patient. Therapeutic methods of the invention contemplate the administration of single anti-PHORl-Al 1 or PHOR1-F5D6 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- PHORl-Al 1 or PHOR1-F5D6 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-PHORl-Al 1 or PHOR1-F5D6 mAbs are administered in their "naked" or unconjugated form, or can have a therapeutic agent(s) conjugated to them:

Anti-PHORl-Al 1 or PHOR1-F5D6 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-PHORl-Al 1 or PHOR1-F5D6 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- PHORl-Al 1 or PHOR1-F5D6 mAb preparation represents an acceptable dosing regimen. Preferably, the initial loading dose is administered as a 90 minute or longer infusion. The

58 la-509107 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 PHORl-Al 1 or PHOR1-F5D6 expression in the patient, the extent of circulating shed PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 in a given sample (e.g. the levels of circulating PHORl-All or PHOR1-F5D6 antigen and/or PHORl-Al 1 or PHOR1-F5D6 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-PHORl-Al 1 or PHOR1-F5D6 antibodies can also be used in anti-cancer therapy as a vaccine for inducing an immune response to cells expressing a PHORl-Al 1- or PHOR1-

F5D6-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-PHORl-Al 1 or PHOR1-F5D6 antibodies that mimic an epitope on a PHORl-Al 1- or PHORl-F5D6-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.) PHORl-All or PHOR1-F5D6 as a Target for Cellular Immune Responses Vaccines and methods of preparing vaccines that contain an immunogenically effective amount of one or more HLA-biήding 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

59 la-509107 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 PHORl-Al 1 or PHOR1-F5D6 antigen, or derives at least some therapeutic benefit when the antigen was tumor-associated. In some embodiments, it may be desirable to combine the class I peptide components with components that induce or facilitate neutralizing antibody and or helper T cell responses directed to the ' target antigen. A preferred embodiment of such a composition comprises class I and class II epitopes in accordance with the invention. An alternative embodiment of such a composition comprises a class I and/or class II epitope in accordance with the invention, along with a cross reactive HTL epitope such as PADRE™ (Epimmune, San Diego, 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.

60 la-509107 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 IC₅₀ of 500 nM or less, often 200 nM or less; and for Class II an IC₅₀ of 1000 nM or less. 3.) Sufficient supermotif bearing-peptides, or a sufficient array of allele-specific motif- bearing peptides, are selected to give broad population coverage. For example, it is preferable to have at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be employed to assess the breadth, or redundancy of, population coverage.

4.) When selecting epitopes from cancer-related antigens it is often useful to select analogs because the patient may have developed tolerance to the native epitope.

5.) Of particular relevance are epitopes referred to as "nested epitopes." Nested epitopes occur where at least two epitopes overlap in a given peptide sequence. A nested peptide sequence can comprise B cell, HLA class I and/or HLA class II epitopes. When providing nested epitopes, a general objective is to provide the greatest number of epitopes per sequence. Thus, an aspect is to avoid providing a peptide that is any longer than the amino terminus of the amino terminal epitope and the carboxyl terminus of the carboxyl terminal epitope in the peptide. When providing a multi-epitopic sequence, such as a sequence comprising nested epitopes, it is generally important to screen the sequence in order to insure that it does not have pathological or other deleterious biological properties.

6.) If a polyepitopic protein is created, or when creating a minigene, an objective is to generate the smallest peptide that encompasses the epitopes of interest. This principle is similar, if not the same as that employed when selecting a peptide comprising nested epitopes. However, with an artificial polyepitopic peptide, the size minimization objective is balanced against the need to integrate any spacer sequences between epitopes in the polyepitopic protein. Spacer amino acid residues can, for example, be introduced to avoid junctional epitopes (an epitope recognized by the immune system, not present in the target antigen, and only created by the marinade 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.

61 la-509107 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 PHORl-Al 1 or PHOR1-F5D6, the PADRE® universal helper T cell epitope (or multiple HTL ' epitopes from PHORl-Al 1 or PHOR1-F5D6), 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 tp guide the codon choice for each amino acid. These epitope-encoding DNA sequences may be directly adjoined, so that when translated, a continuous polypeptide sequence is created. To optimize expression and/or immunogenicity, additional elements can be incorporated into the minigene design. Examples of amino acid sequences that can be reverse translated and included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes, antibody epitopes, a ubiquitination signal sequence, and or an endoplasmic reticulum targeting signal. In addition, HLA presentation of CTL and HTL epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking

62 la-509107 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., JL-2, EL-12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), costimulatory molecules, or for HTL responses, pan-DR binding proteins (PADRE™, 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

63 la-509107 epitopes into the HLA class II pathway, thereby improving HTL induction. In contrast to HTL or CTL induction, specifically decreasing the immune response by co-expression of immunosuppressive molecules (e.g. TGF-β) may be beneficial in certain diseases.

Therapeutic quantities of plasmid DNA can be produced for example, by fermentation in E. coli, followed by purification. Aliquots from the working cell bank are used to inoculate growth medium, and grown to saturation in shaker flasks or a bioreactor according to well-known techniques. Plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion-exchange resins supplied by QIAGEN, Inc. (Valencia, 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 Feigner, et al, Proc. Nat'l Acad. Sci. USA 84:7413 (1987). In addition, peptides and compounds referred to collectively as protective, interactive, non- condensing compounds (PINC) could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.

Target cell sensitization can be used as a functional assay for expression and HLA class I presentation of minigene-encoded CTL epitopes. For example, the plasmid DNA is introduced into a mammalian cell line that is suitable as a target for standard CTL chromium release assays. The transfection method used will be dependent on the final formulation. Electroporation can be used for "naked" DNA, whereas cationic lipids allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS). These cells are then chromium-51 (⁵¹Cr) labeled and used as target cells for epitope- specific CTL lines; cytolysis, detected by ⁵¹Cr release, indicates both production of, and HLA presentation of, minigene-encoded CTL epitopes. Expression of HTL epitopes may be evaluated in an analogous manner using assays to assess HTL activity.

In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations. Transgenic mice expressing appropriate human HLA proteins are immunized with the DNA product. The dose and route of administration are formulation dependent (e.g., IM for DNA in PBS, intraperitoneal (i.p.) for lipid-complexed DNA). Twenty-one days after immunization, splenocytes are

64 la-509107 harvested and restimulated for one week in the presence of peptides encoding each epitope being tested. Thereafter, for CTL effector cells, assays are conducted for cytolysis of peptide-loaded, ⁵¹Cr-labeled target cells using standard techniques. Lysis of target cells that were sensitized by HLA loaded with peptide epitopes, corresponding to minigene-encoded epitopes, demonstrates DNA vaccine function for in vivo induction of CTLs. Immunogenicity of HTL epitopes is 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 (QYIKANSKFIGITE; SEQ ID NO: ), Plasmodiumfalciparum circumsporozoite (CS) protein at positions 378-398 (DIEKKIAKMEKASSVFNVVNS; SEQ ID NO: ), and Streptococcus 18kD protein at positions

65 la-509107 116-131 (GAVDSB GGVATYGAA; SEQ ID NO: ). 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: ), where "X" is either cyclohexylalanine, phenylalanine, or tyrosine, and a is either D-alanine or L-alanine, has been found to bind to most HLA-DR alleles, and to stimulate the response of T helper lymphocytes from most individuals, regardless of their HLA type. An alternative of a pan-DR binding epitope comprises all "L" natural amino acids and can be provided in the form of nucleic acids that encode the epitope.

HTL peptide epitopes can also be modified to alter their biological properties. For example, they can be modified to include D-amino acids to increase their resistance to proteases and thus extend their serum half life, or they can be conjugated to other molecules such as lipids, proteins, carbohydrates, and the like to increase their biological activity. For example, a T helper peptide can be conjugated to one or more palmitic acid chains at either the amino or carboxyl termini.

X.C.3. Combinations of CTL Peptides with T Cell Priming Agents In some embodiments it may be desirable to include in the pharmaceutical compositions of the invention at least one component which primes B lymphocytes or T lymphocytes. Lipids have been identified as agents capable of priming CTL in vivo. For example, palmitic acid residues can be attached to the ε-and α- amino groups of a lysine residue and then linked, e.g., via one or more linking residues such as Gly, Gly-Gly-, Ser, Ser-Ser, or the like, to an immunogenic peptide. The lipidated peptide can then be administered either directly in a micelle or particle, incorporated into a liposome, or emulsified in an adjuvant, e.g., incomplete Freund's adjuvant. In a preferred embodiment, a particularly effective immunogenic composition comprises palmitic acid attached to ε- and α- amino groups of Lys, which is attached via linkage, e.g., Ser-Ser, to the amino terminus of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl- S-glycerylcysteinlyseryl- serine (P₃CSS) can be used to prime virus specific CTL when covalenfly 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

66 la-509107 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 Progenipoietin™ (Pharmacia-Monsanto, St. Louis, MO) or GM-CSF/TL-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 PHORl-Al 1 or PHOR1-F5D6. 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 PHORl-Al 1 or PHOR1-F5D6.

X.D. Adoptive Immunotherapy

Antigenic PHORl-Al 1- or PHORl-F5D6-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 PHORl-Al 1 or PHOR1-F5D6. 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

67 la-509107 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 PHORl-Al 1 or PHOR1-F5D6. 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 PHORl-Al 1- or PHORl-F5D6-associated cancer. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter. The embodiment of the vaccine composition (i.e., including, but not limited to embodiments such as peptide cocktails, polyepitopic polypeptides, minigenes, or TAA-specific CTLs or pulsed dendritic cells) delivered to the patient may vary according to the stage of the disease or the patient's health status. For example, in a patient with a tumor that expresses PHORl- Al 1 or PHOR1-F5D6, a vaccine comprising PHORl-All- or PHORl-F5D6-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 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. Boosting dosages of between about 1.0 μg to about 50,000 μg of peptide pursuant to a boosting regimen over weeks to months may be administered depending upon the patient's response and condition as determined by measuring the specific activity of CTL and HTL obtained from the patient's blood. Administration should continue until at least clinical symptoms or laboratory tests indicate that the neoplasia, has been eliminated or reduced and for a period thereafter. The dosages, routes of administration, and dose schedules are adjusted in accordance with methodologies known in the art.

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

The vaccine compositions of the invention can also be used purely as prophylactic agents. Generally the dosage for an initial prophylactic immunization generally occurs in a unit dosage range

68 la-509107 where the lower value is about 1, 5, 50, 500, or 1000 μg and the higher value is about 10,000; 20,000; 30,000; or 50,000 μg. Dosage values for a human typically range from about 500 μg to about 50,000 μg per 70 kilogram patient. This is followed by boosting dosages of between about 1.0 μg to about 50,000 μg of peptide administered at defined intervals from about four weeks to six months after the initial administration of vaccine. The immunogenicity of the vaccine can be assessed by measuring the specific activity of CTL and HTL obtained from a sample of the patient's blood.

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

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

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

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

A human unit dose form of 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^h 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

69 la-509107 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, e.g., lecithin for intranasal delivery.

XI.) Diagnostic and Prognostic Embodiments of PHORl-All or PHOR1-F5D6.

70 la-509107 As disclosed herein, PHORl-All or PHOR1-F5D6 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). PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 polynucleotides and polypeptides (as well as the PHORl-All or PHORl-F5D6 polynucleotide probes and anti-PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 polynucleotides described herein can be utilized in the same way to detect PHORl-Al 1 or PHOR1-F5D6 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 PHOR1- Al 1 or PHOR1-F5D6 polypeptides described herein can be utilized to generate antibodies for use in detecting PHORl-Al 1 or PHOR1-F5D6 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

71 la-509107 examine a biological sample for the presence of cells expressing PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1- or PHORl-F5D6-expressing cells (lymph node) is found to contain PHORl-Al 1- or PHORl-F5D6-expressing cells such as the PHOR1- Al 1 or PHOR1-F5D6 expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis.

Alternatively PHORl-All or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 or express PHORl-Al 1 or PHOR1-F5D6 at a different level are found to express PHORl-Al 1 or PHOR1-F5D6 or have an increased expression of PHORl-Al 1 or PHOR1-F5D6 (see, e.g., the PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6) 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, PHORl-All or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 polynucleotide fragment is used as a probe to show the expression of PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 polynucleotide shown in FIGURE 2) under conditions of high stringency.

72 la-509107 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. PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 polypeptide shown in FIGURE 2).

As shown herein, the PHORl-Al 1 or PHOR1-F5D6 polynucleotides and polypeptides (as well as the PHORl-Al 1 or PHOR1-F5D6 polynucleotide probes and anti-PHORl-Al 1 or PHOR1-F5D6 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 PHORl-All or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 polynucleotides and polypeptides (as well as the PHORl- Al 1 or PHOR1-F5D6 polynucleotide probes and anti-PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 genes map (see Example 3 below). Moreover, in addition to their use in diagnostic assays, the PHORl-Al 1- or PHORl-F5D6-related proteins and polynucleotides disclosed herein

73 la-509107 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(l-2): 63-9).

Additionally, PHORl-All- or PHORl-F5D6-related proteins or polynucleotides of the invention can be used to treat a pathologic condition characterized by the over-expression of PHORl-Al 1 or PHOR1-F5D6. 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 PHORl-Al 1 or PHORl -F5D6 antigen. Antibodies or other molecules that react with PHORl-Al 1 or PHOR1-F5D6 can be used to modulate the function of this molecule, and thereby provide a therapeutic benefit.

XII.) Inhibition of PHORl-All or PHOR1-F5D6 Protein Function

The invention includes various methods and compositions for inhibiting the binding of PHORl- Al 1 or PHOR1-F5D6 to its binding partner or its association with other protein(s) as well as methods for inhibiting PHORl-Al 1 or PHOR1-F5D6 function.

XII.A.) Inhibition of PHORl-All or PHOR1-F5D6 With Intracellular Antibodies In one approach, a recombinant vector that encodes single chain antibodies that specifically bind to PHORl-Al 1 or PHOR1-F5D6 are introduced into PHORl-Al 1 or PHOR1-F5D6 expressing cells via gene transfer technologies. Accordingly, the encoded single chain anti-PHORl-Al 1 or PHOR1-F5D6 antibody is expressed intracellularly, binds to PHORl-Al 1 or PHOR1-F5D6 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, TDBTECH 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

74 la-509107 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 PHORl-All or PHOR1-F5D6 in the nucleus, thereby preventing its activity within the nucleus. Nuclear targeting signals are engineered into such

PHORl-Al 1 or PHOR1-F5D6 intrabodies in order to achieve the desired targeting. Such PHORl-Al 1 or PHOR1-F5D6 intrabodies are designed to bind specifically to a particular PHORl-Al 1 or PHOR1-F5D6 domain. In another embodiment, cytosolic intrabodies that specifically bind to the PHORl-All or PHOR1-F5D6 protein are used to prevent PHORl-Al 1 or PHOR1-F5D6 from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus (e.g., preventing PHORl-All or PHOR1-F5D6 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 PHORl-All or PHOR1-F5D6 with Recombinant Proteins In another approach, recombinant molecules bind to PHORl-Al 1 or PHOR1-F5D6 and thereby inhibit PHORl-Al 1 or PHOR1-F5D6 function. For example, these recombinant molecules prevent or inhibit PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 specific antibody molecule. In a particular embodiment, the PHORl-Al 1 or PHOR1-F5D6 binding domain of a PHORl-Al 1 or PHOR1-F5D6 binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two PHORl-Al 1 or PHOR1-F5D6 ligand binding domains linked to the Fc portion of a human IgG, such as human IgGl . Such IgG portion can contain, for example, the C_H2 and C_H3 domains and the hinge region, but not the C_H1 domain. Such dimeric fusion proteins are administered in soluble form to patients suffering from a cancer associated with the expression of PHORl-Al 1 or PHOR1- F5D6, whereby the dimeric fusion protein specifically binds to PHORl-Al 1 or PHOR1-F5D6 and blocks PHORl-Al 1 or PHOR1-F5D6 interaction with a binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody linking technologies.

XII.C.) Inhibition of PHORl-All or PHOR1-F5D6 Transcription or Translation The present invention also comprises various methods and compositions for inhibiting the transcription of the PHORl-Al 1 or PHOR1-F5D6 gene. Similarly, the invention also provides methods and compositions for inhibiting the translation of PHORl-All or PHOR1-F5D6 mRNA into protein.

75 la-509107 In one approach, a method of inhibiting the transcription of the PHORl-All or PHOR1-F5D6 gene comprises contacting the PHORl-Al 1 or PHOR1-F5D6 gene with a PHORl-Al 1 or PHOR1-F5D6 antisense polynucleotide. In another approach, a method of inhibiting PHORl-Al 1 or PHOR1-F5D6 mRNA translation comprises contacting the PHORl-Al 1 or PHOR1-F5D6 mRNA with an antisense polynucleotide. In another approach, a PHORl-Al 1 or PHOR1-F5D6 specific ribozyme is used to cleave the PHORl-All or PHOR1-F5D6 message, thereby inhibiting translation. Such antisense and ribozyme based methods can also be directed to the regulatory regions of the PHORl-Al 1 or PHOR1-F5D6 gene, such as the PHORl-All or PHOR1-F5D6 promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a PHORl-Al 1 or PHOR1-F5D6 gene transcription factor are used to inhibit PHORl-Al 1 or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 by interfering with PHORl-Al 1 or PHOR1-F5D6 transcriptional activation are also useful to treat cancers expressing PHOR1- Al 1 or PHOR1-F5D6. Similarly, factors that interfere with PHORl-Al 1 or PHOR1-F5D6 processing are useful to treat cancers that express PHORl-Al 1 or PHOR1-F5D6. 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 PHORl-Al 1 or PHOR1-F5D6 (i.e., antisense, ribozyme, polynucleotides encoding intrabodies and other PHORl-Al 1 or PHOR1-F5D6 inhibitory molecules). A number of gene therapy approaches are known in the art. Recombinant vectors encoding PHORl-Al 1 or PHOR1-F5D6 antisense polynucleotides, ribozymes, factors capable of interfering with PHORl-All or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6 to a binding partner, etc.

76 la-509107 In vivo, the effect of a PHORl-Al 1 or PHOR1-F5D6 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^th 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

77 la-509107 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 PHORl-Al 1- or PHORl-F5D6-related protein or a PHORl-Al 1 or PHOR1-F5D6 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; Identification of Olfactory Receptor Family Members

A degenerate oligo PCR strategy was utilized to identify family members of the G-protein coupled receptor (GPCR), PHOR-1. PHOR-1 RNA expression is predominantly in prostate tissue and is upregulated in prostate cancer. Since PHOR-1 is homologous to a large family of olfactory receptors that are expressed in olfactory epithelium, neurons, and other tissues additional family members can be identified. Two family members were identified and are dislcosed herein: PHORl-Al 1 and PHOR1-F5D6. Materials and Methods

A protein alignment between PHOR-1 and HPRAJ70, another prostate specific olfactory receptor, revealed at least two conserved regions. The conserved protein sequences listed below were used to design

78 la-509107 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.

PHOR-1 Region Conserved Sequence Degenerate Oligo aa 120-127 MAFDRY(I/V)A sense: 5' atg gel ttY gaY Mgl taY Rtl gc 3' aa 237-243 KAFTC(G V)anti-sense: 5 ' DVIc Rca Igt Ice Raa Igc Ytt 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. First strand cDNAs from benign prostatic hyperplasia (BPH), LAPC4AD, LAPC4AI, LAPC9AD, HeLa, kidney, bladder cancer, and the cell lines MD- MBA4355, and LoVo were utilized as templates for the degenerate RT-PCR reactions. The first strand cDNAs were generated from poly A mRNA using Superscript reverse transcriptase (catalogue no. 18089- 011 ; Life Technologies, Rockville Maryland). The first strand cDNAs were diluted to 150 ul for each ug of polyA mRNA used in the reverse transcriptase reaction and 5 ul was used in the RT-PCR reaction.

Master Amp™ buffer A was used for all templates except for MD-MB A4355 which required buffer B for RT-PCR amplification. The sense and anti-sense degenerate oligos were at 1.2 uM and the reaction volume was 50 ul. Thermal cycling conditions consisted of a single denaturation step at 92 C for 1 min followed by 32 cycles of 96 C for 30 sec, 55 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 following two family members were identified with their tissues of origin listed in parenthesis: PHORl-All (LAPC9 AD), PHOR1-F5D6 (Kidney). cDNA sequences and ORFs for these two PHOR-1 family members are listed in Figure 1 A and Figure IB. PHORl-Al 1 is novel and has significant homology to a Marmota olfactory receptor (GenBank Accession AF044033) with 87% identity over 247 bp. PHOR1-F5D6 has significant homology to a human olfactory receptor, family 2, subfamily A, member 4 (GenBank accession XM_027121). Primers for RT-PCR were designed from the PHORl-All and PHOR1-F5D6 sequences to study expression in normal and cancer tissues. (See Figure 6 through Figure 13.)

79 la-509107 Example 2: Full-Length cDNA Cloning of PHORl-All and PHOR1-F5D6

The PHORl-All sequence was used to clone a full-length cDNA with a 312 amino acid ORF from placenta cDNA. This cDNA and corresponding ORF are listed in Figure 2a. The PHOR1-F5D6 sequence was used to clone a full-length cDNA with a 310 amino acid ORF from LAPC-4AD cDNA. The PHOR1-F5D6 cDNA and corresponding ORF are listed in Figure 2 b. The complete protein sequences for these two genes are listed in Figure 3 A and Figure 3B. Protein BLASTs of the two genes show clear homology to olfactory G-protein coupled receptors. PHORl-Al 1 has the highest homology to a Marmota olfactory receptor with 83% identity and 92% similarity over the entire Marmota 237 amino acid sequence (Figure 4). This homology is an indication that PHORl-Al 1 and this Marmota olfactory receptor are orthologues. PHOR1-F5D6 has 100% amino acid homology to an olfactory receptor protein predicted from PAC clone RP5-988G15 (Figure 5).

The PHORl-Al 1 cDNA clone was deposited on , with the American Type

Culture Collection (ATCC, Manassas, VA) as plasmid , and has been assigned Accession No. . The PHOR1-F5D6 cDNA clone was deposited on , with the American

Type Culture Collection (ATCC, Manassas, VA) as plasmid , and has been assigned Accession

No. .

The skilled artisan understands that while methods utilizing degenerate oligonucleotides can incorporate primer sequences into the cloned molecules, the sequences between the primers represent polynucleotide and polypeptide sequences expressed in vivo. Moreover, the skilled artisan understands that these PHOR-1 family intervening sequences can identified simply by a side by side comparison of the primers (see Example 2 below) and the cloned sequence of each family member (see e.g. Figures 1-5).

Example 3: Chromosomal Mapping of PHORl-All and PHOR1-F5D6

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).

80 la-509107 Chromosomal localization of PHORl-All was determined using the GeneBridge4 radiation hybrid panel (Walter et al., 1994, Nat. Genetics 7:22) (Research Genetics, Huntsville Al). The mapping vectors for the 93 radiation hybrid panel DNAs were entered into the mapping program at http://www- genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl to generate the chromosomal localization.The following PCR primers were used to localize PHORl-Al 1 :

FM1A11.1 ACAGCCCTCACAATGTAGCTGTAA

FM1A11.2 AACTCCTTGGCCATGTCTCCTGT

The resulting mapping vector for the 93 radiation hybrid panel DNAs was:

00000000100000110001010010000000100000001100111000110010000000010001100101100000110100 0010001

PHORl-All is localized to chromosome lq43 distal to AFM155XC11.

PHORl-Al 1 is localized to chromosome lq43. Since the hereditary prostate cancer 2 (HPC2) locus is on Iq42.2-q43 (Berthon et al., Am. J. Hum. Genet. 62: 1416-1424, (1998)), PHORl-All is a candidate gene for hereditary prostate cancer. Mutation analysis using SSCP or direct sequencing using prostate cancer patients from the linked families can determine if this gene is the HPC2 gene.

The chromosome localization of PHOR1-F5D6 was determined from the sequencing of this gene on PAC RP5-988G15 from 7q33-q35 (GenBank Accession AC005587) , a region frequently amplified or rearranged in cancer (Arranz E, Martinez-Delgado B, Richart A, Osorio A, Cebrian A, Robledo M, Rivas

C, Benitez J. Cancer Genet Cytogenet 2000 Feb;117(l):41-4; Ong ST, Le Beau MM. Semin Oncol 1998 Aug;25(4):447-60; Johnson E, Cotter FE. Blood Rev 1997 Mar;ll(l):46-55).

Example 4: Expression analysis of PHORl-All and PHORl -F5D6 in normal tissues and patient specimens

Analysis of PHORl-Al 1 by RT-PCR is shown in Figure 6 and Figure 7. Normal tissue expression is restricted to normal testis, prostate and placenta. Analysis of human patient cancer RNA pools shows expression in prostate cancer pool (Figure 6), and in ovarian cancer pool (Figure 7). Extensive northern blot analysis of PHORl-All in 16 human normal tissues confirms the restricted expression observed by RT-PCR (Figure 8). An approximately 3 kb transcript is detected in placenta and prostate. Northern blot analysis shows expression of PHORl-Al 1 in all 4 prostate xenograft tissues, LAPC-4AD,

81 la-509107 LAPC-4AI, LAPC-9AD, and LAPC-9AI (Figure 9). Also, expression is seen in the prostate cancer cell line PC3, and in the bladder cancer cell line J82.

RT-PCR analysis of PHOR1-F5D6 shows restricted expression in normal tissues. Expression is detected in normal ovary and prostate and to lower levels in testis, liver, lung, pancreas, placenta and kidney (Figure 10). In cancer tissues, PHOR1-F5D6 expression is observed in prostate cancer pool, kidney cancer pool, ovarian cancer pool, and in all 4 prostate xenografts tested, LAPC-4AD, LAPC-4AI, LAPC-

9AD, and LAPC-9AI (Figure 10 and Figure 11). Northern blot analysis of PHOR1-F5D6 in normal tissues shows absence of expression in all 16 human tissues tested (Figure 12). Northern blot analysis of PHOR1-

F5D6 on patient tumor specimens shows expression in all 5 kidney tumors tested, but not in normal kidney (Figure 13). The expression detected in normal adjacent tissues (isolated from patients) but not in normal tissues (isolated from a healthy donor) may indicate that these tissues are not fully normal and that PHOR1-

F5D6 may be expressed in early stage tumors.

The restricted expression of PHORl-All and PHOR1-F5D6 in normal tissues and the expression detected cancer suggest that PHORl-Al 1 and PHOR1-F5D6 are potential therapeutic targets and diagnostic markers for human cancers.

Example 5: Production of Recombinant PHORl-All and PHOR1-F5D6 in Prokaryotic Systems

A. In vitro transcription and translation constructs: pCRII: To generate PHORl-Al 1 and PHOR1-F5D6 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 PHORl-Al 1 and PHOR1-F5D6 cDNAs. The pCRII vector has Sp6 and T7 promoters flanking the insert to drive the transcription of PHORl-All and PHOR1-F5D6 RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze the cell and tissue expression of PHORl-Al 1 and PHOR1-F5D6 at the RNA level. Transcribed PHORl-Al 1 and PHOR1-F5D6 RNA representing the cDNA amino acid coding regions of the PHORl-Al 1 and PHOR1-F5D6 genes are used in in vitro translation systems such as the TnT™ Coupled Reticulolysate Sytem (Promega, Corp., Madison, WI) to synthesize PHORl-All and PHOR1-F5D6 proteins.

A. Bacterial Constructs: pGEX Constructs: To generate recombinant PHORl-Al 1 and PHOR1-F5D6 proteins in bacteria that are fused to the Glutathione S-transferase (GST) protein, all or parts of the PHORl-Al 1 and PHOR1- F5D6 cDNA protein coding sequences 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 PHORl-All and PHOR1-F5D6 protein sequences with GST fused at the amino-terminus and a six histidine epitope (6X His) at the carboxyl-terminus. The GST and

82 la-509107 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 6X His 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- 1, may be employed such that it permits cleavage of the GST tag from PHORl-Al 1 and PHOR1-F5D6- 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-l such that the following regions of PHORl-All or PHOR1-F5D6 proteins are expressed as amino-terminal fusions to GST: amino acids 1 to 314 and 1-310 respectively; or any 8, 9, 10, 11, 12,13, 14,15, or more contiguous amino acids from PHORl-Al 1 and PHOR1-F5D6 or analogs thereof. pMAL Constructs: To generate recombinant PHORl-Al 1 and PHOR1-F5D6 proteins that are fused to maltose-binding protein (MBP) in bacterial cells, all or parts of the PHORl-Al 1 and PHORl - F5D6 cDNA protein coding sequences 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 PHORl-Al 1 and PHOR1-F5D6 protein sequences with MBP fused at the amino-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 PHORl-Al 1 and PHOR1-F5D6. 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 PHORl-All or PHOR1-F5D6 proteins are expressed as amino- terminal fusions to MBP: amino acids 1 to 314 and 1-310, respectively; or any 8, 9, 10, 11, 12,13, 14, 15, or more contiguous amino acids from PHORl-All and PHOR1-F5D6 or analogs thereof. pET Constructs: To express PHORl-Al 1 and PHOR1-F5D6 in bacterial cells, all or parts of the PHORl-Al 1 and PHOR1-F5D6 cDNA protein coding sequence are cloned into the pET family of vectors (Novagen, Madison, WI). These vectors allow tightly controlled expression of recombinant PHORl-Al 1 and PHOR1-F5D6 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 ™ 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 PHORl-All or PHOR1-F5D6 proteins are expressed as amino- terminal fusions to NusA : amino acids 1 to 314 and 1-310, respectively; or any 8, 9, 10, 11, 12,13, 14, 15, or more contiguous amino acids from PHORl-All and PHOR1-F5D6 or analogs thereof.

83 la-509107 B. Yeast Constructs: pESC Constructs: To express PHORl-All and PHOR1-F5D6 in the yeast species Saccharomyces cerevisiae for generation of recombinant protein and functional studies, all or parts of the PHORl-Al 1 and PHOR1-F5D6 cDNA protein coding sequences 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 PHORl-All and PHOR1-F5D6. 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-HJS such that the following regions of the PHORl-Al 1 or PHOR1-F5D6 proteins are expressed: amino acids 1 to 314 and 1- 310, respectively; or any 8, 9, 10, 11, 12,13, 14, 15, or more contiguous amino acids from PHORl-Al 1 and PHOR1-F5D6 or analogs thereof. pESP Constructs: To express PHORl-Al 1 and PHORl -F5D6 in the yeast species Saccharomyces pombe, all or parts of the PHORl-Al 1 and PHOR1-F5D6 cDNA protein coding sequences are cloned into the pESP family of vectors. These vectors allow controlled high level of expression of a PHORl-Al 1 and PHOR1-F5D6 protein sequences that are 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 ρESP-1 vector such that the following regions of the PHORl-Al 1 or PHOR1-F5D6 proteins are expressed as amino- terminal fusions to GST: amino acids 1 to 314 and 1-310, respectively; or any 8, 9, 10, 11, 12,13, 14, 15, or more contiguous amino acids from PHORl-Al 1 and PHOR1-F5D6 or analogs thereof.

Example 6: Production of Recombinant PHORl-All and PHOR1-FSD6 in Eukaryotic Systems

A. Mammalian Constructs:

To express recombinant PHORl-Al 1 and PHOR1-F5D6 in eukaryotic cells, the full or partial length PHORl-Al 1 and PHOR1-F5D6 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-PHORl - Al 1 and PHOR1-F5D6 polyclonal serum, described above. pcDNA4 HisMax Constructs: To express PHORl-All and PHOR1-F5D6 in mammalian cells, the PHORl-Al 1 and PHOR1-F5D6 ORF are cloned into pcDNA4/His ax Version A (Invitrogen, Carlsbad, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter and the SP163 translational

84 la-509107 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 PHORl-Al 1 and PHOR1-F5D6 are expressed in this contract, amino acids 1 to 314 and 1 to 310, respectively; or any 8, 9, 10, 11, 12,13, 14,15, or more contiguous amino acids from PHORl-All and PHOR1-F5D6, variants, or analogs thereof. pcDNA3.1 MycHis Constructs: To express PHORl-Al 1 and PHOR1-F5D6 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. The following regions of PHORl-All and PHOR1-F5D6 are expressed in this contract, amino acids 1 to 314 and 1 to 310, respectively; or any 8, 9, 10, 11, 12,13, 14,15, or more contiguous amino acids from PHORl- All and PHOR1-F5D6, variants, or analogs thereof.

PCDNA3.1/CT-GFP-TOPO Construct: To express PHORl-All and PHOR1-F5D6 in mammalian cells and to allow detection of the recombinant proteins using fluorescence, the ORFs with consensus Kozak translation initiation site are cloned into pcDNA3.1/CT-GFP-TOPO (Invitrogen, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the Green Fluorescent Protein (GFP) fused to the C-terminus facilitating non-invasive, in vivo detection and cell biology studies. The ρcDNA3.1CT-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. Additional constructs with a N-terminal GFP fusion are made in pcDNA3.1 NT-GFP-TOPO spanning the entire length of the PHORl-All and PHOR1-F5D6 proteins. The following regions of PHORl-All and PHOR1-F5D6 are expressed in these contracts, amino acids 1 to 314 and 1 to 310,

85 la-509107 respectively; or any 8, 9, 10, 11, 12,13, 14,15, or more contiguous amino acids from PHORl-All and PHOR1-F5D6, variants, or analogs thereof.

PAPtag: The PHORl-Al 1 and PHOR1-F5D6 ORFs are cloned into pAPtag-5 (GenHunter Corp. Nashville, TN). This construct generates an alkaline phosphatase fusion at the C-terminus of the PHOR1- Al 1 and PHOR1-F5D6 proteins while fusing the IgGκ signal sequence to N-terminus. The resulting recombinant PHORl-All and PHOR1-F5D6 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 PHORl-Al 1 and PHOR1-F5D6 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 PHORl-All and PHOR1-F5D6 are expressed in these contracts, amino acids 1 to 314 and 1 to 310, respectively; or any 8, 9, 10, 11, 12,13, 14,15, or more contiguous amino acids from PHORl-All and PHOR1-F5D6, variants, or analogs thereof. ptag5: The PHORl-Al 1 and PHOR1-F5D6 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 PHORl-Al 1 and PHOR1-F5D6 protein while fusing the IgGK signal sequence to the N-terminus. The resulting recombinant PHORl-Al 1 and PHOR1- F5D6 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 PHORl-Al 1 and PHOR1-F5D6 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 PHORl-Al 1 and PHOR1-F5D6 are expressed in these contracts, amino acids 1 to 314 and 1 to 310, respectively; or any 8, 9, 10, 11, 12,13, 14,15, or more contiguous amino acids from PHORl-All and PHOR1-F5D6, variants, or analogs thereof.

PsecFc: The PHORl-Al 1 and PHOR1-F5D6 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 PHORl-Al 1 and PHOR1-F5D6 proteins, while fusing the IgGK signal sequence to N-terminus. The resulting recombinant PHORl-Al 1 and PHOR1-F5D6 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 PHORl-Al 1 and PHOR1-F5D6 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

86 la-509107 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 PHORl-All and PHOR1-F5D6 are expressed in these contracts, amino acids 1 to 314 and 1 to 310, respectively; or any 8, 9, 10, 11, 12,13, 14,15, or more contiguous amino acids from PHORl-All and PHOR1-F5D6, variants, or analogs thereof. pSR Constructs: To generate mammalian cell lines that express PHORl-Al 1 and PHOR1- F5D6 constitutively, the ORFs are cloned into pSRα constructs. Amphotropic and ecotropic retrovirases are generated by transfection of pSRα constructs into the 293T-10A1 packaging line or co-transfection of pSRα and a helper plasmid (containing deleted packaging sequences) into the 293 cells, respectively. The retrovirus can be used to infect a variety of mammalian cell lines, resulting in the integration of the cloned gene, PHORl-Al 1 and PHOR1-F5D6, 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 pSRα constructs are made that fuse an epitope tag such as the FLAG tag to the C- terminus of PHORl-All andPHORl-F5D6 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 pSRα constructs are made to produce both N-terminal and C-terminal GFP and myc/6 HIS fusion proteins of the full-length PHORl-Al 1 and PHOR1-F5D6 proteins. The following regions of PHORl-All and PHOR1-F5D6 are expressed in such constructs, amino acids 1 to 314 and 1 to 310, respectively; or any 8, 9, 10, 11, 12,13, 14,15, or more contiguous amino acids from PHORl-Al 1 and PHOR1-F5D6, variants, or analogs thereof.

Additional Viral Vectors: Additional constructs are made for viral-mediated delivery and expression of PHORl-Al 1 and PHOR1-F5D6. High virus titer leading to high level expression of

PHORl-Al 1 and PHOR1-F5D6 is achieved in viral delivery systems such as adenoviral vectors and herpes amplicon vectors. The PHORl-Al 1 and PHOR1-F5D6 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, PHORl-Al 1 and PHOR1-F5D6 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, NTH 3T3, 293 or rat-1 cells. The following regions of PHORl-Al 1 and PHOR1-F5D6 are expressed in these contracts, amino acids 1 to 314 and 1 to 310, respectively; or any

87 la-509107 8, 9, 10, 11, 12,13, 14,15, or more contiguous amino acids from PHORl-All and PHORl -F5D6, variants, or analogs thereof.

Regulated Expression Systems: To control expression of PHORl-All and PHOR1-F5D6 in mammalian cells, coding sequences of PHORl-All and PHOR1-F5D6 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 PHQR1-A11 and PHOR1-F5D6. These vectors are thereafter used to control expression of PHORl-All and PHOR1-F5D6 in various cell lines such as SCaBER, NIH 3T3, 293 or rat-1 cells. The following regions of PHORl-Al 1 and PHOR1-F5D6 are expressed in these contracts, amino acids 1 to 314 and 1 to 310, respectively; or any 8, 9, 10, 11, 12,13, 14,15, or more contiguous amino acids from PHORl-Al 1 and PHOR1-F5D6, variants, or analogs thereof. B. Baculovirus Expression Systems

To generate recombinant PHORl-Al 1 and PHOR1-F5D6 proteins in a baculovirus expression system, PHORl-Al 1 and PHOR1-F5D6 ORFs are cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag at the N-terminus. Specifically, pBlueBac-PHORl-All and PHOR1-F5D6 is co-transfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperdd) 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 PHORl-Al 1 and PHOR1-F5D6 protein is then generated by infection of HighFive insect cells (Invitrogen) with purified baculovirus. Recombinant PHORl-Al 1 and PHOR1-F5D6 protein can be detected using anti-PHORl-Al 1 and PHOR1-F5D6 or anti-His-tag antibody. PHORl-All and PHOR1-F5D6 protein can be purified and used in various cell-based assays or as immunogen to generate polyclonal and monoclonal antibodies specific for PHORl-Al 1 and PHOR1-F5D6.

The following regions of PHORl-All and PHOR1-F5D6 are expressed in these contracts, amino acids 1 to 314 and 1 to 310, respectively; or any 8, 9, 10, 11, 12,13, 14,15, or more contiguous amino acids from PHORl-All and PHOR1-F5D6, variants, or analogs thereof.

Example 7A Antigenicity Profiles for PHORl All

Figure 14A, Figure 15A, Figure 16A, Figure 17A, and Figure 18A depict graphically five amino acid profiles of the PHORl-Al 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 14A, Hydrophilicity, (Hopp T.P., Woods K.R., 1981. Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828); Figure 15 A, 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,

88 la-509107 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 PHORl-Al 1 protein. Each of the above amino acid profiles of PHORl-Al 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 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 PHORl-Al 1 protein indicated, e.g., by the profiles set forth in Figure

14A, Figure 15 A, 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-PHORl-Al 1 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 PHORl-Al 1 protein. In particular, peptide immunogens of the invention can comprise, a peptide region of at least 5 amino acids of Figure 2 in any whole number increment up to 314 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 2 in any whole number increment up to 314 that includes an amino acid position having a value less than 0.5 in the Hydropathicity profile of Figure 15 A; a peptide region of at least 5 amino acids of Figure 2 in any whole number increment up to 314 that includes an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 16 A; a peptide region of at least 5 amino acids of Figure 2 in any whole number increment up to 314 that includes an amino acid position having a value greater than 0.5 in the Average Flexibility profile on Figure 17 A; and, a peptide region of at least 5 amino acids of Figure 2 in any whole number increment up to 314 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.

89 la-509107 Example 7B. Antigenicity Profiles for PHOR1-F5D6

Figure 14B, Figure.15B, Figure 16B, Figure 17B, and Figure 18B depict graphically five amino acid profiles of the PHOR1-F5D6 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 PHOR1-F5D6 protein. Each of the above amino acid profiles of PHOR1-F5D6 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 PHORl -F5D6 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-PHORl -F5D6 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 PHOR1-F5D6 protein. In particular, peptide immunogens of the invention can comprise, a peptide region of at least 5 amino acids of Figure 2 in any whole number increment up to 310 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 2 in any whole number increment up to 310 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 2 in any whole number increment up to 310 that includes an

90 la-509107 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 2 in any whole number increment up to 310 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 2 in any whole number increment up to 310 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 PHORl-All Polyclonal Antibodies

Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. In addition to immunizing with the full length PHORl-All 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 PHORl-All). For example, PHORl-Al 1 recombinant bacterial fusion proteins or peptides encoding hydrophilic, flexible, beta-turn regions of the PHORl-All sequence, such as amino acids 1-26, 80-95, and amino acids 225-240 of PHORl-Al 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 1-26 of PHORl-All is conjugated to KLH and used to immunize the rabbit. Alternatively the immunizing agent may include all or portions of the PHORl-Al 1 protein, analogs or fusion proteins thereof. For example, the PHORl-Al 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 PHOR1-F5D6 in Prokaryotic Systems" and Current Protocols In Molecular Biology, Volume 2, Unit 16,

91 la-509107 Frederick M. Ausubul et al. eds., 1995; Linsley, P.S., Brady, W., Urnes, M., Grosmaire, L., Damle, N., and Ledbetter, L.(1991) J.Exp. Med. 174, 561-566). In one embodiment, a GST-fusion protein encoding amino acids 80-197 of PHORl-All is produced, purified, and 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 μg, typically 100-200 μg, of fusion protein or peptide conjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits are then injected subcutaneously every two weeks with up to 200 μg, typically 100-200 μg, of the immunogen in incomplete Freund's adjuvant (EFA). 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 PHORl-Al 1 proteins, the full-length PHORl-Al 1 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 PHORl-All in Eukaryotic Systems"). After transfection of the constructs into 293T cells, cell lysates are probed with the anti-PHORl-Al 1 serum and with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) to determine specific reactivity to denatured PHORl-Al 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 PHORl-Al 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 PHORl-Al 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 8B: Generation of PHOR1-F5D6 Polyclonal Antibodies

Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be

92 la-509107 injected in the mammal by multiple subcutaneous or intraperitoneal injections. In addition to immunizing with the full length PHORl -F5D6 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 PHOR1-F5D6).

For example, PHOR1-F5D6 recombinant bacterial fusion proteins or peptides encoding hydrophilic, flexible, beta-turn regions of the PHOR1-F5D6 sequence, such as amino acids 1-23, 80-95, and amino acids 259-274 of PHOR1-F5D6 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 1-26 of PHOR1-F5D6 is conjugated to KLH and used to immunize the rabbit. Alternatively the immunizing agent may include all or portions of the PHOR1-F5D6 protein, analogs or fusion proteins thereof. For example, the PHOR1-F5D6 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, thioredqxin, NusA, or an immunoglobulin constant region (see e.g. the section entitled "Expression of PHOR1-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). In one embodiment, a GST-fusion protein encoding amino acids 80-197 of PHOR1-F5D6 is produced, purified, and 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 μg, typically 100-200 μg, of fusion protein or peptide conjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits are then injected subcutaneously every two weeks with up to 200 μg, typically 100-200 μg, of the

93 la-509107 immunogen in incomplete Freund's adjuvant (IF A). 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 PHOR1-F5D6 proteins, the full-length PHORl -F5D6 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 PHOR1-F5D6 in Eukaryotic Systems"). After transfection of the constructs into 293T cells, cell lysates are probed with the anti-PHORl-F5D6 serum and with anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) to determine specific reactivity to denatured PHOR1-F5D6 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 PHORl -F5D6-expressing cells. Alternatively, specificity of the antiserum is tested by Western blot, immunoprecipitation, and flow cytometric techniques using lysates of cells that endogenously express PHOR1-F5D6.

Example 9A: Generation of PHORl-All Monoclonal Antibodies (mAbs)

In one embodiment, therapeutic mAbs to PHORl-All comprise those that react with epitopes of the protein that would disrupt or modulate the biological function of PHORl-Al 1, for example those that disrupt the interaction of PHORl-All with ligands. Therapeutic mAbs also comprise those which specifically bind epitopes of PHORl-All 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 PHORl-Al 1 protein or regions of the PHORl-Al 1 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

PHORl-All, such as 293T-PHOR1-A11 cells, are used to immunize mice. To generate mAbs to PHORl- All, mice are first immunized intraperitoneally (IP) with, typically, 10-50 μg of protein immunogen or 10⁷ PHORl-Al 1-expressing cells mixed in complete Freund's adjuvant. Mice are then subsequently immunized IP every 2-4 weeks with, typically, 10-50 μg of protein immunogen or 10⁷ cells mixed in

94 la-509107 incomplete Freund's adjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. A DNA- based immunization protocol is also employed in which a mammalian expression vector encoding PHORl- Al 1 sequence is used to immunize mice by direct injection of the plasmid DNA. For example, either pCDNA 3.1 encoding the full length PHORl-Al 1 cDNA, or amino acids 160-197 of PHORl-Al 1 5 (predicted to contain antigenic sequences, see, e.g., Figure 14A, Figure 15A, Figure 16A, Figure 17A or Figure 18 A) 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 and in combination with protein and 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

10 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 PHORl-Al 1 monoclonal antibodies, 293T cells transfected with the pCDNA4-PHORl-All expression vector are used to immunize mice. Balb C mice are initially

15 immunized intraperitoneally with 10⁷293T-PHOR1-A11 cells mixed in complete Freund's adjuvant. Mice are subsequently immunized every two weeks with 10⁷293T-PHOR1-A11 cells mixed in incomplete Freund's adjuvant for a total of three immunizations. Reactivity and specificity of serum to full length PHORl-All protein is monitored by Western blotting and flow cytometry using 293T and RAT1 cells expressing either the neomycin resistance gene or the PHORl-Al 1 cDNA (see e.g., the Example entitled

20. "Production of Recombinant PHORl-Al 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, immunofluorescence, and flow cytometry to identify PHORl-All

25 specific antibody-producing clones.

The binding affinity of a PHORl-Al 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 PHORl-All 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

30 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.

95 la-509107 Example 9B: Generation of PHOR1-F5D6 Monoclonal Antibodies (mAbs)

In one embodiment, therapeutic mAbs to PHOR1-F5D6 comprise those that react with epitopes of the protein that would disrupt or modulate the biological function of PHOR1-F5D6, for example those that disrupt the interaction of PHOR1-F5D6 with ligands. Therapeutic mAbs also comprise those which specifically bind epitopes of PHOR1-F5D6 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 PHOR1-F5D6 protein or regions of the PHOR1-F5D6 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 PHOR1-F5D6, such as 293T-PHOR1-F5D6 cells, are used to immunize mice. To generate mAbs to PHOR1-F5D6, mice are first immunized intraperitoneally (IP) with, typically, 10-50 μg of protein immunogen or 10⁷ PHORl-F5D6-expressing cells mixed in complete Freund's adjuvant. Mice are then subsequently immunized IP every 2-4 weeks with, typically, 10-50 μg of protein immunogen or 10⁷ cells mixed in incomplete Freund's adjuvant. Alternatively, MPL-TDM adjuvant is used in immunizations. A DNA-based immunization protocol is also employed in which a mammalian expression vector encoding PHOR1-F5D6 sequence is used to immunize mice by direct injection of the plasmid DNA. For example, either pCDNA 3.1 encoding the full length PHOR1-F5D6 cDNA, or amino acids 160-197 of PHOR1-F5D6 (predicted to contain antigenic sequences, 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 and in combination with protein and 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 PHOR1-F5D6 monoclonal antibodies, 293T cells transfected ' with the pCDNA4-PHORl-F5D6 expression vector are used to immunize mice. Balb C mice are initially immunized intraperitoneally with 10⁷293T-PHOR1-F5D6 cells mixed in complete Freund's adjuvant.

Mice are subsequently immunized every two weeks with 10⁷293T-PHOR1-F5D6 cells mixed in incomplete Freund's adjuvant for a total of three immunizations. Reactivity and specificity of serum to full length PHOR1-F5D6 protein is monitored by Western blotting and flow cytometry using 293T and RAT1 cells expressing either the neomycin resistance gene or the PHOR1-F5D6 cDNA (see e.g., the Example

96 la-509107 entitled "Production of Recombinant PHORl -F5D6 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, immunofluorescence, and flow cytometry to identify PHOR1-F5D6 specific antibody-producing clones.

The binding affinity of a PHOR1-F5D6 monoclonal antibody is determined using standard technologies. Affinity measurements quantify the strength of antibody to epitope binding and are used to help define which PHOR1-F5D6 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 Binding Assays

HLA class I and class II binding assays using purified HLA molecules are performed in accordance with disclosed protocols (e.g., PCT publications WO 94/20127 and WO 94/03205; Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al, J. Immunol. 154:247 (1995); Sette, et al, Mol. Immunol. 31:813 (1994)). Briefly, purified MHC molecules (5 to 500 nM) are incubated with various unlabeled peptide inhibitors and 1-10 nM ¹²⁵I-radiolabeled probe peptides as described. Following incubation, MHC-peptide complexes are separated from free peptide by gel filtration and the fraction of peptide bound is determined. Typically, in preliminary experiments, each MHC preparation is titered in the presence of fixed amounts of radiolabeled peptides to determine the concentration of HLA molecules necessary to bind 10-20% of the total radioactivity. All subsequent inhibition and direct binding assays are performed using these HLA concentrations.

Since under these conditions [label]<[HLA] and IC₅₀≥[HLA], the measured IC₅₀ values are reasonable approximations of the true K_D values. Peptide inhibitors are typically tested at concentrations ranging from 120 μg/ml to 1.2 ng/ml, and are tested in two to four completely independent experiments.

To allow comparison of the data obtained in different experiments, a relative binding figure is calculated for each peptide by dividing the IC₅₀ of a positive control for inhibition by the IC₅₀ for each tested peptide

(typically unlabeled versions of the radiolabeled probe peptide). For database purposes, and inter-

97 la-509107 experiment comparisons, relative binding values are compiled. These values can subsequently be converted back into IC₅₀ nM values by dividing the IC₅₀ nM of the positive controls for inhibition by the relative binding of the peptide of interest. This method of data compilation is accurate and consistent for comparing peptides that have been tested on different days, or with different lots of purified MHC. - . Binding assays as outlined above may be used to analyze HLA supermotif and or HLA motif- bearing peptides.

Example 11: Identification of HLA Supermotif- and Motif-Bearing CTL Candidate Epitopes HLA vaccine compositions of the invention can include multiple epitopes. The multiple epitopes can comprise multiple HLA supermotifs or motifs to achieve broad population coverage. This example illustrates the identification 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 PHORl-Al 1 or PHOR1-F5D6 set forth in Figures 2 and 3.

Computer searches for epitopes bearing HLA Class I or Class II supermotifs or motifs are performed as follows. All translated PHORl-Al 1 or PHOR1-F5D6 protein sequences are analyzed using a text string search software program to identify potential peptide sequences containing appropriate HLA binding motifs; such programs are readily produced in accordance with information in the art in view of known motif/supermotif disclosures. Furthermore, such calculations can be made mentally.

Identified A2-, A3-, and DR-supermotif sequences are scored using polynomial algorithms to predict their capacity to bind to specific HLA-Class I or Class II molecules. These polynomial algorithms account for the impact of different amino acids at different positions, and are essentially based on the premise that the overall affinity (or ΔG) of peptide-HLA molecule interactions can be approximated as a linear polynomial function of the type:

"ΔG" = ai,- x a_2l- x a₃₍- x a„,- where _y; is a coefficient which represents the effect of the presence of a given amino acid (j) at a given position (0 along the sequence of a peptide of n amino acids. The crucial assumption of this method is that the effects at each position are essentially independent of each other (i.e., independent binding of individual side-chains). When residue j occurs at position i in the peptide, it is assumed to contribute a constant amount j, to the free energy of binding of the peptide irrespective of the sequence of the rest of the peptide.

98 la-509107 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 / 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 supertype cross-reactive peptides

Complete protein sequences from PHORl-Al 1 and or PHOR1-F5D6 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 PHORl-Al 1 and/or PHOR1-F5D6 protein sequences scanned above are 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 PHORl-Al 1 and/or PHOR1-F5D6 proteins are also analyzed for the presence of 8-, 9- 10-, or

11-mer peptides with the HLA-B7-supermotif. Corresponding peptides are synthesized and tested for binding to HLA-B*0702, the molecule encoded by the most common B7-supertype allele (i.e., the prototype B7 supertype allele). Peptides binding B*0702 with IC₅₀ of ≤500 nM are identified using standard methods. These peptides are then tested for binding to other common B7-supertype molecules

99 la-509107 (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 Al 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 PHORl-Al 1 and/or PHOR1-F5D6 proteins can also be performed to identify HLA-A1- and A24-motif-containing sequences.

High affinity and or cross-reactive binding epitopes that bear other motif and/or supermotifs are identified using analogous methodology.

Example 12: Confirmation of Immunogenicity

Cross-reactive candidate CTL A2-supermotif-bearing peptides that are identified as described herein are selected 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 μg/ml DNAse, washed twice and resuspended in complete medium (RPMI-1640 plus 5% AB human serum, non-essential amino acids, sodium pyruvate, L-glutamine and penicillin/streptomycin). The monocytes are purified by plating 10 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. TNFα is added to the DCs on day 6 at 75 ng ml and the cells are used for CTL induction cultures on day 7. Induction of CTL with DC and Peptide: CD8+ T-cells are isolated by positive selection with

Dynal immunomagnetic beads (Dynabeads® M-450) and the detacha-bead® reagent. Typically about 200-

250xl0⁶ PBMC are processed to obtain 24xl0⁶ CD8⁺ T-cells (enough for a 48-well plate culture). Briefly, the PBMCs are thawed in RPMI with 30μg/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

100 .la-509107 are washed 3 times with PBS/AB serum, added to the cells (140μl beads/20xl0⁶ 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 lOOxlO⁶ cells/ml (based on the original cell number) in PBS/AB serum containing lOOμl/ml detacha-bead® reagent and 30 μg/ml DNAse. The mixture is incubated for 1 hour at room temperature with continuous mixing. The beads are washed again with PBS/AB/DNAse to collect the CD8+ T-cells. The DC are collected and centrifuged at 1300 rpm for 5-7 minutes, washed once with PBS with 1% BSA, counted and pulsed with 40μg/ml of peptide at a cell concentration of l-2xl0⁶/ml in the presence of 3μg ml β₂- microglobulin for 4 hours at 20°C. The DC are then irradiated (4,200 rads), washed 1 time with medium and counted again. Setting up induction cultures: 0.25 ml cytokine-generated DC (at lxlO⁵ cells/ml) are co-cultured with 0.25ml of CD8+ T-cells (at 2xl0⁶ cell ml) in each well of a 48-well plate in the presence of 10 ng/ml of IL-7. Recombinant human TL-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 5xl0^δ cells/ml and irradiated at -4200 rads. The PBMCs are plated at 2xl0⁶ 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 lOμg/ml of peptide in the presence of 3 μg/ml β₂ 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 ml with fresh media. The cells are then transferred to the wells containing the peptide-pulsed adherent cells. Twenty four hours later recombinant human IL-10 is added at a final concentration of 10 ng/ml and recombinant human IL2 is added the next day and again 2- 3 days later at 50IU/ml (Tsai et al, Critical Reviews in Immunology 18(l-2):65-75, 1998). Seven days later, the cultures are assayed for CTL activity in a ⁵¹Cr release assay. In some experiments the cultures are assayed for peptide-specific recognition in the in situ IFNγ ELISA at the time of the second restimulation followed by assay of endogenous recognition 7 days later. After expansion, activity is measured in both assays for a side-by-side comparison. Measurement of CTL lytic activity by ⁵¹Cr release.

Seven days after the second restimulation, cytotoxicity is determined in a standard (5 hr) ⁵¹Cr release assay by assaying individual wells at a single E:T. Peptide-pulsed targets are prepared by incubating the cells with lOμg ml peptide overnight at 37°C.

101 la-509107 Adherent target cells are removed from culture flasks with trypsin-EDTA. Target cells are labeled with 200μCi of ^5ICr 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 3.3xl0⁶/ml (an NK- sensitive erythroblastoma cell line used to reduce non-specific lysis). Target cells (100 μl) and effectors (lOOμl) are plated in 96 well round-bottom plates and incubated for 5 hours at 37°C. At that time, 100 μl of supernatant are collected from each well and percent lysis is determined according to the formula: [(cpm of the test sample- cpm of the spontaneous ^5ICr release sample)/(cpm of the maximal ^5ICr release sample- cpm of the spontaneous ⁵¹Cr 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 IFNγ Production as an Indicator of Peptide-specific and Endogenous Recognition Immulon 2 plates are coated with mouse anti-human IFNy monoclonal antibody (4 μg ml 0.1M

NaHCOs, pH8.2) overnight at 4°C. The plates are washed with Ca²⁺, Mg²⁺-free PBS/0.05% Tween 20 and blocked with PBS/10% FCS for two hours, after which the CTLs (100 μl/well) and targets (100 μl/well) are added to each well, leaving empty wells for the standards and blanks (which received media only). The target cells, either peptide-pulsed or endogenous targets, are used at a concentration of lxlO⁶ cells/ml. The plates are incubated for 48 hours at 37°C with 5% C0₂.

Recombinant human EFN-gamma is added to the standard wells starting at 400 pg or 1200ρg/100 microliter/well and the plate incubated for two hours at 37°C. The plates are washed and 100 μl of biotinylated mouse anti-human IFN-gamma monoclonal antibody (2 microgram/ml in PBS/3%FCS/0.05% Tween 20) are added and incubated for 2 hours at room temperature. After washing again, 100 microliter HRP-streptavidin (1 :4000) are added and the plates incubated for one hour at room temperature. The plates are then washed 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 IM 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, 5xl0⁴ CD8+ cells are added to a T25 flask containing the following: lxlO⁶ irradiated (4,200 rad) PBMC (autologous or allogeneic) per ml,

102 la-509107 2xl0⁵ 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, sodium pyruvate, 25μM 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 lxl0⁶/ml and the cultures are assayed between days 13 and 15 at E:T ratios of 30, 10, 3 and 1:1 in the ⁵¹Cr release assay or at lxl0⁶/ml in the in situ IFNγ assay using the same targets as before the expansion.

Cultures are expanded in the absence of anti-CD3⁺ as follows. Those cultures that demonstrate specific lytic activity against peptide and endogenous targets are selected and 5xl0⁴ CD8⁺ cells are added to a T25 flask containing the following: lxlO⁶ autologous PBMC per ml which have been peptide-pulsed with 10 μg/ml peptide for two hours at 37°C and irradiated (4,200 rad); 2xl0⁵ 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.

Immunogenicity of A2 supermotif-bearing peptides A2-suρermotif 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 PHORl-Al 1 and/or PHOR1-F5D6. Briefly, PBMCs are isolated from patients, re-stimulated with peptide-pulsed monocytes and assayed for the ability to recognize peptide-pulsed target cells as well as transfected cells endogenously expressing the antigen.

Evaluation of A*03/A11 immunogenicity

HLA- A3 supermotif-bearing cross-reactive binding peptides are also evaluated for immunogenicity using methodology analogous for that used to evaluate the immunogenicity of the HLA- A2 supermotif peptides.

Evaluation of B7 immunogenicity

Immunogenicity screening of the B7 -supertype cross-reactive binding peptides identified as set forth herein are 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

103 la-509107 Example 13: Implementation of the Extended Supermotif to Improve the Binding Capacity of Native Epitopes by Creating Analogs

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

Peptide engineering strategies are implemented to further increase the cross-reactivity of the epitopes. For example, the main anchors of A2-supermotif-bearing peptides are altered, for example, to introduce a preferred L, I, V, or M at position 2, and I or V at the C-terminus.

To analyze the cross-reactivity of the analog peptides, each engineered analog is initially tested for binding to the prototype A2 supertype allele A*0201, then, if A*0201 binding capacity is maintained, for A2-supertype cross-reactivity.

Alternatively, a peptide is 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 IC₅₀ 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.

104 la-509107 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-supertyρe 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 PHORl-Al 1- and/or PHORl-F5D6-expressing tumors. Other analoging strategies

Another form of peptide analogizing, unrelated to anchor positions, involves the substitution of a cysteine with α-amino butyric acid. Due to its chemical nature, cysteine has the propensity to form disulfide bridges and sufficiently alter the peptide structurally so as to reduce binding capacity. Substitution of α-amino butyric acid for cysteine not only alleviates this problem, but has been shown to improve binding and crossbinding capabilities in some instances (see, e.g., the review by Sette et al, In: Persistent Viral Infections, Eds. R. Ahmed and I. Chen, John Wiley & Sons, England, 1999).

105 la-509107 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 PHORl-All- and/or PHORl -F5D6-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 PHORl-Al 1- and/or PHORl-F5D6-derived, HLA class II HTL epitopes, the PHOR1- All and/or PHOR1-F5D6 antigens are 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 DRl, DR4w4, and DR7, can efficiently select DR cross-reactive peptides.

The PHORl-Al 1- and/or PHORl-F5D6-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: DRl, DR4w4, and DR7. Peptides binding at least two of these three DR molecules are then tested for binding to DR2w2 βl, DR2w2 β2, DR6wl9, 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 DR4wl5, DR5 wl 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. PHORl-Al 1- and/or PHORl-F5D6-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

106 la-509107 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 PHORl-Al 1 and/or PHOR1-F5D6 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 lμM or better, i.e., less than 1 μM. Peptides are found that meet this binding criterion and qualify as HLA class II high affinity binders.

DR3 binding epitopes identified in this manner are included in vaccine compositions with DR supermotif-bearing peptide epitopes.

Similarly to the case of HLA class I motif-bearing peptides, the class II motif-bearing peptides are analoged to improve affinity or cross-reactivity. For example, aspartic acid at position 4 of the 9-mer core sequence is an optimal residue for DR3 binding, and substitution for that residue often improves DR 3 binding.

Example 15: Immunogenicity of PHORl-All- and/or PHORl-F5D6-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 PHORl-Al 1- and/or PHORl-F5D6-expressing tumors.

Example 16: Calculation of phenotypic frequencies of HLA-supertypes in various ethnic backgrounds to determine breadth of population coverage

This example illustrates the assessment of the breadth of population coverage of a vaccine composition comprised of multiple epitopes comprising multiple supermotifs and/or motifs.

In order to analyze population coverage, gene frequencies of HLA alleles are determined. Gene frequencies for each HLA allele are calculated from antigen or allele frequencies utilizing the binomial distribution formulae gf=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=l-(l-Cgf)²].

107 la-509107 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, and A*6801. Although the A3-like supertype may also include A34, A66, and A*7401, these alleles were not included in overall frequency calculations. Likewise, confirmed members of the A2-like supertype family are A*0201, A*0202, A*0203, A*0204, A*0205, A*0206, A*0207, A*6802, and A*6901. Finally, the B7-like supertype-confirmed alleles are: B7, B*3501-03, B51, B*5301, B*5401, B*5501-2, B*5601, B*6701, and B*7801 (potentially also B*1401, B*3504-06, B*4201, and B*5602).

Population coverage achieved by combining the A2-, A3- and B7-suρertypes is approximately 86% in five major ethnic groups. Coverage may be extended by including peptides bearing the Al and A24 motifs.. On average, Al 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. and Rubinstein, A. "A course in game theory" MIT Press, 1994), can be used to estimate what percentage of the individuals in a population comprised of the Caucasian, North American Black, Japanese, Chinese, and Hispanic ethnic groups would recognize the vaccine epitopes described herein. A preferred percentage is 90%. A more preferred percentage is 95%.

108 la-509107 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 ⁵¹Cr labeled Jurkat-A2.1/K target cells in the absence or presence of peptide, and also tested on ⁵¹Cr labeled target cells bearing the endogenously synthesized antigen, i.e. cells that are stably transfected with PHORl-Al 1 and/or PHOR1-F5D6 expression vectors.

The results demonstrate that CTL lines obtained from animals primed with peptide epitope recognize endogenously synthesized PHORl-Al 1 and/or PHOR1-F5D6 antigens. The choice of transgenic mouse model to be used for such an analysis depends upon the epitoρe(s) that are being evaluated. In addition to HLA-A*0201/K^b 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 Conjugated Epitopes In Transgenic Mice This example illustrates the induction of CTLs and HTLs in transgenic mice, by use of a PHORl-

Al 1- and/or PHORl-F5D6-derived CTL and HTL peptide vaccine compositions. The vaccine composition used herein comprise peptides to be administered to a patient with a PHORl-Al 1- and/or PHOR1-F5D6- 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/K^b 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.

109 la-509107 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/K^b chimeric gene (e.g., Vitiello etal, J. Exp. Med. 173:1007, 1991) In vitro CTL activation: One week after priming, spleen cells (30xl0⁶ cells/flask) are co-cultured at 37°C with syngeneic, irradiated (3000 rads), peptide coated lymphoblasts (10xl0⁶ 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 1.5xl0⁶) are incubated at 37°C in the presence of 200 μl of ⁵¹Cr. After 60 minutes, cells are washed three times and resuspended in R10 medium. Peptide is added where required at a concentration of 1 μg/ml. For the assay, 10^{4 51}Cr-labeled target cells are added to different concentrations of effector cells (final volume of 200 μl) in U-bottom 96- well plates. After a six hour incubation period at 37°C, a 0.1 ml aliquot of supernatant is removed from each well and radioactivity is determined in a Micromedic automatic gamma counter. The percent specific lysis is determined by the formula: percent specific release = 100 x (experimental release - spontaneous release)/(maximum release - . spontaneous release). To facilitate comparison between separate CTL assays run under the same conditions, % ⁵¹Cr release data is expressed as lytic units/10⁶ cells. One lytic unit is arbitrarily defined as the number of effector cells required to achieve 30% lysis of 10,000 target cells in a six hour ⁵¹Cr release assay. To obtain specific lytic units/10⁶, the lytic units/10⁶ obtained in the absence of peptide is subtracted from the lytic units/10⁶ obtained in the presence of peptide. For example, if 30% ⁵¹Cr release is obtained at the effector (E): target (T) ratio of 50:1 (i.e., 5xl0⁵ effector cells for 10,000 targets) in the absence of peptide and 5:1 (i.e., 5xl0⁴ effector cells for 10,000 targets) in the presence of peptide, the specific lytic units would be: [(l/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 a PHORl-All- and/or PHORl-F5D6-specific vaccines.

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

110 la-509107 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 PHORl-Al 1 and or PHOR1-F5D6 clearance. The number of epitopes used depends on observations of patients who spontaneously clear PHORl-All and/or PHOR1-F5D6. For example, if it has been observed that patients who spontaneously clear PHORl-Al 1 and or PHOR1-F5D6 generate an immune response to at least three (3) epitopes from PHORl-Al 1 and/or PHOR1-F5D6 antigen respectively, 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 IC₅₀ of 500 nM or less for an HLA class I molecule, or for class II, an IC₅₀ of 1000 nM or less; or HLA Class I peptides with high binding scores 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

111 la-509107 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 PHORl-Al 1 or PHOR1-F5D6, 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 PHORl-Al 1 and or PHOR1-F5D6.

Example 20: Construction of ^<<Minigene" Multi-Epitope DNA Plasmids

This example discusses the construction of a minigene expression plasmid. Minigene plasmids may, of course, contain various configurations of B cell, CTL and or HTL epitopes or epitope analogs as described herein.

A minigene expression plasmid typically includes multiple CTL and HTL peptide epitopes. In the present example, HLA-A2, -A3, -B7 supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif- bearing peptide epitopes are used in conjunction with DR supermotif-bearing epitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearing peptide epitopes derived from PHORl-Al 1 and/or PHOR1-F5D6, are selected such that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from PHORl-Al 1 and/or PHOR1- ' F5D6 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

112 la-509107 accordance with principles disclosed herein. The sequence encodes an open reading frame fused to the Myc and His antibody epitope tag coded for by the pcDNA 3.1 Myc-His vector.

Overlapping oligonucleotides that can, for example, average about 70 nucleotides in length with 15 nucleotide overlaps, are synthesized and HPLC-purified. The oligonucleotides encode the selected peptide epitopes as well as appropriate linker nucleotides, Kozak sequence, and signal sequence. The final multiepitope minigene is assembled by extending the overlapping oligonucleotides in three sets of reactions using PCR. A Perkin/Elmer 9600 PCR machine is used and a total of 30 cycles are performed using the following conditions: 95°C for 15 sec, annealing temperature (5° below the lowest calculated Tm of each primer pair) for 30 sec, and 72°C for 1 min. For example, a minigene is prepared as follows. For a first PCR reaction, 5 μg of each of two oligonucleotides are annealed and extended: In an example using eight oligonucleotides, i.e., four pairs of primers, oligonucleotides 1+2, 3+4, 5+6, and 7+8 are combined in 100 μl reactions containing Pfit polymerase buffer (lx= 10 mM KCL, 10 mM (NH4)₂S0₄, 20 mM Tris-chloride, pH 8.75, 2 mM MgS0₄, 0.1% Triton X-100, 100 μg/ml BSA), 0.25 mM each dNTP, and 2.5 U of Pfu polymerase. The full-length dimer products are gel-purified, and two reactions containing the product of 1+2 and 3+4, and the product of 5+6 and 7+8 are mixed, annealed, and extended for 10 cycles. Half of the two reactions are then mixed, and 5 cycles of annealing and extension carried out before flanking primers are added to amplify the full length product. The full-length product is gel-purified and cloned into pCR-blunt (Invitrogen) and individual clones are screened by sequencing.

Example 21: The Plasmid Construct and the Degree to Which It Induces Immunogenicity The degree to which a plasmid construct, for example a plasmid constructed in accordance with the previous^" Example, is able to induce immunogenicity is 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).

113 la-509107 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/K^b transgenic mice, for example, are immunized intramuscularly with 100 μg of naked cDNA. As a means of comparing the level of CTLs induced by cDNA immunization, a control group of animals is also immunized with an actual peptide composition that comprises multiple epitopes synthesized as a single polypeptide as they would be encoded by the minigene.

Splenocytes from immunized animals are stimulated twice with each of the respective compositions (peptide epitopes encoded in the minigene or the polyepitopic peptide), then assayed for peptide-specific cytotoxic activity in a ⁵¹Cr release assay. The results indicate the magnitude of the CTL response directed against the A2-restricted epitope, thus indicating the in vivo immunogenicity of the minigene vaccine and polyepitopic vaccine.

It is, therefore, found that the minigene elicits immune responses directed toward the HLA-A2 supermotif peptide epitopes as does the polyepitopic peptide vaccine. A similar analysis is also performed using other HLA- A3 and HLA-B7 transgenic mouse models to assess CTL induction by HLA- A3 and

HLA-B7 motif or supermotif epitopes, whereby it is also found that the minigene elicits appropriate immune responses directed toward the provided epitopes.

To 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-A^b- restricted mice, for example, are immunized intramuscularly with 100 μg of plasmid DNA. As a means of comparing the level of HTLs induced by DNA immunization, a group of control animals is also immunized with an actual peptide composition emulsified in complete Freund's adjuvant. CD4+ T cells, i.e. HTLs, are purified from splenocytes of immunized animals and stimulated with each of the respective compositions (peptides encoded in the minigene). The HTL response is measured using a ³H-thymidine incorporation proliferation assay, (see, e.g., Alexander et al. Immunity 1:751-761, 1994). The results indicate the magnitude of the HTL response, thus demonstrating the in vivo immunogenicity of the minigene.

DNA minigenes, constructed as described in the previous Example, can also be 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).

114 la-509107 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 K^b transgenic mice are immunized IM with 100 μg of a DNA minigene encoding the immunogenic peptides including at least one HLA-A2 supermotif-bearing peptide. After an incubation period (ranging from 3-9 weeks), the mice are boosted IP with 10⁷ pfu/mouse of a recombinant vaccinia virus expressing the same sequence encoded by the DNA minigene. Control mice are immunized with 100 μg of DNA or recombinant vaccinia without the minigene sequence, or with DNA encoding the minigene, but without the vaccinia boost. After an additional incubation period of two weeks, splenocytes from the mice are immediately assayed for peptide-specific activity in an ELISPOT assay. Additionally, splenocytes are stimulated in vitro with the A2-restricted peptide epitopes encoded in the minigene and recombinant vaccinia, then assayed for peptide-specific activity in an alpha, beta and/or gamma IFN ELISA.

It is found that the minigene utilized in a prime-boost protocol elicits greater immune responses toward the HLA-A2 supermotif peptides than with DNA alone. Such an analysis can also be performed using HLA-A11 or HLA-B7 transgenic mouse models to assess CTL induction by HLA- A3 or HLA-B7 motif or supermotif epitopes. The use of prime boost protocols in humans is described below in the Example entitled "Induction of CTL Responses Using a Prime Boost Protocol ."

Example 22: Peptide Composition for Prophylactic Uses

Vaccine compositions of the present invention can be used to prevent PHORl-All and/or PHOR1-F5D6 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 PHORl-Al 1- and/or PHOR1-F5D6- 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 50,000 μg, generally 100-5,000 μg, for a 70 kg patient. The initial administration of vaccine is followed by booster dosages at 4 weeks followed by evaluation of the magnitude of the immune response in the patient, by techniques that determine the presence of epitope-specific CTL populations in a PBMC sample. Additional booster doses are administered as required. The composition is found to be both safe and efficacious as a prophylaxis against PHORl-All- and/or PHORl-F5D6-associated disease.

115 la-509107 Alternatively, a composition typically comprising transfecting agents is used for the administration of a nucleic acid-based vaccine in accordance with methodologies known in the art and disclosed herein.

Example 23: Polyepitopic Vaccine Compositions Derived from Native PHORl-All and/or

PHOR1-F5D6 Sequences

Native PHORl-Al 1 and/or PHOR1-F5D6 polyprotein sequences are 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 PHORl-Al 1 and/or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6, . 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.

116 la-509107 Example 24: Polyepitopic Vaccine Compositions From Multiple Antigens

The PHORl-All and/or PHOR1-F5D6 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 PHORl-Al 1 and/or PHOR1-F5D6 and such other antigens. For example, a vaccine composition can be provided as a single polypeptide that incorporates multiple epitopes from PHORl-Al 1 and or PHOR1-F5D6 as well as tumor-associated antigens that are often expressed with a target cancer associated with PHORl-Al 1 and/or PHOR1-F5D6 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 PHORl-Al 1 and/or PHOR1-F5D6. 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, PHORl-Al 1 and/or PHOR1-F5D6 HLA-A*0201- specific CTL frequencies from HLA A*0201-ρositive individuals at different stages of disease or following immunization comprising a PHORl-Al 1 and/or PHOR1-F5D6 peptide containing an A*0201 motif.

Tetrameric complexes are synthesized as described (Musey et al, N. Engl. J. Med. 337:1267, 1997).

Briefly, purified HLA heavy chain (A*0201 in this example) and β2-microglobulin are synthesized by means of a prokaryotic expression system. The heavy chain is modified by deletion of the transmembrane- cytosolic tail and COOH-terminal addition of a sequence containing a BirA enzymatic biotinylation site.

The heavy chain, β2-microglobulin, and peptide are refolded by dilution. The 45-kD refolded product is isolated by fast protein liquid chromatography and then biotinylated by BirA in the presence of biotin

(Sigma, St. Louis, 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 μl of cold phosphate-buffered saline. Tri-color analysis is

117 la-509107 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-ρositive 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 PHORl-Al 1 and/or PHOR1-F5D6 epitope, and thus the status of exposure to PHORl-Al 1 and/or PHOR1-F5D6, 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 PHORl-Al 1- and/or PHORl-F5D6-associated disease or who have been vaccinated with a PHORl- Al 1 and/or PHOR1-F5D6 vaccine. For example, the class I restricted CTL response of persons who have been vaccinated may be analyzed. The vaccine may be any PHORl-Al 1 and/or PHOR1-F5D6 vaccine. PBMC are collected from vaccinated individuals and HLA typed. Appropriate peptide epitopes of the invention that, optimally, bear supermotifs to provide cross-reactivity with multiple HLA supertype family members, are then used for analysis of samples derived from individuals who bear that HLA type. PBMC from vaccinated individuals are separated on Ficoll-Histopaque density gradients (Sigma

Chemical Co., St. Louis, MO), washed three times in HBSS (GIBCO Laboratories), resuspended in RPMI- 1640 (GIBCO Laboratories) supplemented with L-glutamine (2mM), penicillin (50U/ml), streptomycin (50 μg/ml), and Hepes (lOmM) containing 10% heat-inactivated human AB serum (complete RPMI) and plated using microculture formats. A synthetic peptide comprising an epitope of the invention is added at 10 μg/ml to each well and HBV core 128-140 epitope is added at 1 μg ml to each well as a source of T cell help during the first week of stimulation.

In the microculture format, 4 x 10⁵ PBMC are stimulated with peptide in 8 replicate cultures in 96- well round bottom plate in 100 μl/well of complete RPMI. On days 3 and 10, 100 UL of complete RPMI and 20 U/ml final concentration of rIL-2 are added to each well. On day 7 the cultures are transferred into a 96-well flat-bottom plate and restimulated with peptide, rIL-2 and 10⁵ irradiated (3,000 rad) autologous feeder cells. The cultures are tested for cytotoxic activity on day 14. A positive CTL response requires two or more of the eight replicate cultures to display greater than 10% specific ⁵¹Cr release, based on comparison with non-diseased control subjects as previously described (Rehermann, et al, Nature Med.

118 la-509107 2:1104,1108, 1996; Rehermann et al, J. Clin. Invest. 97:1655-1665, 1996; and Rehermann et al. J. Clin. Invest. 98: 1432-1440, 1996).

Target cell lines are autologous and allogeneic EB V-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-.2670-2678¹, 1992).

Cytotoxicity assays are performed in the following manner. Target cells consist of either allogeneic HLA-matched or autologous EB V-transformed B lymphoblastoid cell line that are incubated overnight with the synthetic peptide epitope of the invention at 10 μM, and labeled with 100 μCi of ^5ICr (Amersham Corp., Arlington Heights, EL) for 1 hour after which they are washed four times with HBSS. Cytolytic activity is determined in a standard 4-h, split well ⁵¹Cr release assay using U-bottomed

96 well plates containing 3,000 targets/well. Stimulated PBMC are tested at effector/target (E/T) ratios of 20-50:1 on day 14. Percent cytotoxicity is determined from the formula: 100 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 PHORl-Al 1 and/or PHOR1-F5D6, or a PHORl-Al 1 and/or PHOR1-F5D6 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 1.5xl0⁵ cells/well and are stimulated with 10 μg/ml synthetic peptide of the invention, whole PHORl-Al 1 and or PHOR1-F5D6 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 μCi ³H-thymidine is added to each well and incubation is continued for an additional 18 hours. Cellular DNA is then harvested on glass fiber mats and analyzed for ³H-thymidine incorporation. Antigen-specific T cell proliferation is calculated as the ratio of ³H-thymidine incorporation in the presence of antigen divided by the ³H-thymidine incorporation in the absence of antigen.

Example 27: Induction Of Specific CTL Response In Humans A human clinical trial for an immunogenic composition comprising CTL and HTL epitopes of the invention is set up as an IND Phase I, dose escalation study and carried out as a randomized, double-blind, placebo-controlled trial. Such a trial is designed, for example, as follows:

A total of about 27 individuals are enrolled and divided into 3 groups:

119 la-509107 Group I: 3 subjects are injected with placebo and 6 subjects are injected with 5 μg of peptide composition;

Group II: 3 subjects are injected with placebo and 6 subjects are injected with 50 μg peptide composition; Group III: 3 subjects are injected with placebo and 6 subjects are injected with 500 μg of peptide composition.

After 4 weeks following the first injection, all subjects receive a booster inoculation at the same dosage.

The endpoints measured in this study relate to the safety and tolerability of the peptide composition as well as its immunogenicity. Cellular immune responses to the peptide composition are an index of the intrinsic activity of this the peptide composition, and can therefore be viewed as a measure of biological efficacy. The following summarize the clinical and laboratory data that relate to safety and efficacy endpoints.

Safety: The incidence of adverse events is monitored in the placebo and drug treatment group and assessed in terms of degree and reversibility.

Evaluation of Vaccine Efficacy: For evaluation of vaccine efficacy, subjects are bled before and after injection. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll- Hypaque density gradient centrifugation, aliquoted in freezing media and stored frozen. Samples are assayed for CTL and HTL activity. The vaccine is found to be both safe and efficacious.

Example 28: Phase II Trials In Patients Expressing PHORl-All and/or PHOR1-F5D6

Phase II trials are performed to study the effect of administering the CTL-HTL peptide compositions to patients having cancer that expresses PHORl-Al 1 and/or PHOR1-F5D6. The main objectives of the trial are to determine an effective dose and regimen for inducing CTLs in cancer patients that express PHORl-Al 1 and/or PHOR1-F5D6, 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.

120 la-509107 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 PHORl-Al 1 and/or PHOR1-F5D6. 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 PHORl-Al 1- and/or PHORl-F5D6-associated disease.

Example 29: Induction of CTL Responses Using a Prime Boost Protocol A prime boost protocol similar in its underlying principle to that used to 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 μg) can also be administered using a gene gun. Following an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-10⁷ to 5xl0⁹ 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 PHORl-Al 1 and/or PHOR1-F5D6 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

121 la-509107 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 PHORl-Al 1 and/or PHOR1-F5D6 ρrotein(s) 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 Progenipoietin™ are injected into patients without purification of the DC. The total number of PBMC that are administered often ranges from 10⁸ to 10¹⁰. Generally, the cell doses injected into patients is based on the percentage of DC in the blood of each patient, as determined, for example, by immunofluorescence analysis with specific anti-DC antibodies. Thus, for example, if 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 Progenipoietin™ 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 PHORl-All and/or PHOR1-F5D6 antigens can be induced by incubating, in tissue culture, the patient's, or genetically compatible, CTL or HTL precursor cells together with a source of APC, such as DC, and immunogenic peptides. After an appropriate incubation time (typically about 7-28 days), in which the precursor cells are activated and expanded into effector cells, the cells are infused into the patient, where they will destroy (CTL) or facilitate destruction (HTL) of their specific target cells, i.e., tumor cells.

Example 31: An Alternative Method of Identifying 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

122 la-509107 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, i.e., PHORl-All and/or PHOR1-F5D6. 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 PHORl-Al 1 and/or PHOR1-F5D6 to isolate peptides corresponding to PHORl-Al 1 and or PHOR1-F5D6, respectively, that have been presented on the cell surface. Peptides obtained from such an analysis will bear motif(s) that correspond to binding to the single HLA allele that is expressed in the cell.

As appreciated by one in the art, one can perform a similar analysis on a cell bearing more than one HLA allele and subsequently determine peptides specific for each HLA allele expressed. Moreover, one of skill would also recognize that means other than transfection, such as loading with a protein antigen, can be used to provide a source of antigen to the cell.

Example 32: Complementary Polynucleotides

Sequences complementary to the PHORl-Al 1- and/or PHORl-F5D6-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring PHORl-All and/or PHOR1-F5D6, respectively. 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 PHORl-Al 1 and/or PHOR1-F5D6. 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 PHORl-Al 1- and/or PHORl-F5D6-encoding transcript(s).

123 la-509107 Example 33: Purification of Naturally-occurring or Recombinant PHORl-All and/or PHOR1-F5D6 Using Corresponding PHORl-All and/or PHOR1-F5D6 Specific Antibodies

Naturally occurring or recombinant PHORl-Al 1 and/or PHOR1-F5D6 is substantially purified by immunoaffinity chromatography using antibodies specific for PHORl-Al 1 and/or PHOR1-F5D6. An immunoaffinity column is constructed by covalently coupling anti-PHORl-Al 1 and/or PHOR1-F5D6 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 PHORl-Al 1 and/or PHOR1-F5D6 are passed over theimmunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of PHORl-Al 1 and/or PHOR1-F5D6 (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody/PHORl-Al 1 and/or PHOR1-F5D6 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 PHORl-All and/or PHOR1-

F5D6

PHORl-All and/or PHOR1-F5D6, or biologically active fragments thereof, are labeled with 121 -I 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 PHORl-All and/or PHOR1-F5D6, washed, and any wells with labeled PHORl-Al 1 and/or PHOR1-F5D6 complex are assayed. Data obtained using different concentrations of PHORl-Al 1 and/or PHOR1-F5D6 are used to calculate values for the number, affinity, and association of PHORl-Al 1 and/or PHOR1-F5D6 with the candidate molecules, respectively.

Example 35: In Vivo Assay for PHORl-All and PHOR1-F5D6 Tumor Growth Promotion

The effect of PHORl-Al 1 and PHOR1-F5D6 on tumor cell growth is confirmed in vivo by, respective, gene overexpression in tumor-bearing mice. For example, SCID mice are injected SQ on each flank with 1 x 10⁶ of either PC3, TSUPR1, DU145 cells or other prostate, bladder and kidney cell line containing tkNeo empty vector PHORl-Al 1 or PHOR1-F5D6. At least two strategies may be used: (1) Constitutive PHORl-Al 1 or PHOR1-F5D6 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

124 la-509107 immunoglobulin promoter, provided such promoters are compatible with the host cell systems. (2) Regulated expression under control of an inducible vector system, such as ecdysone, tet, etc., are used provided such promoters are compatible with the host cell systems.

Tumor volume is then monitored at the appearance of palpable tumors and is followed over time to confirm that PHORl-Al 1-expressing or PHORl-F5D6-exρressing cells, respectively, grow at a faster rate and that tumors produced by PHORl-Al 1 or PHORl-F5D6-expressing cells have altered aggressiveness (e.g. enhanced metastasis, vascularization, reduced responsiveness to chemotherapeutic drugs).

Additionally, mice are implanted with 1 x 10⁵ of the same cells orthotopically to confirm that PHORl-Al 1 or PHOR1-F5D6, respectively, have an effect on local growth in the prostate and on the ability of the cells to metastasize, specifically to lungs, lymph nodes, and bone marrow. Also see Saffran et al, "Anti-PSCA mAbs inhibit tumor growth and metastasis formation and prolong the survival of mice bearing human prostate cancer xenografts" PNAS 10:1073-1078.

The assay also determines that PHORl-Al 1 or PHOR1-F5D6 have inhibitory effects on therapeutic compositions, such as for example, PHORl-Al 1 or PHOR1-F5D6 intrabodies, PHORl-Al 1 or PHORl -F5D6 antisense molecules and ribozymes.

Example 36: PHORl-All and/or PHOR1-F5D6 Monoclonal Antibody-mediated Inhibition of Prostate Tumors In Vivo The significant expression of PHORl-Al 1 and PHOR1-F5D6, in cancer tissues, together with their restricted expression in normal tissues along with its expected cell surface expression makes PHORl- Al 1 and PHOR1-F5D6 excellent targets for antibody therapy. Moreover, PHORl-All and PHOR1-F5D6 are targets for T cell-based immunotherapy. Thus, the therapeutic efficacy of anti-PHORl-Al 1 and anti- PHOR1-F5D6 mAbs in human prostate cancer xenograft mouse models is confirmed by using androgen- independent LAPC-4 and LAPC-9 xenografts (Craft, R, et al,. Cancer Res, 1999.59(19): p. 5030-6) and the androgen independent recombinant cell line PC3-121P2A3 (see, e.g., Kaighn, M.E., et al, Invest Urol, 1979. 17(1): p. 16-23). Similarly, the therapeutic effects of anti-PHORl-Al 1 and anti-PHORl -F5D6 mAbs on kidney cancer are confirmed using xenograft models. As indicated in Example 36, PHORl-Al 1 and PHOR1-F5D6 are predicted to be cell surface proteins. Antibody efficacy on tumor growth and metastasis formation is confirmed, e.g., in a mouse orthotopic prostate and kidney cancer xenograft model. The antibodies can be unconjugated or can be conjugated to a therapeutic modality, as appreciated in the art. Anti-PHORl-Al 1 and anti-PHORl-F5D6 mAbs inhibit formation of both the androgen-dependent LAPC-9 and androgen-independent PHORl-All or PHOR1-F5D6 expressing PC3 tumor xenografts. Anti-PHORl-Al 1 and anti-PHORl-F5D6 mAbs also

125 la-509107 retard the growth of established orthotopic tumors and prolonged survival of tumor-bearing mice. These results indicate the utility of anti-PHORl-Al 1 and anti-PHORl-F5D6 mAbs in the treatment of local and advanced stages of prostate cancer. (See, e.g., Saffran, D., et al., PNAS 10:1073-1078.

Administration of the anti-PHORl-Al 1 and anti-PHORl-F5D6 mAbs retards established orthotopic tumor growth and inhibits metastasis to distant sites, resulting in a significant prolongation in the survival of tumor-bearing mice. These studies confirm that PHORl-Al 1 and PHOR1-F5D6 are attractive targets for immunotherapy and demonstrate the therapeutic efficacy of anti-PHORl-Al 1 and anti-PHORl- F5D6mAbs for the treatment of local and metastatic prostate and kidney cancer.

Tumor inhibition using multiple unconjugated mAbs

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. Subcutaneous (s.c.) tumors are generated by injection of 1 x 10⁶ cells such as LAPC-9, PC3, 3T3 or A-498 cells expressing or lacking PHORl-Al 1 and PHOR1-F5D6 cells mixed at a 1:1 dilution with Matrigel (Collaborative Research) in the right flank of male SCID mice. To confirm 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.

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-μl 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 confirm the effect of anti-PHORl-Al 1 and anti-PHORl -F5D6 mAbs on established orthotopic tumors, i.p. antibody injections are started when PSA levels reach 2-80 ng/ml.

Anti- PHORl-All and PHOR1-F5D6 mAbs Inhibit Growth of PHORl-All and PHOR1-F5D6- Expressing Cancer Tumors

The effect of anti- PHORl-All and PHOR1-F5D6 mAbs on tumor formation is confirmed by use of the prostate and kidney orthotopic model. As compared with the s.c. tumor model, the orthotopic model, which requires injection of tumor cells directly in the mouse prostate or kidney, results in a local tumor growth, development of metastasis in distal sites, deterioration of mouse health, and subsequent death

126 la-509107 (Saffran, D., et al., PNAS supra; Fu, X., et al., Int J Cancer, 1992. 52(6): p. 987-90; Kubota, T., J Cell Biochem, 1994. 56(1): p. 4-8). The features make the orthotopic model more representative of human disease progression and allow monitoring of the therapeutic effect of PHORl-All and PHOR1-F5D6 mAbs, respectively, on clinically relevant end points. For example, 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) 50-2000μg, usually 200-500μg, of anti-PHORl- Al 1 or anti- PHOR1-F5D6 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 and kidney cancer models is the ability to study the development of metastases. Formation of metastasis in mice bearing established orthotopic tumors is determined by IHC analysis on lung sections using an antibody against a prostate-specific or kidney- specific cell-surface protein such as STEAP-1 for prostate xenografts (Hubert, R.S., etal, Proc Natl Acad Sci U S A, 1999. 96(25): p. 14523-8). Mice bearing established orthotopic tumors are administered ■ 1000μg⁽ injections of either anti-PHORl-Al 1, anti-PHORl-F5D6 mAb or PBS over a 4-week period. Mice in both groups are allowed to establish a high tumor burden, to ensure a high frequency of metastasis formation in mouse lungs. Mice then are sacrificed and their prostate, kidney and lungs are analyzed for the presence of cancer cells by IHC analysis.

These studies demonstrate a broad anti-tumor efficacy of anti-PHORl-Al 1 and anti-PHORl- F5D6 antibodies, respectively, on initiation and progression of prostate and kidney cancer in xenograft mouse models. Thus, the effect of anti-PHORl-All and anti-PHORl-F5D6 mAbs, respectively, on major clinically relevant end points including tumor growth, prolongation of survival, and health is confirmed.

Example 37A: Comparison of PHORl-All to known sequences

The PHORl-All protein has 314 amino acids with calculated molecular weight of 34.76 kDa, and pi of 8.86. PHORl-Al 1 is a cell surface protein, predicted to have 7 transmembranes that span the cytoplamic membrane, with the N-terminus predicted to be extracellular, and the C-terminus to be intracellular.

At the protein level, PHORl-Al 1 shows best homology to a human olfactory receptor of the B2 family (gi 14423800) [http://www.ncbi.nlm.nih.gov/entrez], with 83% identity and 92% homology. PHORl-Al 1 also shows homology to another family of olfactory receptors, namely human olfactory receptor 2Wl(gil4423828) with 60% identity and 78% homology.

127 la-509107 Example37B: Comparison of PHOR1-F5D6 to known sequences

The PHOR1-F5D6 protein consists of 310 amino acids with calculated molecular weight of 34.8 kDa, and pi of 9.18. Like PHORl-All, PHOR1-F5D6 is a cell surface protein, predicted to have 7 transmembrane domains that span the cytoplamic membrane, with the N-terminus predicted to be extracellular, and the C-terminus to be intracellular.

PHOR1-F5D6 shows best homology to the human olfactory receptor of the 2A sub-family (gi 13929212) [http://www.ncbi.nlm.nih.gov/entrez], with 100% identity and 100% homology.

Olfactory receptor genes represent approximately 1% of genomic coding sequence in mammals, representing approximately 1000 genes and pseudogenes (Lane et al, Proc Natl Acad Sci U S A. 2001, 98:7390). Olfactory receptors are G-protein-coupled receptors that recognize and respond to specific ligands, including odorant molecules, neuropeptides, phospholipids, etc (Civelli O, et al. Trends Neurosci. 2001, 24:230; Schoneberg T et al, Biochim Biophys Acta. 1999, 1446:57). Most olfactory receptors transmit signals from the cell membrane by activating cAMP-dependent pathways and associating with protein kinases and surface receptors (Pilpel Y et al, Essays Biochem 1998;33:93; Liebmann C, and Bohmer FD, Curr Med Chem 2000, 7:911). In humans, a subset of olfactory GPCR genes is expressed in testis and/or prostate (Goto T et al, Mol Hum Reprod. 2001, 7:553; Xu L et al, Cancer Res. 2000, 60:6568) indicating that olfactory receptors recognize signaling molecules necessary for reproduction and play a role in chemotaxis of spermatozoa towards the oocyte.

The tumor-associated expression of GPCR has been studied in patient samples (Xu L et al, Cancer Res. 2000, 60:6568; Bais C et al, Nature. 1998, 391:86). Such studies have reported increased expression and/or activation of GPCR in tumor samples. For example, the prostate specific GPCR, PSGR is over- expressed in prostate cancer (Xu L et al, Cancer Res. 2000, 60:6568), and KSHV-GPCR is constitutively active in Kaposi's sarcoma (Arvanitakis L et al, Nature 1997, 385:347). Over-expression of several GPCRs, including KSHV-GPCR and G2A, in engineered cells resulted in transformation indicating that these GPCRs function as oncogenes (Zhon I et al, Oncogene. 2000,19:3866). In addition, these and other GPCRs have been shown to regulate essential biological functions including cell growth and angiogenesis (Liu Y et al, J Clin Invest. 2000, 106:951; Bais C et al, Nature. 1998, 391:86). Accordingly, based on these data, PHORl-All and PHOR1-F5D6 control tumor growth and progression by regulating cell proliferation, mitogenesis, transformation and vessel formation. Example 38: Identification of Signal Transduction Pathways

Many mammalian proteins have been reported to interact with signaling molecules and to participate in regulating signaling pathways (J Neurochem. 2001; 76:217-223). In particular, GPCR have been shown to regulate several MAPK and phospholipid cascades (Liebmann C and Bohmer FD, Curr Med

128 la-509107 Chem. 2000, 7:911). Using immunoprecipitation and Western blotting techniques, proteins that associate with PHORl-All and/or PHOR1-F5D6 are identified, and used to mediate signaling events. Several pathways known to play a role in cancer biology are regulated by 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 PHOR-1 to regulate of these pathways is confirmed. Cells expressing PHORl-Al 1 or PHOR1-F5D6 and cells lacking these genes are either left untreated or stimulated with cytokines, androgen and anti-integrin antibodies. Cell lysates are analyzed using anti-phosphos-specific antibodies (Cell Signaling, Santa Cruz Biotechnology) in order to detect phosphorylation and regulation of ERK, p38, AKT, PI3K, PLC and other signaling molecules. Using the . same Western blotting approach, antibodies generated against PHORl-Al 1 and PHOR1-F5D6 are found to have the ability to modulate the activity of one. or more signaling pathways. When PHORl-Al 1 and/or PHORl -F5D6 play a role in the regulation of signaling pathways, whether individually or communally, they are used as a target for diagnostic, preventative and therapeutic purposes.

To conform that PHORl-All and/or PHOR1-F5D6 directly or indirectly activate 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/ELKl; MAPK/SAPK; growth/differentiation

3 AP-l-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

PHOR-1-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.

129 la-509107 Signaling pathways activated by PHORl-Al 1 and or PHOR1-F5D6 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, prognostic, preventative and therapeutic purposes.

Example 39: Involvement in Tumor Progression

PHORl-Al 1 and or PHOR1-F5D6 contribute to the growth of cancer cells, as has been shown for other GPCRs (Liu Y et al, J Clin Invest. 2000, 106:951; Bais C et al, Nature. 1998, 391:86). The role of these genes in tumor growth is conformed 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 PHORl-Al 1 or PHOR1-F5D6. Parental cells lacking either of the genes of interest and cells expressing the gene are evaluated for cell growth using a well-documented proliferation assay (Fraser SP, et al., Prostate 2000;44:61, Johnson DE, et al., Anticancer Drugs 1996, 7:288).

To confirm the role of each gene in the transformation process, colony forming assays are used. Parental NIH3T3 cells lacking PHORl-Al 1 or PHOR1-F5D6 are compared to NHI-3T3 cells expressing PHORl-Al 1 or PHOR1-F5D6, using a soft agar assay under stringent and more permissive conditions (Song, Z., et al. Cancer Res. 2000; 60:6730).

To confirm the role of PHORl-All and PHOR1-F5D6 in invasion and metastasis of cancer cells, the well-established Transwell Insert System assay (Becton Dickinson) is used (Cancer Res. 1999; 59:6010). Control cells, including prostate, colon, bladder and kidney cell lines lacking PHORl-All or PHOR1-F5D6 are compared to cells expressing the PHORl-Al 1 or PHOR1-F5D6. 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. In another iteration of this assay the role of PHORl-Al 1 and PHOR1-F5D6 in invasion and metastasis of cancer cells is confirmed, whereby recombinant forms of PHORl-Al 1 and PHOR1-F5D6 are evaluated for their ability to induce cell migration and invasion. In this embodiment, calcein loaded cells are incubated in media alone or in the presence of protein products encoded by PHORl-Al 1 or PHOR1-F5D6. Invasion is determined by measuring the fluorescence of cells in the lower chamber of the Transwell system.

Similarly, the role of PHORl-Al 1 and PHOR1-F5D6 in invasion and metastasis of cancer cells is confirmed by use of antibodies directed to products of PHORl-Al 1 and PHOR1-F5D6 are evaluated for their effect on invasion, migration and tumor progression.

PHORl-Al 1 and/or PHOR1-F5D6 play a role in cell cycle and apoptosis as shown for other GPCRs including G2A, PAFR and KSHV-GPCR (Zhon I et al, Oncogene. 2000,19:3866; Bussolati et al,

130 la-509107 Am J Pathol. 2000, 157:1713; Montaner S et al, Cancer Res. 2001, 61:2641). Parental cells and cells expressing PHORl-All and/or PHOR1-F5D6 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 PHORl-Al 1 or PHOR1-F5D6, 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.

These protocols confirm that PHORl-Al 1 and PHOR1-F5D6 play a role in cell growth, transformation, invasion or apoptosis. Thsu, these genes and their products are used as targets for diagnostic, preventative and therapeutic purposes.

Example 40: Involvement in Angiogenesis

Angiogenesis, or new capillary blood vessel formation, is necessary for tumor growth (Hanahan D, Folkman J. Cell. 1996, 86:353; Folkman J. Endocrinology. 1998 139:441). As mentioned above, several GPCRs have been shown to regulate blood vessel formation and angiogenesis (Bussolati et al, Am J Pathol. 2000, 157:1713; Montaner S et al, Cancer Res. 2001, 61:2641). Several assays have been developed to measure angiogenesis in vitro and in vivo, including two tissue culture assays, namely endothelial cell tube formation and endothelial cell proliferation. Using these assays as well as in vitro neo-vascularization, it is confirmed that PHORl-Al 1 and PHOR1-F5D6 each modulate angiogenesis. For example, endothelial cells and cell lines are plated on an artificial basement membrane, such as matrigel, in the presence or absence the proteins encoded by PHORl-Al 1 or PHOR1-F5D6, respectively. The effect of PHORl-Al 1 or PHOR1-F5D6 on vessel formation is confirmed using light microscopy.

In another embodiment, the role PHORl-Al 1 and PHOR1-F5D6 in angiogenesis is confirmed by use of endothelial cells engineered to express PHORl-Al 1 or PHOR1-F5D6, when these cells are used in tube formation and proliferation assays.

The effect of PHORl-Al 1 and PHOR1-F5D6 is also confirmed in animal models in vivo. For example, cells either expressing or lacking PHORl-Al 1 or PHOR1-F5D6 are implanted subcutaneously in immunocompromized mice. Endothelial cell migration and angiogenesis are evaluated 5-15 days later using immunohistochemistry techniques, confirming the role of PHORl-Al 1 and PHOR1-F5D6 in angiogenesis. When products of PHOR-1 affect angiogenesis, they are used as targets for diagnostic, preventative and therapeutic purposes

131 la-509107 Example 41: Regulation of Transcription

In accordance with the present invention, PHORl-All and PHOR1-F5D6 play a role in transcriptional regulation of eukaryotic genes. Their regulation of gene expression is confirmed by gene ^■ expression in cells expressing or lacking PHORl-Al 1 and/or PHOR1-F5D6. For this purpose, two types of protocols are used.

In the first protocol, RNA from parental cells that do not express PHORl-Al 1 or PHOR1-F5D6 and cells that do, respectively are extracted and hybridized to commercially available gene arrays (Clontech) (Smid-Koopman E et al. Br J Cancer. 2000. 83:246). Resting cells as well as cells treated with FBS or androgen are compared. Differentially expressed genes are identified in accordance with procedures known in the art. The differentially expressed genes, from the cells that express PHORl-All and PHOR1-F5D6, relative to parental cells that do not express the genes of the present invention, respectively, are then mapped to biological pathways (Chen K et al. Thyroid. 2001. 11:41.).

In a second protocol, specific transcriptional pathway activation by a gene of the inevtion is conformed 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 confirm the role PHORl-All and PHORl -F5D6 in pathway activation and as positive and negative modulators of pathway activation. When PHORl-Al 1 and/or PHOR1-F5D6 play a role in gene regulation, they are used as a target ^v for diagnostic, prognostic, preventative and therapeutic purposes.

Example 42: Subcellular Localization and Cell Binding

The cellular location of PHORl-All and PHORl -F5D6 is confirmed using subcellular fractionation techniques widely used in cellular biology (Storrie B, et al. Methods Enzymol. 1990;182:203- 25). A variety of cell lines, including prostate, kidney and bladder cell lines as well as cell lines engineered to express PHORl-All or PHORl-F5D6 are separated into nuclear, cytosolic and membrane fractions. Gene expression and location in nuclei, heavy membranes (lysosomes, peroxisomes, and mitochondria), light membranes (plasma membrane and endoplasmic reticulum), and soluble protein fractions are confirmed by Western blotting techniques.

Alternatively, 293T cells are transfected with an expression vector encoding individual genes, HIS-tagged (PCDNA 3.1 MYC/EHS, Invitrogen) and the subcellular localization of these genes is determined as described above. In short, the transfected cells are harvested and subjected to a differential

132 ^' la-509107 subcellular fractionation protocol (Pemberton, P. A. et al, 1997, J of Histochemistry and Cytochemistry, 45:1697-1706.) Location of the HIS-tagged genes is followed by Western blotting.

Using anti-PHORl-Al 1 and anti-PHORl-F5D6 antibodies, cellular localization is demonstrated by immunofluorescence and immunohistochemistry. For example, cells expressing or lacking PHORl-All or PHOR1-F5D6, respectively, are adhered to a microscope slide and stained with anti-PHORl-Al 1 or anti-PHORl-F5D6 specific Ab. Cells are incubated with an FITC-coupled secondary anti-species Ab, and analyzed by fluorescent microscopy. Alternatively, cells and tissues lacking or expressing PHORl-Al 1 or PHOR1-F5D6, respectively, are analyzed by IHC as described in section above.

When products of PHORl-Al 1 or PHOR1-F5D6 are localized to specific cell compartments, they are used as a target for diagnostic, preventative and therapeutic purposes.

Example 43: Protein and Ion Transporter Function

Using a modified rhodamine retention assay (Davies J et al. Science 2000, 290:2295; Leith C et al. Blood 1995, 86:2329) it is determine that PHORl-Al 1 and PHOR1-F5D6 function as protein transporters. Cell lines, such as prostate, colon, bladder and kidney cancer and normal cells, expressing or lacking a gene of the invention 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. 1994. 1191:384).

Information obtained from such experiments confirms that PHORl-Al 1 and PHOR1-F5D6 serve 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 also confirms that PHORl-Al 1 and/or PHOR1-F5D6 function in transporting ions and other small molecules. When PHORl-All or PHOR1-F5D6 function as a transporter, the gene(s) is used as a target for preventative, prognostic and therapeutic purposes as well as to evaluate drug sensitivity/resistance. To confirm that PHORl-Al 1 and PHOR1-F5D6 function as an ion channels, FACS analysis and electrophysiology techniques are used (Gergely L, et al, Clin Diagn Lab Immunol. 1997; 4:70; Skryma R, et al., J Physiol. 2000, 527: 71). Using FACS analysis and commercially available indicators (Molecular Probes), parental cells and cells expressing a gene of the invention are compared for their ability to transport calcium, sodium or potassium. Prostate, colon, bladder and kidney or other normal cells and tumor cell lines are used in these procedures. 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 procedures provides a mechanism by which cancer cells are regulated. This is particularly true in the case of calcium, as calcium channel inhibitors have been reported

133 la-509107 to induce the death of certain cancer cells, including prostate cancer cell lines (Batra S, et al., Prostate 1991, 19: 299).

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 confirm the function of PHORl-All and PHOR1-F5D6 as ion channels (Schweitz H. et al. Proc. Natl. Acad. Sci. 1994. 91:878; Skryma R. et al. Prostate. 1997. 33:112).

Using any of the assays listed above, the effect of antibodies directed against PHORl-Al 1 and PHOR1-F5D6 on ion and protein transport is confirmed. Similarly, these assays can be used to identify and evaluate small molecules that modulate ion and protein transport. When PHORl-Al 1 and/or PHOR1- F5D6 function as an ion channel, they are used as a target for diagnostic, preventative and therapeutic purposes.

Example 44: Protein-Protein Interactions

Several GPCRs, including olfactory receptors, have been shown to associate with essential proteins that regulate their function, including G protein-coupled receptor kinases (GRKs), receptor tyrosine kinases (RTKs), integrin-based focal adhesions, etc (Penn RB, Trends Cardiovasc Med. 2000,10:81; Luttrell L et al, Curr Opin Cell Biol. 1999, 11:177. Using immunoprecipitation techniques as well as two yeast hybrid systems, proteins are identified that associate with PHORl-Al 1 and PHOR1- F5D6. Immunoprecipitates from cells expressing and cells lacking PHORl-Al 1 or PHOR1-F5D6, respectively, are compared for specific protein-protein associations. Studies comparing PHORl-Al 1 and PHOR1-F5D6 positive to 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 protein-protein interactions.

In addition, protein-protein interactions are studied using two yeast hybrid methodology (Curr Opin Chem Biol. 1999, 3:64). A vector carrying a library of proteins fused to the activation domain of a transcription factor is introduced into yeast expressing a PHOR-1 -DNA-binding domain fusion protein and a reporter construct. Protein-protein interaction is detected by calorimetric reporter activity. Specific association with effector molecules and transcription factors conforms for one of skill the mode of action of PHORl-All and PHOR1-F5D6, 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 PHORl-All and or PHOR1-F5D6.

134 la-509107 When PHORl-Al 1 or PHOR1-F5D6 associates with proteins or small molecules they are used as a target for diagnostic, prognostic, preventative and therapeutic purposes.

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.

135 la-509107 TABLES

TABLE I: Tissues that Express PHORl-All or PHOR1-F5D6 When Malignant

PHORl-All: Tissues that express the gene when malignant

Prostate

Ovary

Bladder

PHOR1-F5D6: Tissues that express the gene when malignant

Prostate

Kidney

Ovary

TABLE II: AMINO ACID ABBREVIATIONS

136 la-509107

137 la-509107 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.chmanualblosum62.html )

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

4 0 -2 -1 -2 0 -2 -1 -1 -1 -1 -2 -1 -1 -1 1 0 0 -3 -2 A 9 -3 -4 -2 -3 -3 -1 -3 -1 -1 -3 -3 -3 -3 -1 -1 -1 -2 -2 C

6 2 -3 -1 -1 -3 -1 -4 -3 1 -1 0 -2 0 -1 -3 -4 -3 D

5 -3 -2 0 -3 1 -3 -2 0 -1 2 0 0 -1 -2 -3 -2 E

-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

138 la-509107 TABLE IV A

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

139 la-509107 TABLE IV C

MOTIFS 1° anchor 1 1° anchor 6

DR4 preferred FMY /VW M T I VSTCPAL/M MH MH deleterious W R WDE

DRl preferred MFLiY Y PAMQ VMAΥSPLIC M AVM deleterious C CH FD CWD GDE D

DR7 preferred MFLIVWY M W A IVMSAC7/P M TV deleterious C G GRD N G

DR3 MOTIFS 1° anchor 1 1° anchor 4 1° anchor 6 motif a LIVMFY D preferred motif b LIVMFAY DNQEST KRH

DR MFLIVWY VMSTACPL7

Jupermotif

Italicized residues indicate less preferred or "tolerated" residues.

TABLE IV D

POSITION

C-term

SUPERMOTIFS

Al 1° Anchor 1° An TΪLVMS FW

A2 , 1° Anchor 1° An UVMATQ LIVM

A3 preferred 1° Anchor YFW(4/5 YFW (3/5) YFW (4/5) P (4/5) 1° An VSMAT/ / ) R deleterious DE (3/5); DE (4/5) P (5/5)

A24 1° Anchor 1° An YF ZVZΛtr FIYW

B7 preferred FWY (5/5) 1° Anchor FWY(4/5 FWY (3/5) 1 "Anc LIVM (3/5) P ) VB F deleterious DE (3/5); DE(3/5) 0(4/5) QN(4/5) DE(4/5)

P(5/5);

B27 1° Anchor 1 "Anc SHK FYLW

< o u o c ^•8 «

< <

3

TABLE IV E

4 C-ter or C-terminus

Al preferred GFYW 1 "Anchor DEA YFW DEQN YFW TAnchor -mer STM Y deleterious DE RHKLIVMP G A

Al preferred GRHK ASTCLIVM 1 "Anchor GSTC ASTC LΓVM DE 1 "Anchor -mer DEAS Y deleterious A RHKDEPY DE PQN RHK PG GP

FW

Al preferred YFW 1 "Anchor DEAQN YFWQN PASTC GDE 1 "An 0-mer STM deleterious GP RHKGLIVM DE RHK QNA RHKYFW RHK

Al preferred YFW STCLIVM 1 "Anchor YFW PG YFW TAn 0-mer ΌΈAS deleterious RHK RHKDEPY G PRHK QN FW

A2.1 preferred YFW l°Anchor YFW STC YFW l°Anchor -mer IMTVQAT YUM AT deleterious DEP DERKH RKH DERKH

C-ter

A2.1 preferred AYFW T Anchor LVIM FYWL 1 "An 10-mer IMTVQAT VIM VLI deleterious DEP DE RKHA RKH DERKH RKH

A3 preferred RHK T Anchor YFW PRHKYFW YFW 1 "Anchor

LMVISATF KYRff A

CGD deleterious DEP DE

All preferred 1 "Anchor YFW YFW YFW YFW 1 "Anchor

VTLMISAG KRYH

NCDF deleterious DEP G

A24 preferred YFWRHK l°Anchor STC YFW YFW 1 "Anchor

9-mer YFWM FLIW deleterious DEG DE G QNP DERHK AQN

A24 preferred 1 "Anchor YFWP TAn 10-mer YFWM FL deleterious GDE QN RHK DE QN DEA

A3101 preferred RHK 1 "Anchor YFW YFW YFW AP 1 "Anchor MVTA 7S RK

C-ter deleterious DEP DE ADE DE DE DE

A3301 preferred TAnchor YFW AYFW TAnchor

MVALF/SΓ RK deleterious GP DE

A6801 preferred YFWSTC TAnchor YFWIIVM YFW TAnchor AVTMSLI RK deleterious GP DEG RHK

B0702 preferred RHKFWY TAnchor RHK RHK RHK RHK PA TAnchor P LMFWYAIV deleterious DEQNP DEP DE DE GDE QN DE

B3501 preferred FWYLrVM TAnchor FWY FWY TAnchor P LMFWYΓV

A deleterious AGP G G

B51 preferred LIVMFWY TAnchor FWY STC FWY G FWY TAnchor

P LIVFWYAM deleterious AGPDERH DE G DEQN GDE KSTC

C-ter

B5301 preferred LIVMFWY TAnchor FWY STC FWY LIVMFWY FWY TAnchor

P IMFWY L V deleterious AGPQN G RHKQN DE

B5401 preferred FWY TAnchor FWYLIVM LIVM ALIVM FWYAP TAnchor

P ATIV MF WY deleterious GPQNDE GDESTC RHKDE DE QNDGE DE

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

147 la-509107

148 la-509107

149 la-509107

150 la-509107

151 la-509107

152 la-509107

153 la-509107

154 la-509107

155 la-509107

156 la-509107

157 la-509107

158 la-509107

159 la-509107

160 la-509107

161 la-509107

162 la-509107

163 la-509107

164 la-509107

165 la-509107

166 la-509107

167 la-509107

168 la-509107

169 la-509107

170 la-509107

171 la-509107

172 la-509107

173 la-509107

174 la-509107

175 la-509107

176 la-509107

177 la-509107

178 la-509107

179 la-509107

180 la-509107

181 la-509107

182 la-509107

183 la-509107

184 la-509107

185 la-509107

186 la-509107

187 la-509107

188 la-509107

189 la-509107

190 la-509107

191 la-509107

192 la-509107

193 la-509107

194 la-509107

195 la-509107

196 la-509107

197 la-509107

198 la-509107

199 la-509107

200 la-509107

201 la-509107

202 la-509107 Table XlXa: Motifs and Post-translational modifications of the PHORl-All Protein:

N-glycosylation site (Number of matches: 2) 1 5-8 NGST 2 42-45 NTTI

Protein kinase C phosphorylation site (Number of matches: 4)

1 18-20 SDR

2 163-165 TLR 3 291-293 TLR

4 232-234 SGR

Casein kinase II phosphorylation site (Number of matches: 3) 1 49-52 SRLD 2 67-70 SFLD

3 266-269 SSQD

N-myristoylation site (Number of matches: 9)

1 3-8 GTNGST

2 6-11 GSTQTH

3 87-92 GCDKTI

4 108-113 GGVECL

5 150-155 GCGVAN

6 152-157 GVANSL

7 239-244 GTCGSH

8 242-247 GSHLTV

9 263-268 GASSSQ

lidi ition site

306-309 LGKR

G-protein coupled receptors family 1 signature 110-126 VECLLLAVMAYDRCVAI

203 la-509107 Table XlXb Motifs and post-translational modifications of the PHOR1-F5D6 Protein

7 tiansmembrane GPCRl 40..289(6.2e-25)

N-glycosylation site (Number of matches: 2)

1 4-7 NITS

2 41-44 NGTI

Protein kinase C phosphorylation site (Number of matches: 4)

1 7-9 SIR

2 136-138 TWR 3 290-292 SLR

4 299-301 TLK

Casein kinase II phosphorylation site 7-10 SIRE

N-myristoylation site (Number of matches: 5) 1 42-47 GTILGL 2 151-156 GVLLSL

3 201-206 GAISGL

4 248-253 GLVYGT

5 305-310 GVERAL

204 la-509107

205 la-509107

206 la-509107 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(l-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. Figure Table XXII shows the alignment of the splice variants with the PHOR1-F5D6 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 PHOR1-F5D6. Thus, for each splice variant, a variant's reading frame that encodes the longest single contiguous peptide homology between PHOR1-F5D6 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

207 la-509107 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(l-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 l;249(l):l-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(l-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 PHOR1-F5D6 maps is upregulated in a particular cancer, the splice variants of PHOR1-F5D6 are upregulated as well. Disclosed herein is that PHOR1-F5D6 has a particular expression profile. Splice variants of PHOR1-F5D6 that are structurally and/or functionally similar to PHOR1-F5D6 share this expression pattern, thus serving as tumor-associated markers/antigens.

Table XXIA. Nucleotide sequence of splice variant A for PHOR1-F5D6.

1 GTCATTCAAC ATTTATTCAA CCAAAAATAC TAAGTCAGCT CTATACAAAC TAATGGA

AGG

^'61 NTACAGCTAT GCAAATATAG AACACTAAAG TNTTACATGA CAGATGTATG AGTAGTG AAA

121 TGGTGAAAAA TCAGACAATG GTCACAGAGT TCCTCCTACT GGGATTTNTC CTGGGCC

CAA

181 GGATTCANAT GCTCCTNTTT GGGCTCTTCT CCCTGTTCTA TGTCTTCACC CTGCTGG GGA 241 ATGGGACCAT CCTGGGGGCT CATCTCACTG GACTCCAGAC TCCACACCCC CATGTAC

TTC

301 TTCCTCTCAC AACTGGGCCG TCGTCAACAT CGGCTAT

208 la-509107 Table XXIIA. Nucleotide sequence alignment of variant A with PHOR1-F5D6. Score = 258 bits (134), Expect = 6e-66 Identities = 177/195 (90%), Gaps = 2/195 (1%) Strand = Plus / Plus PHOR1-F5D6: 23 tcagagagttcctcctactgggatttcccgttggcccaaggattcagatgctcctctttg 82

I I I I I

Variant A : 142 tcacagagttcctcctactgggatttntcctgggcccaaggattcanatgctcctntttg 201

PHOR1-F5D6 : 83 ggctcttctccctgttctacgtcttcaccctgctggggaacgggaccatact- ggggctc 141 I II II 1111111111 II 111 II

Variant A : 202 ggctcttctccctgttctatgtcttcaccctgctggggaatgggaccatcctgggggctc 261

PHOR1-F5D6 : 142 atctcactggactccagactgcacgcccccatgtacttcttcctctcacacct- ggcggt 200 I I I 1 1 1 I I I I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I I I I I Variant A : 262 atctcactggactccagactccacacccccatgtacttcttcctctcacaactgggccgt 321

PHOR1-F5D6: 201 cgtcgacatcgccta 215

M M M U M I I I

Variant A : 322 cgtcaacatcggcta 336

Table XXIIIA. Longest amino acid sequence alignment of variant A and PHORl-F5D6.

Score = 55.1 bits (131), Expect = 4e-06 Identities = 27/36 (75%), Positives = 29/36 (80%) Frame = +3

PHOR1-F5D6: 1 MGDNITSIREFLLLGFPVGPRIQMLLFGLFSLFYVF 36 M N T + EFLLLGF +GPRI MLLFGLFSLFYVF

Variant A : 120 MVKNQTMVTEFLLLGFXLGPRIX LLFGLFSLFYVF 227

209 la-509107 Table XXIVA. Peptide sequences from the translation of the nucleotide se uence of variant A.

Note: Frame 3 gives the longest subsequence that is identical with

PHOR1-F5D6 amino acid sequence. In this Table each (*) indicates a single unknown amino acid.

Table XXIB. Nucleotide sequence of splice variant B for PHOR1-F5D6.

1 AGACAATCCT TATTAGATGG TCATTTTACT TCCACTTTTG GTACGACATT CTTTCTT ccc

61 ATGATGTGTC AATGGCTCTA TGTATCCTAT CATTGGTGAC ACAATCCCTT CTATAAC

ACT

121 GGATACTTAC AATCAGTAAT TAACTTAATG TGTAGCTCAA TCACTAATGT TAAAAGT TTA 181 TCTTTTAAAA ATGACTAAAT TCATAAAATA ATGTCTAGGT GTTTTTTGAC AATCTGG

TCC

241 TAAGTGATCT TTTTCTTTTT CACAGGGAAA TGGGGGACAA TCAGACAACG GTCACAG

AGT

301 TCCTCCTACT GGGATTTCCC GTGGGCCCAA GGATTCAGAT GCTCCTCTTT GGGCTCT TCT

361 CCCTGTTCTA CGTCTTCACC CTGCTGGGGA ACGGGACCAT ACTGGGGCTC ATCTCAC

TGG

421 ACTCCAGACT GCACGCCCCC ATGTACTTCT TCCTCTCACA CCTGGCGGTC GTCGACA TCG 481 CCTACGCCTG CAACACGGTG CCCCGGATGC TGGTGAACCT CCTGCATCCA GCCAAGC

CCA

541 TCTCCTTTGC GGGCCGCATG ATGCAGACCT TTCTGTTTTC CACTTTTGCT GTCACAG AAT

601 GTCTCCTCCT GGTGGTGATG TCCTATGATC TGTACGTGGC CATCTGCCAC CCCCTCC GAT

661 ATTTGGCCAT CATGACCTGG AGAGTCTGCA TCACCCTCGC GGTGACTTCC TGGACCA CTG

721 GAGTCCTTTT ATCCTTGATT CATCTTGTGT TACTTCTACC TTTACCCTTC TGTAGGC CCC 781 AGAAAATTTA TCACTTTTTT TGTGAAATCT TAA

210 la-509107 Table XXIIB. Nucleotide sequence alignment of variant B with PHOR1-F5D6. Score = 963 bits (501), Expect = 0.0 Identities = 526/542 (97%) Strand = Plus / Plus

PHOR1-F5D6: 1 atgggagacaatataacatccatcagagagttcctcctactgggatttcccgttggccca 60

Mill IIIMI III I III 11111 II 11111 II 11 II I II 111111

Variant B : 270 atgggggacaatcagacaacggtcacagagttcctcctactgggatttcccgtgggccca 329

PHOR1-F5D6: 61 aggattcagatgctcctctttgggctcttctccctgttctacgtcttcaccctgctgggg 120

II I II I^' 1111111 II 11111 II II II 111 II 1111111111111 II II 111 II II I II 11

Variant B : 330 aggattcagatgctcctctttgggctcttctccctgttctacgtcttcaccctgctgggg 389

PHOR1-F5D6: 121 aacgggaccatactggggctcatctcactggactccagactgcacgcccccatgtacttc 180

MMMIMMMMMMMMMMMMMMMMMMMMMMIMMMM

Variant B : 390 aacgggaccatactggggctcatctcactggactccagactgcacgcccccatgtacttc 449

PHOR1-F5D6: 181 ttcctctcacacctggcggtcgtcgacatcgcctacgcctgcaacacggtgccccggatg 240

Variant B : 450 ttcctctcacacctggcggtcgtcgacatcgcctacgcctgcaacacggtgccccggatg 509

PHOR1-F5D6: 241 ctggtgaacctcctgcatccagccaagcccatctcctttgcgggccgcatgatgcagacc 300 MMMMMMMMMMMMMMMMMMMMMMMMMMMMMM

Variant B : 510 ctggtgaacctcctgcatccagccaagcccatctcctttgcgggccgcatgatgcagacc 569

PHORl-F5D6: 301 tttctgttttccacttttgctgtcacagaatgtctcctcctggt'ggtgatgtcctatgat 360

VIaIr1i1a1n1tIIB11 :1 I 5I7101111 II II 11111 II 11 II I II I II II II 1111 II 111111111 II tttctgttttccacttttgctgtcacagaatgtctcctcctggtggtgatgtcctatgat 629

PHORl-F5D6: 361 ctgtacgtggccatctgccaccccctccgatatttggccatcatgacctggagagtctgc 420

I Ml I i 11111 i 111111111111111 II 111111 M 11111 E 11111 M 111 II I IM I

211 la-509107 Variant B : 630 ctgtacgtggccatctgccaccccctccgatatttggccatcatgacctggagagtctgc 689

PHOR1-F5D6: 421 atcaccctcgcggtgacttcctggaccactggagtccttttatccttgattcatcttgtg 480

MMMMIIIMMMMMMMMMIMMMMMMMMMIMIMMMM

Variant B : 690 atcaccctcgcggtgacttcctggaccactggagtccttttatccttgattcatcttgtg 749

PHORl-F5D6: 481 ttacttctacctttacccttctgtaggccccagaaaatttatcacnnnnnnngtgaaatc 540

MMMIMMMMIMMMMMMMMMMMIMMM

Variant B : 750 ttacttctacctttacccttctgtaggccccagaaaatttatcactttttttgtgaaatc 809

PHOR1-F5D6: 541 tt 542 Variant B : 810 t ItI 811

Table XXIIIB. Amino acid sequence alignment of variant B and PHOR1-F5D6, Score = 358 bits (777), Expect = 3e-97 Identities = 120/181 (66%) Frame = +1 / +3

PHOR1-F5D6: 1 MGDNITSIREFLLLGFPVGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPYF 180

MGDN T++ EFLLLGFPVGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYF Variant B : 270

MGDNQTTVTEFLLLGFPVGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYF 449

PHOR1-F5D6: 181 FLSHLAWDIAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTFAVTECLLLWMSYD 360

FLSHLAWDIAYACNTVPRMLVNLLHPAKPISFAGRM QTFLFSTFAVTECLLLWMSYD Variant B : 450

FLSHLAWDIAYACNTVPRMLVNLLHPAKPISFAGRMMQTFLFSTFAVTECLLLWMSYD 629

PHOR1-F5D6: 361 LYVAICHPLRYLAIMT RVCITLAVTSWTTGVLLSLIHLVLLLPLPFCRPQKIYHFFCEI 540 LYVAICHPLRYLAIMT RVCITLAVTSWTTGVLLSLIHLVLLLPLPFCRPQKIYHFFCEI Variant B : 630 LYVAICHPLRYLAIMT RVCITLAVTS TTGVLLSLIHLVLLLPLPFCRPQKIYHFFCEI -809

PHOR1-F5D6: 541 L 543

L Variant B : 810 L 812

212 la-509107 Table XXIVB. Peptide sequences from the translation of the nucleotide sequence of variant B.

Open reading Amino acid sequences frame

Frame 1 RQSLLDGHFTSTFGTTFFLPMMCQ LYVSYHW*HNPFYNTGYLQSVINL CSSITNV KSLSFKND*IHKIMSRCFLTI S*VIFFFFTGK GTIRQRSQSSSY DFPWAQGFRC SSLGSSPCSTSSPCWGTGPYWGSSHWTPDCTPPCTSSSHTWRSSTSPTPATRCPGCW* TSCIQPSPSPLRAA*CRPFCFPLLLSQNVSS W*CPMICTWPSATPSDIWPS*PGES . ASPSR*LPGPLESFYP*FILCYFYLYPSVGPRKFITFFVKS*

Frame 2 DNPY*CVNGSMYPIIGDTIPSITLDTYNQ*LT*CVAQSLML VYLLKMTKFIK*CLG VF*QSGPK*SFSFSQGNGGQSDNGHRVPPTGISRGPKDSDAPLWALLPVLRLHPAGE RDHTGAHLTGLQTARPHVLLPLTPGGRRHRLRLQHGAPDAGEPPASSQAHLLCGPHD ADLSVFHFCCHRMSPPGGDVL*SVRGHLPPPPIFGHHDLESLHHPRGDFLDHWSPFI LDSSCVTSTFTLL*APENLSLFL*NL

Frame 3 TILIRWSFYFHFWYDILSSHDVSMALCILSLVTQSLL*H ILTISN*LNV*LNH*C* KFIF*K*LNS*NNV*VFFDNLVLSDLFLFHREMGDNQTTVTEFLLLGFPVGPRIQML LFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYFFLSHLAWDIAYACNTVPRMLV NLLHPAKPISFAGRMMQTFLFSTFAVTECLLLWMSYDLYVAICHPLRYLAI T RV CITLAVTSWTTGVLLSLIHLVLLLPLPFCRPQKIYHFFCEIL

Note : Frame 3 gives the longest subsequence that is identical with

PHOR1-F5D6 amino acid sequence . In this Table each ( * ) indicates a single unknown amino acid.

213 la-509107 Table XXIC . Nucleotide sequence of splice variant C for PHOR1-F5D6.

1 CANGTTTAAA ANCCCGGGTG CTTGGGGNCC AAACCCCCNA ATTTCCGNAA GGNCGGG TCN

61 ACCCCAGNTG GTTTCCANCA GGTCCAGNGG GTTCCCAATT TTTTCCCAGA TGCAGGC TGC

121 CCTTAAACCC CGGGNAACAC ACCTTTATAT CCCAGGTAGG ACTGGCCAAA ATTAAAC TGG

181 GGACTGGCCC AAAACCGTGC CTTTCCTCAC TTTAATCTCA CTAAAGTGGA TAAGACT CAA 241 GTTATTTTGT TCTTGCAATG GCATTGACAA ATGTTTGCAC CAAAAACCAT GTTGAAG TTC

301 ATTAAGGAAA CTGTGATCCA AGATCCAAGG TCAAAAAAAC AAATTCATCA ATTCAGC

ACA

361 CCACCAACTC ACAGGCTAAG CATCTTACTG CTAATTCATT GATGCTGCCA TTTGTCA AGT

421 GCCAAATTGA ATTATTGATT TGTCAATAAT TTCCTTCCGT TGGTTACTTA TATAGTA

TAT

481 TGCAATTCTT GTTGCTGAAG TCAGCTACAC TTTTTCTATT TGAAAAACAA TTTCTTG CAT 541 TTGGGATTTC AGGTATAGTG ATTGTTACAA ATATGAAGGA CTTGAATTAA CAGCAAG TT

601 TCAAGTAAAA CTTTACTTAT GTATAACTGA ATGAGTTCTT AAAGACATTT ACTAACA

ATT

661 TTCCACAAAC TAAAAATTTA TAAAACAATA AATAAAATAG ACTTTAAAAA AAAGCGT GTC

721 ACACAGCTGC TTGTTTTTTG TTTGTTTCTT TGTTTGTTTT TTAGTAGTGA AATGGTG

AAA

781 AATCAGACAA TGGTCACAGA GTTCCTCCTA CTGGGATTTC TCCTGGGCCC AAGGATT CAG 841 ATGCTCCTCT TTGGGCTCTT CTCCCTGTTC TATGTCTTCA CCCTGCTGGG GAATGGG ACC

901 ATCCTGGGGC TCATCTCACT GGACTCCAGA CTCCACACCC CCATGTACTT CTTCCTC TCA

961 CACCTGGCCG TCGTCAACAT CGCCTATGCC TGCAACACAG TGCCCCAGAT GCTGGTG AAC

1021 CTCCTGCATC CAGCCAAGCC CATCTCCTTT GCTGGCTGCA TGACATAGAC CTTTCTC TTT

1081 TTGAGTTTTG CACATACTGA ATGCCTCCTG TTGGTGCTGA TGTCCTACGA TCGGTAC GTG 1141 GCCATCTGCC ACCCTCTCCG ATATTTCATC ATCATGACCT GGAAAGTCTG CATCACT CTG

1201 GCCATCACTT CCTGGACATG TGGCTCCCTC CTGGCTATGG TCCATGTGAG CCTCATC CTA

1261 AGACTGCCCT TTTGTGGGCC TCGTGAAATC AACCACTTCT TCTGTGAAAT CCTGTCT GTC

1321 CTCAGGCTGG GCTGTGCTGA TACCTGGCTC AACCAGGTGG TCATCTTTGC GCCTGCA TGT

1381 TCATCCTGGT GGGACCACTC TGCCTGGTGC T

214 la-509107 Table XXIIC . Nucleotide sequence alignment of variant C with PHOR1-F5D6 Score = 562 bits (292), Expect = e-157 Identities = 380/424 (89%) Strand = Plus / Plus

PHOR1-F5D6: 23 tcagagagttcctcctactgggatttcccgttggcccaaggattcagatgctcctctttg 82

V MarMiant C : 794 ^{lιmi M} tcacagagttcctcctactgggatttctcctgggcccaaggattcagatgctcctctttg 853

PHOR1-F5D6: 83 ggctcttctccctgttctacgtcttcaccctgctggggaacgggaccatactggggctca 142 MMMMMMMMIM 1111 II II 11 II I II 11 II I IIIMIM

Variant C : 854 ggctcttctccctgttctatgtcttcaccctgctggggaatgggaccatcctggggctca 913

PHOR1-F5D6: 143 tctcactggactccagactgcacgcccccatgtacttcttcctctcacacctggcggtcg 202

MMMMMMMMIM III II I II I II 1111 II 11 II II 1111

Variant C : 914 tctcactggactccagactccacacccccatgtacttcttcctctcacacctggccgtcg 973

PHOR1-F5D6: 203 tcgacatcgcctacgcctgcaacacggtgccccggatgctggtgaacctcctgcatccag 262

Variant C ,, „: 9ι7,4 ,1, ii, mill^{11111 ,iM} tcaacatcgcctatgcctgcaacacagtgccccagatgctggtgaacctcctgcatccag 1033

PHOR1-F5D6: 263 ccaagcccatctcctttgcgggccgcatgatgcagacctttctgttttccacttttgctg 322 MMMMMMMMIM III 111 II 11111 1111

Variant C : 1034 ccaagcccatctcctttgctggctgcatgacatagacctttctctttttgagttttgcac 1093

PHOR1-F5D6: 323 tcacagaatgtctcctcctggtggtgatgtcctatgatctgtacgtggccatctgccacc 382

Vlalrlilant C : 109l4lllll ' ^M" atactgaatgcctcctgttggtgctgatgtcctacgatcggtacgtggccatctgccacc 1153

PHOR1-F5D6: 383 ccctccgatatttggccatcatgacctggagagtctgcatcaccctcgcggtgacttcct 442

215 la-509107 Variant C : 1154 ctctccgatatttcatcatcatgacctggaaagtctgcatcactctggccatcacttcct 1213

PHOR1-F5D6: 443 ggac 446 Variant C : 1214 glglalcl 1217

Table XXIIIC. Amino acid sequence alignment of variant C and PHOR1-F5D6. Score = 320 bits (694), Expect = 8e-86 Identities = 136/204 (66%) Frame = +1 / +1 PHOR1-F5D6: 1

MGDNITSIREFLLLGFPVGPRIQ LLFGLFSLFYVFTLLGNGTILGLISLDSRLHAPMYF 180

M N T + EFLLLGF +GPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLH

PMYF

Variant C : 772 MVKNQTMVTEFLLLGFLLGPRIQMLLFGLFSLFYVFTLLGNGTILGLISLDSRLHTPMYF 951

PHOR1-F5D6 : 181

FLSHLAWD IAYACNTVPRMLVNLLHPAKPI SFAGRMMQTFLFSTFAVTΞCLLLW S YD 360 FLSHLAW+IAYACNTVP+MLVNLLHPAKPISFAG M TFLF +FA

TECLLLV+ SYD Variant C : 952 FLSHLAWNIAYACNTVPQMLVNLLHPAKPISFAGCMT*TFLFLSFAHTECLLLVLMSYD 1131

PHOR1-F5D6: 361 LYVAICHPLRYLAIMTWRVCITLAVTSWTTGVLLSLIHLVLLLPLPFCRPQKIYHFFCEI 540

YVAICHPLRY IMTW+VCITLA+TS T G LL H L L LPFC P++I HFFCEI Variant C : 1132

RYVAICHPLRYFIIMTWKVCITLAITS TCGSLLAMVHVSLILRLPFCGPREINHFFCΞI 1311

PHOR1-F5D6: 541 LAVLKLACADTHINENMVLAGAIS 612 L+VL+L CADT +N+ ++ A A S

Variant C : 1312 LSVLRLGCADTWLNQWIFAPACS 1383

216 la-509107 Table XXIVC. Peptide sequences from the translation of the nucleotide se uence of variant C .

Note: Frame 1 gives the longest subsequence that is identical with

217 la-509107

Claims

CLAIMS:

1. A method for monitoring PHORl-Al 1 or PHOR1-F5D6 gene products in a biological sample from a patient who has or who is suspected of having cancer, the method comprising: determining the status of PHORl-Al 1 or PHOR1-F5D6 gene products expressed by cells in a tissue sample from an individual; comparing the status so determined to the status of PHORl-Al 1 or PHOR1-F5D6 gene products in a corresponding normal sample; and, identifying the presence of aberrant PHORl-Al 1 or PHOR1-F5D6 gene products in the sample relative to the normal sample.

2. A method of monitoring the presence of cancer in an individual comprising: performing the method of claim 1 whereby the presence of elevated PHORl-Al 1 or PHOR1-F5D6 mRNA or protein expression in the test sample relative to the normal tissue sample provides an indication of the presence or status of a cancer.

3. The method of claim 2, wherein the cancer occurs in a tissue set forth in Table I.

4. A composition comprising: a substance that modulates the status of PHORl-Al 1 or PHOR1-F5D6 or a molecule that is modulated by PHORl-Al 1 or PHOR1-F5D6 and thereby modulates the status of a cell that expresses PHORl-All or PHORl-F5D6.

5. The composition of claim 4, further comprising a pharmaceutically acceptable carrier.

6. A pharmaceutical composition that comprises the composition of claim 4 in a human unit dose form.

7. A composition of claim 4 that comprises a PHORl-Al 1- or PHORl-F5D6-related protein.

8. A composition of claim 4 that comprises an antibody or fragment thereof that specifically binds to a PHORl-Al 1- or PHORl-F5D6-related protein.

218 la-509107

9. A composition of claim 4 that comprises a polynucleotide that encodes a single chain monoclonal antibody that immunospecifically binds to a PHORl-Al 1- or PHORl -F5D6-related protein.

10. A composition of claim 4 that comprises a polynucleotide comprising a PHORl-Al 1- or PHORl-F5D6-related protein coding sequence.

11. A composition of claim 4 that comprises an antisense polynucleotide complementary to a polynucleotide having a PHORl-Al 1 or PHOR1-F5D6 coding sequence.

12. A pharmaceutical composition of claim 4 that comprises a ribozyme capable of cleaving a polynucleotide having PHORl-Al 1 or PHOR1-F5D6 coding sequence and a physiologically acceptable carrier.

13. A method of inhibiting growth of cancer cells that expresses PHORl-Al 1 or PHOR1- F5D6, the method comprising: administering to the cells the composition of claim 4.

14. A method of claim 13 of inhibiting growth of cancer cells that express PHORl-Al 1 or PHOR1-F5D6, the method comprising steps of: administering to said cells an antibody or fragment thereof that specifically binds to a PHORl-

All- or PHORl-F5D6-related protein.

15. A method of treating a patient with a cancer that expresses PHORl-All or PHOR1- F5D6, the method comprising steps of: administering to said patient a vector that comprises the composition of claim 9, such that the vector delivers the single chain monoclonal antibody coding sequence to the cancer cells and the encoded single chain antibody is expressed intracellularly therein.

16. A method of claim 13 of inhibiting growth of cancer cells that express PHORl-Al 1 or PHOR1-F5D6, the method comprising steps of: administering to said cells a polynucleotide comprising a PHORl-All- or PHORl-F5D6-related protein coding sequence.

219 la-509107

17. A method of claim 13 of inhibiting growth of cancer cells that express PHORl-Al 1 or PHOR1-F5D6, the method comprising steps of: administering to said cells an antisense polynucleotide complementary to a polynucleotide having a PHORl-Al 1 or PHOR1-F5D6 coding sequence.

18. A method of treating a patient with a cancer that expresses PHORl-Al 1 or PHOR1- F5D6, the method comprising steps of: identifying that the patient has a cancer the cells of which express PHORl-Al 1 or PHOR1-F5D6; administering to the patient a pharmaceutical composition of claim 12 that comprises a ribozyme capable of cleaving a polynucleotide having a PHORl-Al 1 or PHOR1-F5D6 coding sequence.

19. A method of generating a mammalian immune response directed to PHORl-Al 1 or PHOR1-F5D6, the method comprising: exposing cells of a mammal's immune system to an immunogenic portion of a PHORl-Al 1- or PHORl-F5D6-related protein or a nucleotide sequence that encodes said protein, whereby an immune response is generated to PHORl-Al 1 or PHOR1-F5D6.

20. A method of delivering a cytotoxic agent to a cell that expresses PHORl-Al 1 or PHOR1- F5D6, said method comprising: providing a cytotoxic agent conjugated to an antibody or fragment thereof that specifically binds to PHORl-All or PHORl-F5D6; and, exposing the cell to the antibody-agent conjugate.

21. A method of inducing an immune response to a PHORl-Al 1 or PHOR1-F5D6 protein, said method comprising: providing a PHORl-Al 1- or PHORl-F5D6-related protein that comprises at least one T cell or at least one B cell epitope; contacting the epitope with an immune system T cell or B cell respectively, whereby the immune system T cell or B cell is induced.

22. The method of claim 21, wherein the immune system cell is a B cell, whereby the induced B cell generates antibodies that specifically bind to the PHORl-Al 1- or PHORl-F5D6-related protein. '

220 la-509107

23. The method of claim 21, wherein the immune system cell is a T cell that is a cytotoxic T cell (CTL), whereby the activated CTL kills an autologous cell that expresses the PHORl-Al 1 or PHOR1- F5D6 protein.

24. The method of claim 21, wherein the immune system cell is a T cell that is a helper T cell

(HTL), whereby the activated HTL secretes cytokines that facilitate the cytotoxic activity of a CTL or the antibody producing activity of a B cell.

25. An antibody or fragment thereof that specifically binds to a PHORl-Al 1- or PHOR1- F5D6-related protein.

26. The antibody or fragment thereof of claim 25, which is monoclonal.

27. A recombinant protein comprising the antigen-binding region of a monoclonal antibody of claim 26.

28. The antibody or fragment thereof of claim 25, which is labeled with a detectable marker.

29. The recombinant protein of claim 27, which is labeled with a detectable marker.

30. The antibody fragment of claim 25, which is an Fab, F(ab')2, Fv or sFv fragment.

31. The antibody of claim 25 , which is a human antibody.

32. The recombinant protein of claim 27, which comprises murine antigen binding region residues and human constant region residues.

33. A non-human transgenic animal that produces an antibody of claim 25.

34. A hybridoma that produces an antibody of claim 26.

35. A single chain monoclonal antibody that comprises the variable domains of the heavy and light chains of a monoclonal antibody of claim 26.

221 la-509107

36. A vector comprising a polynucleotide that encodes a single chain monoclonal antibody of claim 35 that immunospecifically binds to a PHORl-All- or PHORl-F5D6-related protein.

37. An assay for detecting the presence of a PHORl-Al 1- or PHORl-F5D6-related protein or polynucleotide in a biological sample from a patient who has or who is suspected of having cancer, comprising steps of: contacting the sample with an antibody or another polynucleotide, respectively, that specifically binds to the PHORl-Al 1- or PHORl -F5D6-related protein or polynucleotide, respectively; and, determining that there is a complex of the antibody and PHORl-Al 1- or PHORl-F5D6-related protein or the another polynucleotide and PHORl-Al 1- or PHORl-F5D6-related polynucleotide.

38. The assay in accordance with claim 37 for detecting the presence of a PHORl-Al 1- or PHORl-F5D6-related protein or polynucleotide in a biological sample from a patient who has or who is suspected of having cancer, comprising the steps of: obtaining a sample from a patient who has or who is suspected of having cancer.

39. The assay of claim 37 for detecting the presence of a PHORl-Al 1 or PHOR1-F5D6 polynucleotide in a biological sample, comprising: contacting the sample with a polynucleotide probe that specifically hybridizes to a polynucleotide encoding a PHORl-Al 1- or PHORl-F5D6-related protein having the amino acid sequence of Figure 3; and, j detecting the presence of a hybridization complex formed by the hybridization of the probe with PHORl-All or PHOR1-F5D6 polynucleotide in the sample, wherein the presence of the hybridization complex indicates the presence of PHORl-All or PHOR1-F5D6 polynucleotide within the sample.

40. An assay for detecting the presence of PHORl-Al 1 or PHOR1-F5D6 mRNA in a biological sample from a patient who has or who is suspected of having cancer, said method comprising:

(a) producing cDNA from the sample by reverse transcription using at least one primer;

(b) amplifying the cDNA so produced using PHORl-All or PHOR1-F5D6 polynucleotides as sense and antisense primers, wherein the PHORl-Al 1 or PHOR1-F5D6 polynucleotides used as the sense and antisense primers are capable of amplifying the PHORl-Al 1 or PHOR1-F5D6 cDNA contained within the plasmid pPHORl-Al 1- or PHOR1-F5D6-C as deposited with American Type Culture Collection as Accession No. PTA-1893; and

(c) detecting the presence of the amplified PHORl-Al 1 or PHOR1-F5D6 cDNA.

222 la-509107

41. A composition comprising a polynucleotide from position number 201 through number 2375 of Figure 2.

42. The composition of claim 41, wherein T is substituted with U.

43. A composition comprising the amino acid sequence or nucleic acid sequence of Figure 2.

44. The composition of claim 43, wherein T is substituted with U.

45. A composition comprising a polynucleotide that encodes a PHORl-Al 1- or PHOR1- F5D6-related protein that is at least 90% homologous to the entire amino acid sequence shown in Figure 2.

46. An analog peptide of eight, nine ten or eleven contiguous amino acids of Figure 2.

47. A polynucleotide that encodes an analog peptide of claim 46.

48. The composition of claim 45, wherein the polynucleotide encodes a PHORl-Al 1- or PHORl-F5D6-related protein that is at least 90% identical to the entire amino acid sequence shown in FIGURE 2.

49. A composition comprising a polynucleotide that encodes at least one peptide set forth in Tables V-XVIII.

50. A composition comprising a polynucleotide that encodes a peptide region of at least 5 amino acids of Figure 2 in any whole number increment up to 725 that includes an amino acid position selected from: an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of Figure 14, an amino acid position having a value less than 0.5 in the Hydropathicity profile of Figure 15; an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 16; an amino acid position having a value greater than 0.5 in the Average Flexibility profile on Figure 17; or an amino acid position having a value greater than 0.5 in the Beta-turn profile of Figure 18.

51. ' A composition comprising a polynucleotide that is fully complementary to a polynucleotide of claim 41.

223 la-509107

52. A composition comprising a polynucleotide that is fully complementary to a polynucleotide of claim 42.

53. A composition comprising a polynucleotide that is fully complementary to a polynucleotide of claim 43.

54. A composition comprising a polynucleotide that is fully complementary to a polynucleotide of claim 44.

55. A composition comprising a polynucleotide that is fully complementary to a polynucleotide of claim 45.

56. A composition comprising a polynucleotide that is fully complementary to a polynucleotide of claim 48.

57. A composition comprising a polynucleotide that is fully complementary to a polynucleotide of claim 49.

58. A composition comprising a polynucleotide that is fully complementary to a polynucleotide of claim 48.

59. A composition comprising a polynucleotide that encodes a PHORl-Al 1- or PHOR1- F5D6-related protein whose sequence is encoded by the cDNAs contained in the plasmid designated pPHORl-Al 1- or PHOR1-F5D6-C deposited with American Type Culture Collection as Accession No. PTA-1893.

60. A composition comprising a polypeptide at least 90% homologous to Figure 2.

61. The composition of claim 60, wherein the polypeptide is at least 90% identical to Figure

2.

62. The composition of claim 61, wherein the polypeptide comprises Figure 2.

224 la-509107

63. A composition comprising a CTL polypeptide epitope from Figure 2.

64. The composition of claim 63, wherein the CTL epitope comprises a polypeptide selected from Tables V-XVIII.

65. A composition comprising a peptide region of at least 5 amino acids of Figure 2 in any whole number increment up to 725 that includes an amino acid position selected from: an amino acid position having a value greater than 0.5 in the Hydrophilicity profile of Figure 14, an amino acid position having a value less than 0.5 in the Hydropathicity profile of Figure 15; an amino acid position having a value greater than 0.5 in the Percent Accessible Residues profile of Figure 16; an amino acid position having a value greater than 0.5 in the Average Flexibility profile on Figure 17; or an amino acid position having a value greater than 0.5 in the Beta-turn profile of Figure 18.

66. A composition comprising a PHORl-Al 1- or PHORl-F5D6-related protein whose sequence is encoded by the cDNAs contained in the plasmid designated as containing the pPHORl-Al 1 or

PHOR1-F5D6 gene deposited with American Type Culture Collection as Accession No. .or

Accession No. , respectively.

225 la-509107