PT1753871E - Antibodies and related molecules that bind to psca proteins - Google Patents

Description

DESCRIPTION

ANTIBODIES AND RELATED MOLECULES CONNECTING

PSCA PROTEINS

FIELD OF THE INVENTION The invention described herein relates to antibodies, as well as binding fragments thereof and molecules engineered from them, which bind to proteins, designated PSCA. The invention further relates to diagnostic, prognostic, prophylactic and therapeutic methods and compositions useful for the treatment of cancers that express PSCA.

BACKGROUND OF THE INVENTION Cancer is the second leading cause of human death following coronary disease. Across the world, millions of people die from cancer each year. In the United States alone, as reported by the American Cancer Society, cancer causes the death of more than half a million people annually, with more than 1.2 million new cases diagnosed each year. While heart disease deaths have been decreasing significantly, those resulting from cancer are generally increasing. At the beginning of the next century, cancer is predicted to become the leading cause of death.

Throughout the world, several cancers stand out as the leading causes of death. In particular, carcinomas of the lung, prostate, breast, colon, pancreas, ovary, and bladder represent the leading causes of cancer death. These and virtually all other cancers share a common lethal characteristic. With very few exceptions, the metastatic disease of a carcinoma is fatal. In addition, 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 awareness of the potential for recurrence or treatment failure. Many cancer patients experience physical impairments after treatment. In addition, many cancer patients experience a recurrence.

Around the world, 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 males. In the United States alone, more than 30,000 men die annually from 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 remain the main treatment modalities. Unfortunately, these treatments are ineffective for many and are usually associated with undesirable consequences.

In the face of the diagnosis, the lack of a prostatic tumor marker that can accurately detect tumors in early and localized phase remains a significant limitation in the diagnosis and management of this disease. Although 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. LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer xenografts that survived passage in several mice with severe combined immunodeficiency (SCID) and exhibited the ability to mimic the transition from androgen dependence to independence of androgens (Klein et al., 1997, Nat Med. 3: 402). The most recently identified prostate cancer markers include PCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate specific membrane antigen (PSM) (Pinto et al. Clin Cancer Res 2 September 1996 (9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad Sci USA, Dec 7, 1999; 96 (25): 14523-8) and cell antigen (PSA) (Reiter et al., 1998, Proc Natl Acad Sci USA 95: 1735).

While previously identified PSA, PSM, PCTA and PSCA markers facilitated efforts to diagnose and treat prostate cancer, there is a need for the identification of additional markers and therapeutic targets for prostate cancers and related to further improve the diagnosis and treatment of prostate cancer. therapy. Renal cell carcinoma (RCC) accounts for approximately 3 percent of adult malignancies. Once the adenomas reach the size of 2 to 3 cm, there is the malignant potential. In adults, the two main malignant renal tumors are renal cell adenocarcinoma and transitional cell carcinoma of the renal pelvis or ureters. 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 leading 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 can be approached aggressively in appropriate patients with a possibility for long-lasting responses. Nonetheless, there remains a need for effective therapies for these patients.

Of all new cancers in the United States, bladder cancer accounts for approximately 5 percent in men (fifth most common neoplasm) and 3 percent in women (the eighth most common neoplasm). The incidence is declining slowly, simultaneously with an aging population. In 1998, there were 54,500 estimated 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 for women. The historical ratio of men to women of 3: 1 may be declining relative 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). Incidence and mortality from bladder cancer increase with age and will be a growing problem as the population becomes older. Most bladder cancers recur in the bladder. Bladder cancer is managed with a combination of transurethral bladder resection (TUR) and intravesical chemotherapy or immunotherapy. The recurrent and multifocal nature of bladder cancer highlights the limitations of TUR. Most muscle-invasive cancers are not cured by TUR alone. Radical cystectomy and urinary diversion is the most effective means of eliminating cancer but carries an undeniable impact on urinary and sexual function. There continues to be a significant need for treatment modalities that are beneficial to patients with bladder cancer.

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 cases of rectal cancer. Colorectal cancers are the third most common cancers in men and women. The incidence rates decreased significantly during 1992-1996 (-2.1% per year). Research suggests that these declines were due to screening and removal of polyps, increasing the progression of polyps to invasive cancers. There were an estimated 56,300 deaths (47,700 colon cancer, 8,600 rectal cancer) in 2000, accounting for about 11% of all cancer deaths in the United States.

Surgery is currently the most common form of therapy for colorectal cancer, and for cancers that have not spread, and it is often curative. Chemotherapy, or chemotherapy plus radiation, is provided before or after surgery to most patients whose cancer has deeply pierced the wall of the intestine or has spread to the lymph nodes. A permanent colostomy (creation of an abdominal opening for the elimination of body waste) is occasionally required for colon cancer and is rarely necessary for rectal cancer. There remains 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 cancer diagnoses in the United States. 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, lung cancer mortality declined significantly among men (-1.7% per year) while rates for women were still increasing significantly (0.9% per year). Since 1987, more women have died each year from lung cancer than breast cancer, which for 40 years was the leading cause of cancer death in women. The decline in lung cancer incidence and death rates most likely resulted from the decline in smoking rates over the previous 30 years; however, the decline in smoking patterns among women lagged behind that of men. Worryingly, although declines in adult tobacco use have slowed down, tobacco use in youth has increased again.

Treatment options for lung and bronchial cancer are determined by the type and stage of cancer and include surgery, radiation therapy, and chemotherapy. For many localized cancers, surgery is usually the treatment of choice. Since the disease usually dispersed at the time it is discovered, radiation therapy and chemotherapy are usually needed in combination with surgery. Chemotherapy alone or combined with radiation is the treatment of choice for small cell lung cancer; in this regimen, a large percentage of patients experience remission, which in some cases is long-lasting. There is, however, a continuing need for effective treatment and diagnostic approaches for lung and bronchial cancers.

An estimated 182,800 new invasive cases of breast cancer were reported among women in the United States during 2000. In addition, an estimated 1,400 new cases of breast cancer were diagnosed in men in 2000. After increasing about 4 % per year in the 1980s, breast cancer incidence rates in women stabilized in the 1990s by about 110.6 cases per 100,000. In the United States alone, there were an estimated 41,200 deaths (40,800 women, 400 men) in 2000 due to breast cancer. Breast cancer is ranked second among cancer deaths in women. According to the most recent data, mortality rates declined significantly during 1992-1996 with the largest decreases in young women, both black and Caucasian. These decreases were probably the result of early detection and improved treatment.

Taking into account medical circumstances and patient preferences, breast cancer treatment 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 hormonal 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 following mastectomy. Recently, such reconstruction has been performed at the same time as mastectomy. Local excision of ductal carcinoma in situ (DCIS) with adequate amounts of surrounding normal breast tissue may prevent local recurrence of DCIS. Radiation in the breast and / or tamoxifen may reduce the likelihood that DCIS will occur in the remaining breast tissue. This is important because DCIS, if left untreated, can progress to invasive breast cancer. Nevertheless, there are serious side effects or sequelae associated with these treatments. There is, as such, a need for effective 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 gynecological cancers. During 1992-1996, incidence rates for ovarian cancer were declining significantly. As a result of 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 the treatment options for ovarian cancer. Surgical removal usually includes removal of one or both of the ovaries, fallopian tubes (salpingo-oophorectomy), and uterus (hysterectomy). In some very early tumors, only the ovary involved will be removed, especially in young women who wish to have children. In advanced disease, an attempt is made to remove intra-abdominal disease to enhance the effect of chemotherapy. There continues to be an important need for effective treatment options for ovarian cancer.

There were an estimated 28,300 new cases of pancreatic cancer in the United States in 2000. Over the past 20 years, pancreatic cancer rates have declined in men. Rates among women have remained about constant but may be starting 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, drop in mortality rates among men (about -0.9% per year) while rates have increased slightly among women. Surgery, radiation therapy, and chemotherapy are the treatment options for pancreatic cancer. These treatment options can extend survival and / or alleviate the symptoms in many patients but are not likely to produce the cure for most. There is a significant need for additional therapeutic and diagnostic options for cancers. These include the use of antibodies, vaccines, and small molecules as treatment modalities. Additionally, there is also a need to use these modalities as research tools for diagnosing, detecting, monitoring, and improving the prior art in all areas of cancer treatment and studies. The therapeutic utility of monoclonal antibodies (mAbs) (G. Kohler and C. Milstein, Nature 256: 495-497 (1975)) has been performed. Monoclonal antibodies have now been approved as transplantation therapies, cancer, infectious diseases, cardiovascular disease and inflammation. Different isotypes have different effector functions. Such differences in function are reflected in different 3-dimensional structures for the various immunoglobulin isotypes (P.M. Alzari et al., Annual Rev. Immunol., 6: 555-580 (1988)).

Because mice are convenient for immunization and recognize most human antigens as exogenous, mAbs against human targets with therapeutic potential have typically been of murine origin. However, murine mAbs have inherent disadvantages as human therapeutics. They require more frequent dosing since mAbs have a shorter circulating half-life in humans than human antibodies. More critically, repeated administration of murine antibodies to the human immune system causes the human immune system to respond by recognizing the mouse protein as exogenous and generating a human anti-mouse antibody (HAMA) response. Such a HAMA response may result in an allergic reaction and rapid elimination of the murine antibody from the system rendering the murine antibody treatment useless. To avoid such effects, attempts have been made to create human immune systems in mice.

Initial attempts were hoped to create transgenic mice capable of responding to antigens with antibodies having human sequences (See Bruggemann et al., Proc Natl Acad Sci USA 86: 6709-6713 (1989)), but were limited by the amount of DNA that could be stably maintained by available cloning vehicles. The use of yeast artificial chromosome (YAC) cloning vectors paved the way for the introduction of large germline fragments of human Ig locus into transgenic mammals. Essentially a majority of the genes of the human V, D, and J regions disposed with the same spacing found in the human genome and the human constant regions were introduced into mice using YACs. Such a strain of transgenic mice is known as XenoMouse® mice and is commercially available from Abgenix, Inc. (Fremont CA). US 2004/0018571 discusses prostate antigen antigen (PSCA) antibodies and presents data related to inhibition of LAPC-9 tumor growth by anti-PSCA monoclonal antibodies, including the antibody designated 1G8. WO 01/40309 discloses isolated anti-PSCA antibodies that internalize upon binding to PSCA in an in vivo mammal, an isolated nucleic acid encoding an anti-PSCA antibody, and compositions comprising an anti-PSCA antibody and a carrier.

Ross, S. et al. (2002, Cancer Res., 62, pp. 2546-2553) discusses an analysis of PSCA expression in normal urogenital tissues, benign prostatic hyperplasia, prostatic intraepithelial neoplasia, and primary and metastatic adenocarcinoma of the prostate, and features anti- Monoclonal PSCAs. A number of anti-PSCA antibodies are identified as 6F8, 8D11 and 5F2. EP 1514876 discusses two anti-PSCA monoclonal antibodies, 1G8 and 3E6, and establishes the hypothesis that anti-PSCA monoclonal antibodies can be used in the immunotherapy of prostate cancer. US 6,258,939 discloses a number of monoclonal antibodies, including those designated 1G8, 2H5, 3C5, and 4A10. The patent suggests that these antibodies can recognize different epitopes on the PSCA target protein.

Saffran, D. C. et al. (2001, Proc Natl Acad Sci USA, 98, pp. 2658-2663) discusses the antibodies designated 1G8, 2H5, 3C5, and 4A10, and the evaluation of the therapeutic efficacy of anti-PSCA mAbs in mouse models of human prostate cancer xenografts.

SUMMARY OF THE INVENTION The invention provides antibodies as well as binding fragments thereof and molecules engineered therefrom, which bind to PSCA proteins and polypeptide fragments of PSCA proteins. The invention comprises monoclonal and polyclonal antibodies, murine and other mammalian antibodies, chimeric antibodies, humanized and fully human antibodies, and antibodies labeled with a detectable label or therapeutic agent. In certain embodiments, there is a condition that the complete nucleic acid sequence of Figure 3 is not encoded and / or that the full amino acid sequence of Figure 2 is not prepared. In certain embodiments, the full nucleic acid sequence of Figure 3 is encoded and / or the full amino acid sequence of Figure 2 is prepared, any of which is in unit dosage forms for the respective human.

There are disclosed herein methods for detecting the presence and status of PSCA polynucleotides and proteins in various biological samples, as well as methods for identifying cells expressing PSCA. A disclosure provides methods for monitoring the PSCA gene products in a hematological tissue or sample having or suspected of having some forms of growth dysregulation such as cancer. The invention further provides various therapeutic and immunogenic compositions and strategies for the treatment of PSCA-expressing cancers such as tissue cancers listed in Table I, including therapies directed to the inhibition of transcription, translation, processing, or function of PSCA as well as vaccines against cancer . In one aspect the invention provides compositions and uses therefor for the treatment of cancer expressing PSCA in a human subject wherein the composition comprises a carrier suitable for human use and a human unit dose of one or more than one agent inhibits the production of the PSCA function. Preferably, the carrier is a carrier only human. In another aspect of the invention, the agent is a fraction that is immunoreactive with the PSCA protein. Non-limiting examples of such fractions include, but are not limited to, antibodies (such as single chain, monoclonal, polyclonal, humanized, chimeric, or human antibodies), functional equivalents thereof (whether naturally occurring or synthetic), and combinations of the same. The antibodies may be conjugated to a diagnostic or therapeutic fraction. It is disclosed herein that the agent may be a small molecule as defined herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. The cDNA and amino acid sequence of PSCA (also called " PSCA v.l " or " PSCA variant 1 ") is shown in Figure 1A. The initial methionine is underlined. The open reading frame extends from nucleic acid 18-389 including the stop codon. The cDNA and amino acid sequence of PSCA variant 2 (also called " PSCA v.2 ") is shown in Figure 1B. The codon for the initial methionine is underlined. The open reading frame extends from nucleic acid 56-427 including the stop codon.

The cDNA and amino acid sequence of PSCA variant 3 (also called " PSCA v.3 ") is shown in Figure 1C. The codon for the initial methionine is underlined. The open reading frame extends from nucleic acid 423-707 including the stop codon. The cDNA and amino acid sequence of variant 4 of

PSCA (also called " PSCA v.4 ") is shown in Figure 1D. The codon for the initial methionine is underlined. The open reading frame extends from nucleic acid 424-993 including the stop codon. The cDNA and amino acid sequence of variant 5

PSCA (also called " PSCA v.5 ") is shown in Figure 1E. The codon for the initial methionine is underlined. The open reading frame extends from nucleic acid 910-1479 including the stop codon. The cDNA and amino acid sequence of variant 6

PSCA (also called " PSCA v.6 ") is shown in Figure 1F. The codon for the initial methionine is underlined. The open reading frame extends from nucleic acid 83-427 including the stop codon.

Figure 1G. PSCA variants of PSCA v.2, PSCA v.7 through v.18. The proteins of PSCA v.7 through v.18 have 123 amino acids. PSCA variants v.7 through v.18 are variants with a single nucleotide difference of PSCA v.2, and encode the same protein as v.2. Although these SNP variants are shown separately, they may also occur in any combinations and in any of the transcript variants listed above in Figures IA to 1F.

Figure 1H. SNCA variants of PSCA v.4, PSCA v.19 through v.30. PSCA proteins v.19 through v.30 have 189 amino acids. PSCA variants v.19 through v.30 are variants with a single nucleotide difference of PSCA v.4. PSCA proteins v.9, v.10, v.ll v.24 and v.25 differ from PSCA v.l by one amino acid. PSCA v.23, v.28, v.29 and v.30 encode the same protein as v.4. Although these SNP variants are shown separately, they can also occur in any combination and in any of the variants of transcript v.3 and v.4. Figure II. Expression of PSCA variants. (11 (a)) primers were designed to differentiate between PSCA variants v.l / v.2 / v.4, PSCA v.3 and PSCA v.5. The primers A and B indicated by small arrows above the exons in the figure result in a PCR product of 425 bp for PSCA v1 / v2 / v4, a 300 bp PCR product for PSC v3 and a PCR product of 910 bp for PSCA v.5. (11 (b)) First-strand cDNA was prepared from the bladder, brain, heart, kidney, liver, lung, prostate, spleen, skeletal muscle, testis, pancreas, colon, normal stomach, prostate cancer clusters, cancer bladder cancer, renal cancer, colon cancer, lung cancer, ovarian cancer, breast cancer, cancer metastases and pancreatic cancer. Standardization was performed by PCR using primers for actin. Semi-quantitative PCR using the specific primer variants was performed in 30 cycles of amplification. The results show expression of PSCA v.5 primarily in mammary tissue, cancer metastasis, and pancreatic cancer, and to a lower level in colon cancer and lung cancer. The PSCA v1 / v.2 / v.4 PCR product has been detected in prostate cancer, bladder cancer, renal cancer, colon cancer, lung cancer, ovarian cancer, breast cancer, cancer metastasis, and cancer. pancreas. Among normal tissues, the PSCA v1 / v.2 / v.4 PCR product was detected only in the prostate, stomach and to a lesser extent in the kidney and lung, whereas PS CA v.5 was not detected in any normal tissue . The PCR product of PSCA v.3 was not detected in any of the samples tested. Figure 1J. Expression of PSCA v.4 and PSCA v.5. 1J (a) Initiators were designed to differentiate between PSCA v.4 and PSCA v.5 as indicated by the arrows marked B and C in the figure. Specific primers for PSCA v.4 led to a 460 bp PCR product, whereas primers specific for PSCA v.5 led to a 945 bp PCR product of size. 1J (b) First-strand cDNA was prepared from the bladder, brain, heart, kidney, liver, lung, prostate, spleen, skeletal muscle, testis, pancreas, colon, normal stomach, prostate cancer clusters, bladder cancer , and multi-xenograft pooling (prostate cancer xenografts, renal cancer and bladder cancer). Standardization was performed by PCR using primers for actin. Semi-quantitative PCR using the specific primer variants was performed in 30 cycles of amplification. The results show expression of PSCA v.4 in prostate cancer, bladder cancer, and multi-xenograft, kidney and normal prostate clustering. PSCA v.5 was detected only in normal prostate and bladder cancer.

Figure 2. Amino acid sequences of antibodies to PSCA. Figure 2A. The VH amino acid sequence of Hal-4.117. The heavy chain constant region is underlined. Figure 2B. The VL amino acid sequence of Hal-4.117. The light chain constant region is underlined. Figure 2C. The VH amino acid sequence of Hal-4.120. Figure 2D. The VL amino acid sequence of Hal-4.120. The light chain constant region is underlined. Figure 2E. The VH amino acid sequence of Hal-5.99. The heavy chain constant region is underlined. Figure 2F. The VL amino acid sequence of Hal-5.99. The light chain constant region is underlined. Figure 2G. The VH amino acid sequence of Hal-4.121. The heavy chain constant region is underlined. Figure 2H. The amino acid sequence of VL c.5 of Hal-4.121. The light chain constant region is underlined. Figure 21. The amino acid sequence of VL c.26 of Hal-4.121. The light chain constant region is underlined. Figure 2J. The H-amino acid sequence of Hal-1.16. The heavy chain constant region is underlined. Figure 2K. The VL amino acid sequence of Hal-1.16. The light chain constant region is underlined. Figure 2L. The VH amino acid sequence of Hal-4.5. The heavy chain constant region is underlined. Figure 2M. The VL amino acid sequence of Hal-4.5. The light chain constant region is underlined. Figure 2N. The VH amino acid sequence of Hal-4.40. The heavy chain constant region is underlined. Figure 20. The VL amino acid sequence of Hal-4.40. The light chain constant region is underlined. Figure 2P. The VH amino acid sequence of Hal-4.37. The heavy chain constant region is underlined. Figure 2Q. The VL amino acid sequence of Hal-4.37. The light chain constant region is underlined. Figure 2R. The H-amino acid sequence of Hal-1.43. The heavy chain constant region is underlined. Figure 2S. The VL amino acid sequence of Hal-1.43. The light chain constant region is underlined. Figure 2T. The VH amino acid sequence of Hal-1,152. The heavy chain constant region is underlined. Figure 2U. The VL amino acid sequence of Hal-1,152. The light chain constant region is underlined.

Figure 3. Amino acid and nucleotide sequences of antibodies to PSCA. Figure 3A. The VH cDNA and amino acid sequence of Hal-4.117. The heavy chain constant region is underlined. Figure 3B. The cDNA and amino acid sequence of VL of Hal-4.117. The light chain constant region is underlined. Figure 3C. The VH cDNA and amino acid sequence of Hal-4.120. The heavy chain constant region is underlined. Figure 3D. The cDNA and amino acid sequence of VL of Hal-4.120. The light chain constant region is underlined. Figure 3E. The VH cDNA and amino acid sequence of Hal-5.99. The heavy chain constant region is underlined. Figure 3F. The cDNA and amino acid sequence of VL of Hal-5.99. The light chain constant region is underlined. Figure 3G. The VH cDNA and amino acid sequence of Hal-4.121. The heavy chain constant region is underlined. Figure 3H. The cDNA and amino acid sequence of VL c.5 of Hal-4.121. The light chain constant region is underlined. Figure 31. The cDNA and amino acid sequence of VL c.26 of Hal-4.121. The light chain constant region is underlined. Figure 3J. The cDNA and amino acid sequence of Hal-1.16 VH. The heavy chain constant region is underlined. Figure 3K. The cDNA and amino acid sequence of VL of Hal-1.16. The light chain constant region is underlined. Figure 3L. The VH cDNA and amino acid sequence of Hal-4.5. The heavy chain constant region is underlined. Figure 3M. The cDNA and amino acid sequence of VL of Hal-4.5. The light chain constant region is underlined. Figure 3N. The VH cDNA and amino acid sequence of Hal-4.40. The heavy chain constant region is underlined. Figure 30. The cDNA and amino acid sequence of VL of Hal-4.40. The light chain constant region is underlined. Figure 3P. The VH cDNA and amino acid sequence of Hal-4.37. The heavy chain constant region is underlined. Figure 3Q. The cDNA and amino acid sequence of VL of Hal-4.37. The light chain constant region is underlined. Figure 3R. The cDNA and amino acid sequence of Hal-1.43 VH. The heavy chain constant region is underlined. Figure 3S. The cDNA and amino acid sequence of VL from Hal-1.43. The light chain constant region is underlined. Figure 3T. The cDNA and amino acid sequence of Hal-1,152 VH. The heavy chain constant region is underlined. Figure 3U. The cDNA and amino acid sequence of VL from Hal-1,152. The light chain constant region is underlined.

Figure 4. Alignment of antibodies to PSCA with the germline V-D-J sequences. Figure 4A. VH alignment of Hal-4.117 (SEQ ID NO: 13) with human VH4-31. Figure 4B. VL alignment of Hal-4.117 (SEQ ID NO: 14) with human LI 9 Figure 4C. Alignment of VH from

Hal-4,120 (SEQ ID NO: 15) with human VH4-31 Figure 4D.

VL alignment of Hal-4,120 (SEQ ID NO: 16) with human Figure 4E. Hal-5.99 VH Alignment (SEQ ID NO: 17) with human VH4-34 Figure 4F. AlL alignment of Hal-5.99 (SEQ ID NO: 18) with human A27. Figure 4G.

VH alignment of Hal-4.121 (SEQ ID NO: 19) with human VH4-34. Figure 4H. VL c.5 alignment of Hal-4.121 (SEQ ID NO: 20) with human 08. Figure 41. VL c.26 alignment of Hal-4.121 (SEQ ID NO: 21) with human A3.

Figure 4J. Hal-1.16 VH Alignment (SEQ ID NO: 22) with human VH6-1. Figure 4K. VL alignment of Hal-1.16 (SEQ ID NO: 23) with human B3. Figure 4L.

VH alignment of Hal-4.37 (SEQ ID NO: 28) with human VH4-31. Figure 4M. VL alignment of Hal-4.37 (SEQ ID NO: 29) with human 02.

Figure 5. Expression of PSCA protein in murine, rat and human cell lines. The indicated murine, rat, and human cell lines were infected with retroviruses carrying the human PSCA cDNA and a neomycin resistance or control virus with only the neomycin resistance gene. Stable recombinant cell lines were selected in the presence of G418. PSCA expression was determined by staining FACS with the anti-PSCA mAb 1G8 (5æg / ml). The FACS profile is shown in each cell line demonstrating a fluorescent shift in the PSCA-infected line indicative of PSCA expression on the cell surface. These lines are useful in the development of MAb as immunogens, MAb screening reagents, and in functional assays.

Figure 6. Purification of PSCA protein from E. coli. The E. coli strain BL21 pLysS was transformed with the pET-21b vector encoding amino acids 21-94 of the PSCA cDNA. PSCA protein was expressed by induction of log phase cultures with IPTG and purified by affinity chromatography from the soluble and insoluble fractions of the lysed bacteria. Coomassie blue stained gels of the eluted fractions are shown. This protein is useful as a MAb and pAb immunogen and as an antibody screening reagent. Figure 7. Purification of recombinant glycosylated PSCA protein expressed from 293T cells. Cells

293T were transfected with psecTag2 vector carrying a PSCA cDNA encoding amino acids 28-100. A cell line secreting stable recombinant PSCA was created through hygromycin B drug selection. The PSCA protein present in the conditioned culture medium was purified by affinity chromatography using MAb 1G8. A Coomassie blue stained SDS-PAGE gel of the low pH eluted fractions is shown. The broad molecular weight smear of the protein demonstrates the glycosylation of recombinant PSCA protein as observed in endogenously expressed PSCA.

Figure 8. Purification of GST-PSCA protein from E. coli. The E. coli strain BL21 DE3 was transformed with pGEX-2T encoding amino acids 18-98 of PSCA fused to glutathione-S-transferase (GST). GST-PSCA was induced with isopropyl-beta-D-thiogalactopyranoside (IPTG) from log phase cultures and purified from bacteria lysed by glutathione agarose matrix affinity chromatography. A gel stained with Coomasie blue of the glutathione eluted fractions containing GST-PSCA is shown. The intact GST-PSCA fusion protein and a minor degradation product containing GST is indicated. This protein is useful as a MAb and pAb immunogen and as an Ac screening reagent.

Figure 9. Screening for antibodies to human PSCA by FACS. The concentration of antibody from the supernatants was determined by ELISA. 50 μΐ / well of (pure) was added to 96-well FACS plates and serially diluted. Cells expressing PSCA were added (endogenous and recombinant, 50,000 cells / well) and the mixture was incubated at 4øC for two hours. After incubation, the cells were washed with FACS Buffer and further incubated with 100 μl of detection antibody (anti-hlgG-PE) for 45 minutes at 4 ° C. At the end of the incubation, the cells were washed with FACS Buffer, fixed with formaldehyde and analyzed using FACscan. Data were analyzed using the CellQuest Pro software. The filled histograms represent data from negative control cells and unfilled histograms indicate data from PSCA positive cells.

Figure 10. Relative affinity classification of PSCA MAbs by FACS. 21 serial 1: 2 dilutions of each PSCA antibody were incubated with SW780 cells (50,000 cells per well) overnight at 4 ° C (final concentrations of MAb ranged from 40 nM to 0.038 pM). At the end of the incubation, the cells were washed and incubated with anti-IgG-PE detection antibody. After washing the unbound secondary antibodies, the cells were analyzed by FACS and the mean fluorescence intensity of each point was obtained using the CellQuest Pro software. Affinity was calculated with the Graphpad Prism software using a Sigmoid Dose-Response equation slope). A representative FACS analysis illustrating the binding titre for the MAb of PSCA 4.121 is shown in the figure.

Figure 11. Expression of cynomolgus and murine monkey PSCA in 293T cells and recognition by human anti-PSCA MAbs. 293T cells were transiently transfected with pCDNA3.1 vector carrying the murine PSCA cDNA, the PSCA simian cDNA, or with empty (neo) vector. Two days post-transfection, the cells were harvested and stained with anti-PSCA mAbs Hal-4.117 or murine MAb 1G8 (5æg / ml). The FACS profile demonstrating that the MAb Hal-4.117 created for the human PSCA protein binds to both the murine and simian PSCA proteins expressed in 293T cells is shown. Murine Mab 1G8 binds to PSCA simian but not to murine PSCA. Such results demonstrate the ability to select MAbs that cross-react with antigen from other species. MAbs with cross-reaction will be useful for studying expression and toxicity in these species.

Figure 12. Internalization of PSCA after incubation with MAb 4.121. The MAb of PSCA 4.121 was incubated with PC3-PSCA cells at 4 ° C for 90 min to allow binding of the antibodies to the cell surface. The cells were then divided into two aliquots and incubated at 37 ° C (to allow for the internalization of the antibodies) or 14 ° C (control without internalization). After incubation at 37 ° C or 4 ° C, the remaining PSCA 4.121 MAb bound to the cell surface was removed with an acidic wash. Permeabilization and subsequent incubation with a secondary detection antibody allowed the detection of PSCA 4.121 MAb. Cells were analyzed using FACS or observed under the fluorescence microscope. Approximately 30% of the MAb of PSCA 4,121 was internalized after incubation at 37 ° C for two hours. Figure 13. Antibodies to PSCA mediate saporin-dependent death in cells expressing PSCA. B300.19 cells (750 cells / well) were seeded in a 96-well plate on day 1. The next day an equal volume of medium containing a 2x concentration of the indicated primary antibody together with an excess of 2x of anti-human polyclonal antibody (Hum-Zap) or anti-goat (Goat-Zap) conjugated to saporin toxin (Advanced Targeting Systems, San Diego, CA) was added to each well. Cells were allowed to incubate for 5 days at 37 degrees C. At the end of the incubation period, MTS (Promega) was added to each well and incubation was continued for an additional 4 hours. The OD at 450 nm was determined. The results in Figure 13 (A) show that the antibodies to PSCA HA1-4.121 and HA1-4.117 mediate saporin-dependent cytotoxicity in B300.19-PSCA cells while a non-specific IgG1 antibody control had no effect. The results in Figure 13 (B) show that addition of a secondary saporin conjugated antibody that did not recognize human Fc, failed to mediate cytotoxicity.

Figure 14. Complement mediated cytotoxicity of PSAs MAbs. Antibodies to PSCA (0-50 pg / ml) were diluted with RHB buffer (RPMI 1640, Gibco Life Technologies, 20 mM HEPES). Cells expressing B300.19-PSCA were washed with RHB buffer and resuspended at a density of 106 cells / ml. In a typical assay, 50 μΐ of PSCA antibody, 50 μΐ rabbit complement serum (Cedarlane, Ontario, Can), and 50 μΐ of a cell suspension were added together to a bottom 96 well tissue culture plate plan. The mixture was incubated for 2 h at 37øC in a 5% CO2 incubator to facilitate complement-mediated cell lysis. 50 μΐ Alamar Blue (Biosource Intl.

Camarillo, CA) were added to each well and the incubation continued for an additional 4-5 h at 37 ° C. Fluorescence in each well was read using a 96-well fluorometer with excitation at 530 nm and emission at 590 nm. The results show that antibodies to CA PS having an IgG1 (HA1-4.121) or IgG2 (HA1-5.99.1) isotype but not an IgG4 isotype (HA1-6.46) were able to mediate complement dependent lysis of target cells.

Figure 15. Generation of F (ab ') 2 fragments of MAbs Hal-4.121 via pepsin digestion. 20 mgs of MAb Hal-4.121 in 20 mM sodium acetate buffer pH 4.5 were incubated with and without immobilized pepsin (Pierce, Rockford IL) for the indicated periods of time. Intact MAb and digested Fc fragments were removed by protein A chromatography. A non-reduced, undigested and intact MAb SDS-PAGE stained gel was shown, unaltered aliquots of digested material taken at the indicated times, and a reduced sample of the final digested F (ab ') 2 product.

Figure 16. Binding of recombinant anti-PSCA human mAb to PSCA by flow cytometry. (16A) 293T cells were transfected with expression constructs encoding the light and heavy chains of anti-PSCA human mAb. The supernatant was collected after 48 hours and tested for binding to PSCA. (16B) anti-PSCA human mAb was purified from hybridoma supernatant and used for PSCA binding assays. Binding to PSCA was tested as follows. PC3 or PC3-PSCA cells were incubated with the human anti-PSCA mAbs described above for 30 minutes on ice. The cells were washed and incubated with PE-conjugated anti-human Ig antibody for 30 minutes on ice. Cells were washed and then tested by flow cytometry.

Figure 17. Detection of PSCA protein by immunohistochemistry. Expression of the PSCA protein in tumor specimens from cancer patient was detected using the HA1-4,117 antibody. Paraffin-embedded formalin-fixed tissues were cut into 4 micron sections and mounted on glass slides. The sections were dewaxed, rehydrated and treated with antibody recovery solution (Antigen Retrieval Citra Solution; BioGenex, 4600 Norris Canyon Road, San Ramon, CA, 94583) at elevated temperature. The sections were then incubated in fluorescein-conjugated anti-PSCA monoclonal antibody, Hal-4.117, for 16 hours at 4øC. The slides were washed three times in buffer and further incubated with rabbit anti-fluorescein antibody for 1 hour and, after washing in buffer, immersed in DAKO EnVision + ™ peroxidase-conjugated goat anti-rabbit immunoglobulin goat secondary antibody (DAKO Corporation , Carpenteria, CA) for 30 minutes. The sections were then washed in buffer, developed using the DAB kit (SIGMA Chemicals), counterstained using hematoxylin, and analyzed by light field microscopy. The results show PSCA expression in prostate adenocarcinoma tumor cells (Panel A, Panel B), transitional bladder carcinoma (Panel C) and ductal adenocarcinoma of the pancreas (Panel D). These results indicate that PSCA is expressed in human cancers and that antibodies directed against this antigen are useful as diagnostic reagents. Figure 18. PSCA Mab Hal-4,120 inhibits the growth of subcutaneous prostate cancer xenografts. LAPC-9AI tumor cells (2.0 x 106 cells) were injected subcutaneously into male SCID mice. The mice were randomized into groups (n = 10 in each group) and the treatment was started intraperitoneally (i.p.) in the

Day 0 with HA1-4.120 or isotype control Mab as indicated. The animals were treated twice weekly for a total of 7 doses by day 28 of the study. Tumor growth was monitored using one gauge measurements every 3 to 4 days as indicated. The results show that the human anti-PSCA monoclonal antibody Hal-4.120 significantly inhibited the growth of human prostate cancer xenografts implanted subcutaneously in SCID mice (p <0.05).

Figure 19. PSCA Mab Hal-5.99 inhibits the growth of established prostate cancer xenografts in SCID mice. LAPC-9AI tumor cells (2.0 x 106 cells) were injected subcutaneously into male SCID mice. When the tumor volume reached 50 mm 3, the mice were randomized into groups (n = 10 in each group) and the treatment was started intraperitoneally (i.p.) with HA1-5.99.1 or isotype control Mab as indicated. The animals were treated twice weekly for a total of 5 doses by day 14 of the study. Tumor growth was monitored using one gauge measurements every 3 to 4 days as indicated. The results show that the fully human anti-PSCA monoclonal antibody Hal-5.99 significantly inhibited the growth of established androgen independent human prostate cancer xenografts implanted subcutaneously in SCID mice (p <0.05).

Figure 20. PSCA Mab Hal-4.121 inhibits the growth of established subcutaneous androgen dependent human prostate cancer xenografts. LAPC-9AD tumor cells (2.5 x 106 cells) were injected subcutaneously into male SCID mice. When the tumor volume reached 40 mm 3, the mice were randomized into groups (n = 10 in each group) and treatment was started intraperitoneally (i.p.) with increasing concentrations of HA1-4.121 or isotype control Mab as indicated. The animals were treated twice weekly for a total of 7 doses by day 21 of the study. Tumor growth was monitored using one gauge measurements every 3 to 4 days as indicated. The results of this study demonstrate that HA1-4,121 inhibited the growth of subcutaneous human androgen dependent prostate cancer xenografts established in SCID mice. The results were statistically significant for the 300æg dose group at days 14, 17 and 21 (p <0.05, Kruskal-Wallis test, bilateral at a = 0.05) and at the dose group of 700æg on days 10, 14, 17 and 21 (p <0.05, Kruskal-Wallis test, bilateral with a = 0.05).

Figure 21. Androgen dependent patient derived LAPC-9AD tumor cells (2.0 x 105 cells) were injected into the dorsal lobes of the prostates of male SCID mice. The tumors were allowed to grow for approximately 10 days, at which time the mice were randomized into clusters. Treatment with 500æg of human HA1-4,117, HA1-4,121 or isotype control MAb was initiated 10 days after tumor implantation. Antibodies were administered intraperitoneally twice weekly in a total of 7 doses. Four days after the last dose, the animals were sacrificed and the primary tumors were excised and weighed. The results show that the human anti-PSCA monoclonal antibodies Hal-4.121 (p <0.01) and Hal-4.117 (p &lt; 0.05) significantly inhibited the growth of orthotopically implanted LAPC-9AD prostate cancer xenografts in SCID mice .

Figure 22. PSCA mAb HA1-4.121 prolongs the survival of SCID mice with orthotally-established human androgen dependent prostate tumors. LAPC-9AD tumor cells derived from patients, androgen dependent (2.0 x 106 cells) were injected into the dorsal lobes of the prostates of male SCID mice. The tumors were allowed to grow for approximately 9 days, at which time the mice were randomized into clusters. Randomized animals in the survival groups include 11 mice in the isotype control MAb and 12 mice in the HA1-4,121 treatment group. The animals were treated i.p. with 1000æg of Hal-4.121 or 1000æg of isotype control MAb twice weekly for a total of 9 doses. The results demonstrated that HA1-4.121 significantly (log-rank test: p <0.01) significantly prolonged survival of SCID mice with human androgen dependent prostate tumors. Two mice in the HA1-4,121 treated group remained free of palpable tumors 110 days after the last treatment.

Figure 23. Potential inhibition of prostate tumor growth with combination therapy of HA1-4.21 and taxotere. LAPC-9AI tumor cells (2 x 106 cells per animal) were injected subcutaneously into male SCID mice. When the tumor volume reached 65 mm3, the animals were randomized and assigned to four different groups (n = 10 in each group) as indicated. Starting at day 0, Hal-4.121 or isotype control MAb were administered i.p. twice a week at a dose of 500æg for a total of 6 doses. The last dose was given on day 17. Taxotere was given intravenously at a dose of 5 mg / kg on days 0, 3 and 7. Tumor growth was monitored every 3-4 days using one gauge measurements. The results of this study demonstrate that HA1-4,121 as the sole agent inhibited the growth of androgen-independent prostate xenografts in SCID mice by 45% compared to the control antibody treatment alone at day 28 (ANOVA / Tukey's test: p < 0.05). Administration of the taxotere isotype control MAb inhibited tumor growth by 28% compared to the control antibody treatment alone, which was not statistically significant. Administration of HA1-4,121 in combination with taxotere potentiated the effect and resulted in inhibition of 69% tumor growth compared to the control antibody alone (ANOVA / Tukey's test: p <0.01). A statistically different difference was also demonstrated when the combination group of HA1-4,121 plus taxotere was

compared to the HA1-4.121 or isotype control MAb plus taxotere groups (ANOVA / Tukey's test: p <0.05). Figure 24. Human PSCA MAbs inhibit the growth of pancreatic cancer xenografts in human SCID / HPAC mice. Pancreatic cancer cells (2x106 / mouse) were injected subcutaneously into immunodeficient SCID ICID mice (Taconic Farm, Germantown, NI). Mice were randomized into groups (n = 10 animals / group) and treatment with indicated human PSCA monoclonal antibody initiated the same day. Antibodies (500 mg / mouse) were administered intraperitoneally twice weekly for a total of 8 doses. The results demonstrated that the anti-PSCA monoclonal antibodies Hal-4.121, Hal-4.117 and Hal-1.16 significantly inhibited the growth of human pancreatic cancer xenografts implanted subcutaneously in SCID mice. Statistical analyzes were performed using a t-test (bilateral, a = 0.05).

Figure 25. PSA MAB HA1-4.121 inhibits the growth of orthotopically implanted pancreatic tumors in SCID mice. HPAC cells (3.0 x 106 cells) were orthopedically implanted into the pancreas of SCID mice. Mice were randomly assigned to three groups (n = 9 in each group) as indicated. Treatment with HA1-4,121 (250æg or 1000æg) isotype control Mab (1000æg) was started on the day of implantation. Antibodies were given i.p twice a week for a total of 10 doses. Thirty days after the last dose, the animals were sacrificed and the primary tumors were excised and weighed. The results of this study demonstrate that HA1-4,121 significantly inhibited the orthotopic growth of human pancreatic cancer xenografts in SCID mice at both dose levels examined. Treatment with 250æg and 1000æg of AGS-PSCA inhibited tumor growth by 66% and 70%, respectively (Kruskal-Wallis / Tukey's test: p <0.01 and p <0.01, respectively).

Figure 26. MAb for PSCA HAL-4.121 inhibits metastasis. At necropsy, visible metastases in the lymph nodes and distal organs were observed in the control antibody treated group. No visible metastases were observed in both groups treated with HAL-4.121. Lymph nodes, lungs and livers were removed from all animals and examined histologically for the presence of metastatic tumor. Cuts of the lungs and lymph nodes removed from each animal were stained for human cytokeratin and the number of metastases was determined microscopically. Histological analysis results demonstrated a significant reduction in lymph node (LN) metastasis in animals treated with HAL-4.121 (p = 0.0152 as detected by Fisher's exact test). Incidence of metastases and invasion was also significantly decreased in animals treated with both concentrations of HAL-4.121 (p = 0.0152 as detected by Fisher's exact test). The number of lung metastases decreased significantly in mice treated with the 1.0 mg dose of HAL-4.121 alone (p = 0.0498 as detected by the Fisher's exact test).

Figure 27. MAbs for human PSCA inhibit the growth of SW780 bladder tumors in SCID mice. Human bladder cancer cells SW780 (2x106 / mouse) were

injected subcutaneously into immunodeficient SCID ICID mice (Taconic Farm, Germantown, NI). Mice were randomized into groups (n = 10 animals / group) and treatment with indicated human PSCA MAb initiated on the same day. Antibodies (250 mg / mouse) were administered intraperitoneally twice weekly for a total of 7 doses. The results showed that HA1-4.117 (p = 0.014), HA1-4.37 (p = 0.0056), HA1-1.78 (p = 0.001), Hal-5.99 (p = 0.0002) and HA1-4.5 (p = 0.0008) significantly inhibited the growth of SW780 bladder tumors implanted subcutaneously in SCID mice. Statistical analyzes were performed using a t-test (bilateral, oi = 0.05). DETAILED DESCRIPTION OF THE INVENTION Design of Sections I.) Definitions

II. ) PSCA polynucleotides

II.A.) Uses of PSCA Polynucleotides II.A.1.) Monitoring of Genetic Abnormalities II.A.2.) Forms of Anti-Sensitive Realization II.A.3.) Initiators and Pairs of Initiators II.A.4. ) Isolation of Nucleic Acid Molecules

Coding PSCA II. A.5.) Recombinant Nucleic Acid Molecules and Host-Vector Systems

III. ) Proteins related to PSCA III. A.) Forms of Protein Realization Carrying Reasons

III.B.) Expression of Proteins Related to PSCA III.C.) Modifications of Proteins Related to PSCA

III.D.) Protein Uses Related to CA PS

IV. ) Antibodies to PSCA

V.) Cellular Immune Responses against PSCA

SAW. ) Transgenic animals for PSCA

VII. ) Methods for PSCA VIII Detection. ) Methods for Monitoring the Status of Genes Related to PSCA and its Products

IX. ) Identification of Molecules Interacting with PSCA X.) Therapeutic Methods and Compositions X.A.) Anti-cancer Vaccines X.B.) PSCA as a Target for Therapeutics based on X.C. Antibodies. PSCA as a Target for Cellular Immune Responses X.C.l. Minigenes 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. Vaccinal Compositions Comprising DC Pulsed with CTL and / or HTL X. D. Peptides. ) Vaccine Immunotherapy X.E.) Administration of Vaccines for Purposes

Therapeutic or Prophylactic

XI. ) Ways of Diagnosis and Prognosis of PSCA

XII. ) Inhibition of Protein Function of PSCA XII.A.) Inhibition of PSCA with Intracellular Antibodies XII.B.) Inhibition of PSCA with Recombinant Proteins XII.C.) Inhibition of Transcription or Translation of PSCA XII.D.) General Considerations for Strategies

Therapies

XIII. ) Identification, Characterization and Use of PSCA Modulators

XIV. ) iRNA and therapeutic use of small interfering RNAs (siRNAs) XV. ) Definitions: Unless defined otherwise, all technical terms, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those skilled in the art to those skilled in the art. which the invention refers. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for rapid reference, and the inclusion of such definitions herein shall not necessarily be construed as representing a substantial difference from that which is generally understood in the art . Many of the techniques and procedures described or referenced herein are well understood and customarily employed using conventional methodology by those skilled in the art, such as, for example, the widely used molecular cloning methodologies described in Sambrook et al. , Molecular Cloning: A Laboratory Manual 2nd. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY As appropriate, procedures involving the use of commercially available kits are generally carried out according to the manufacturer's protocols and / or parameters unless otherwise indicated. contrary.

The terms &quot; advanced prostate cancer &quot;, &quot; locally advanced prostate cancer &quot;, &quot; advanced disease &quot; and &quot; locally advanced disease &quot; means prostate cancers that have spread through the prostatic capsule, and are intended to include stage C disease under the American Urological Association (AUA), Cl-C2 stage disease under the Whitmore-Jewett system, and stage T3 - T4 and N + under the TNM system (tumor, nodules, metastasis). In general, surgery is not recommended for patients with locally advanced disease, and these patients have substantially less favorable outcomes compared to patients who have clinically localized (organ-confined) prostate cancer. Locally advanced disease is clinically identified by palpable evidence of hardening beyond the lateral margin of the prostate, or asymmetry or hardening above the prostate base. Locally advanced prostate cancer is currently pathologically diagnosed following radical prostatectomy if the tumor invades or penetrates the prostatic capsule, extends to the surgical margin, or invades the seminal vesicles. &quot; Change the native glycosylation pattern &quot; is intended, for purposes herein, to mean eliminating one or more carbohydrate moieties found in the native sequence PSCAs (either by removal of the underlying glycosylation site or by removal of glycosylation by chemical and / or enzymatic means), and / or adding one or more glycosylation sites that are not present in the native sequence PSCA. Additionally, the phrase includes qualitative changes in the glycosylation of native proteins, involving a change in the nature and proportions of the various carbohydrate fractions present. The term &quot; analog &quot; refers to a molecule that is structurally similar or shares similarity or corresponding attributes with another molecule (e.g., a PSCA-related protein). For example, an analog of the PSCA protein can be specifically bound by an antibody or T cell that binds specifically to PSCA. The term &quot; antibody &quot; is used in the broadest sense unless clearly indicated otherwise. Therefore, an &quot; antibody &quot; may be naturally occurring or man-made such as monoclonal antibodies produced by conventional hybridoma technology. Anti-PSCA 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. As used herein, the term &quot; antibody &quot; refers to any form of antibody or fragment thereof that specifically binds to PSCA and / or exhibits the desired biological activity and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., antibodies bispecific antibodies), and antibody fragments provided that they bind specifically to PSCA and / or exhibit the desired biological activity. Any specific antibody may be used in the methods and compositions provided herein. Thus, in one embodiment the term &quot; antibody &quot; encompasses a molecule comprising at least one variable region from a light chain immunoglobulin molecule and at least one variable region from a heavy chain molecule which in combination form a specific binding site for the target antigen. In one embodiment, the antibody is an IgG antibody. For example, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. Antibodies useful in the present methods and compositions may be generated in cell culture, phage or various animals, including but not limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, apes. Thus, in one embodiment, an antibody of the present invention is a mammalian antibody. Phage techniques may be used to isolate an early antibody or to generate variants with altered specificity or avidity characteristics. Such techniques are routine and are well known in the art. In one embodiment, the antibody is produced by recombinant means known in the art. For example, a recombinant antibody can be produced by transfecting a host cell with a vector comprising a DNA sequence encoding the antibody. One or more vectors may be used to transfect the DNA sequence expressing at least one VH region and one VL in the host cell. Exemplary depictions of recombinant means of generating and producing antibodies include Delves, ANTIBODY PRODUCTION: ESSENTIAL TECHNIQUES (Wiley, 1997); Shephard, et al., MONOCLONAL ANTIBODIES (Oxford University Press, 2000); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (Academic Press, 1993); CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons, most recent edition). An antibody of the present invention may be modified by recombinant means to increase the effectiveness of the antibody to mediate the desired function. Thus, it is within the scope of the present invention that the antibodies may be modified by substitutions using recombinant means. Typically, the substitutions will be conservative substitutions. For example, at least one amino acid in the constant region of the antibody may be substituted with a different residue. See, for example, U.S. Patent No. 5,624,821, U.S. Patent No. 6,194,551, Application No. WO 9958572; and Angal, et al., Mol. Immunol. 30: 105-08 (1993). Modification at amino acids includes deletions, additions, amino acid substitutions. In some cases, such changes are made to reduce undesirable activities, for example, complement dependent cytotoxicity. Antibodies are often labeled by the covalent or non-covalent junction of a substance that provides a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the patent and scientific literature. These antibodies can be screened for normal and defective PSCA binding. See, for example, ANTIBODY ENGINEERING: A PRACTICAL APPROACH (Oxford University Press, 1996). Suitable antibodies with the desired biological activities may be identified by the following in vitro assays including, but not limited to: proliferation, migration, adhesion, growth in soft agar, angiogenesis, cell-cell communication, apoptosis, transport, signal transduction, and the following in vivo assays such as inhibition of tumor growth. Antibodies provided herein may also be useful in diagnostic applications. As capture or non-neutralizing antibodies, they may be screened for the ability to bind to specific antigen without inhibiting binding to the receptor or biological activity of the antigen. As neutralizing antibodies, the antibodies may be useful in competitive binding assays. They can also be used to quantify the PSCA or its receptor.

An &quot; antibody fragment &quot; 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 unique anti-PSCA antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-PSCA antibody compositions with polyepitopic specificity. The antibody of the present methods and compositions may be monoclonal or polyclonal. An antibody may be in the form of an antigen-binding antibody fragment comprising a Fab fragment, F (ab ') 2 fragment, a single-stranded variable region, and the like. Fragments of intact molecules can be generated using methods well known in the art include enzymatic and recombinant digestion media.

As used herein, any form of &quot; antigen &quot; can be used to generate an antibody that is specific for PSCA. Thus, the triggering antigen may be a single epitope, multiple epitopes, or whole protein alone or in combination with one or more immunogenicity enhancing agents known in the art. The triggering antigen may be an isolated full length protein, a cell surface protein (for example, immunizing with cells transfected with at least a portion of the antigen), or a soluble protein (for example, immunizing with only the portion of the extracellular domain of the protein). The antigen can be produced in a genetically modified cell. The DNA encoding the antigen may be genomic or non-genomic (e.g., cDNA) and encodes at least a portion of the extracellular domain. As used herein, the term &quot; portion &quot; refers to the minimum number of amino acids or nucleic acids, as appropriate, to constitute an immunogenic epitope of the antigen of interest. Any suitable genetic vectors for transformation of the cells of interest may be employed, including, but not limited to, adenoviral vectors, plasmids, and non-viral vectors such as cationic lipids. In one embodiment, the antibody of the methods and compositions herein specifically binds to at least a portion of the extracellular domain of the PSCA of interest.

Antibodies or antigen-binding fragments thereof provided herein may be conjugated with a &quot; bioactive &quot;. As used herein, the term &quot; bioactive agent &quot; refers to any synthetic or naturally occurring compound that binds the antigen and / or potentiates or mediates a desired biological effect to potentiate the toxins that kill the cells.

In one embodiment, the linker fragments useful in the present invention are biologically active fragments. As used herein, the term &quot; biologically active &quot; refers to an antibody or antibody fragment that is capable of binding to the desired ANTIPHENIC epitope and directly or indirectly exerting a biological effect. Direct effects include, but are not limited to, modulation, stimulation, and / or inhibition of a growth signal, modulation, stimulation, and / or inhibition of an anti-apoptotic signal, modulation, stimulation, and / or inhibition of an apoptotic or necrotic signal, modulation, stimulation, and / or inhibition of the ADCC cascade, and modulation, stimulation, and / or inhibition of the CDC cascade.

Bispecific antibodies &quot; are also useful in the present methods and compositions. As used herein, the term &quot; bispecific antibody &quot; refers to an antibody, typically a monoclonal antibody, having binding specificities for at least two different antigenic epitopes. In one embodiment, the epitopes are from the same antigen. In another embodiment, the epitopes are from two different antigens. Methods for obtaining bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly using the coexpression of two light chain / immunoglobulin heavy chain pairs. See, for example, Milstein et al., Nature 305: 537-39 (1983). Alternatively, bispecific antibodies may be prepared using chemical binding. See, for example, Brennan, et al., Science 229: 81 (1985). Bispecific antibodies include fragments of bispecific antibodies. See, for example, Hollinger, et al., Proc. Natl. Acad. Know. USA 90: 6444-48 (1993), Gruber, et al., J. Immunol. 152: 5368 (1994).

The monoclonal antibodies herein specifically include &quot; chimeric &quot; in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder of the chain (s) is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, provided that they bind specifically to the target antigen and / or exhibit the desired biological activity (US Pat. 4,816,567, and Morrison et al., Proc Natl Acad Sci USA 81: 6851-6855 (1984)). The term &quot; Chemotherapeutic Agent &quot; refers to all chemical compounds that are effective in inhibiting tumor growth. Non-limiting examples of chemotherapeutic agents include alkylating agents; for example, nitrogen mustards, ethyleneimine compounds and alkyl sulfonates; antimetabolites; for example, folic acid, purine or pyrimidine antagonists; mitotic inhibitors; for example, vinca alkaloids and podophyllotoxin derivatives, cytotoxic antibiotics, compounds that damage or interfere with the expression of DNA, and growth factor receptor antagonists. In addition, the chemotherapeutic agents include cytotoxic agents (as defined herein), antibodies, biological molecules and small molecules. The term &quot; codon optimized sequence &quot; refers to nucleotide sequences that have been optimized for a particular host species by substituting any codons having a frequency of use of less than about 20%. Sequences that have been optimized for expression in a given host species by elimination of spurious polyadenylation sequences, deletion of exon / intron splicing signals, elimination of transposon-like repeats and / or GC content optimization in addition to codon optimization are referred to herein as &quot; enhanced expression sequences &quot;.

A &quot; combinatorial library &quot; is a collection of various chemical compounds generated by chemical synthesis or biological synthesis by combining a series of &quot; building blocks &quot; chemicals such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide library (e.g., mutein), is formed by combining a set of building blocks called amino acids in all possible forms for a given length of a compound (or the number of amino acids in a polypeptide compound). Numerous chemical compounds are synthesized by such a combinatorial mixture of chemical building blocks (Gallop et al., J. Med. Chem. 37 (9): 1233-1251 (1994)). The preparation and screening of combinatorial libraries are well known to those skilled in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, for example, U.S. Patent No. 5,010,175, Furka, Pept.Prot.Res.37: 487-493 (1991), Houghton et al., Nature, 354: 84-88 (1991)), peptoids (PCT Publication No. WO 91/19735), coded peptides (PCT Publication No. WO 93/20242), random bio- oligomers PCT No. WO 92/00091), benzodiazepines (US Patent No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc Nat Nat. Acad Sci USA 90: 6909-6913 (1993)), vinylogenic polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114: 6568 (1992)), non-peptidic peptidomimetics with a Beta-D-Glucose structure (Hirschmann et al., J. Amer Chem. Soc. 114: 9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116: 2661 (1994)), oligocarbamates al., Science 261: 1 303 (1993)), and / or peptidyl phosphonates (Campbell et al. , J. Org. Chem. 59: 658 (1994)). See, generally, Gordon et al., J. Med. Chem. 37: 1385 (1994), nucleic acid libraries (see, for example, Stratagene, Corp.), peptide nucleic acid libraries (see, for example, U.S. Patent 5,539,083), libraries of antibodies (see, for example, Vaughn et al., Nature Biotechnology 14 (3): 309-314 (1996), and PCT / US96 / 10287), carbohydrate libraries (see, for example, , Liang et al., Science 274: 1520-1522 (1996), and U.S. Patent No. 5,593,853), and libraries of small organic molecules (see, for example, benzodiazepines, Baum, et al., Jan 18, page 33 (1993), isoprenoids, U.S. Patent 5,569,588; thiazolidinones and metathiazanones, U.S. Patent No. 5,549,974; pyrrolidines, U.S. Patent Nos. 5,525,735 and 5,519,134; morpholino, US Pat. No. 5,506,337, benzodiazepines, U.S. Patent No. 5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commercially available (see, for example, 357 NIPS, 390 NIPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A, Applied Biosystems, Foster City, CA; , Plus, Millipore, Bedford, NIA). A number of well known robotic systems have also been developed for solution phase chemists. These systems include automated workstations such as the automatic synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems using robotic arms (Zymate H, Zymark Corporation, Hopkinton, Mass., Orca, Hewlett-Packard, Palo Alto, Calif.), Which mimic the synthetic synthetic operations performed by a chemist. Any of the above devices is suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they may function as discussed herein will be apparent to those skilled in the relevant art. In addition, numerous combinatorial libraries are themselves commercially available (see, for example, ComGenex, Princeton, NJ; Asinex, Moscow, UK; Tripos, Inc., St. Louis, MO, ChemStar, Ltd, Moscow, UK , 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

As used herein, the term &quot; conservative substitution &quot; refers to amino acid substitutions that are known to those skilled in the art and can be made generally without altering the biological activity of the resulting molecule. Those skilled in the art recognize that, in general, substitutions of a single amino acid in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson, et al., MOLECULAR BIOLOGY OF THE GENE, The Benjamin / Cummings Pub. Co., p 224 (4th Edition, 1987)). Such exemplary substitutions are preferably made according to those set forth in Table (s) III (a-b). For example, such changes include replacing any of isoleucine (I), valine (V), and leucine (L) with any of these other hydrophobic amino acids; aspartic acid (D) by glutamic acid (E) and vice versa; glutamine (Q) by asparagine (N) and vice versa; and serine (S) by threonine (T) and vice versa. Other substitutions may 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) may often be interchangeable, such as may alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can often be exchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are often interchangeable at locations where the significant characteristic of the amino acid residue is their charge and the distinct pKs of these two amino acid residues are not significant. Still other changes can be considered &quot; conservative &quot; in particular environments (see, for example, Table III (a) herein, pages 13-15 "Biochemistry" 2nd ED Lubert Stryer ed (Stanford University), Henikoff et al., PNAS 1992 Vol 89 10915 Lei et al., J Biol Chem 1995 May 19; 270 (20): 11882-6). Other substitutions are also permissible and may be determined empirically or in accordance with known conservative substitutions. The term &quot; cytotoxic agent &quot; refers to a substance that inhibits or prevents expression activity in cells, cell function and / or causes destruction of cells. The term is intended to include radioactive isotopes, chemotherapeutic agents, chemotherapeutic agents such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof. Examples of cytotoxic agents include, but are not limited to, auristatins, auristatin and, auromycins, maytansinoids, yttrium, bismuth, ricin, ricin A chain, combrestatin, duocarmycins, dolostatins, doxorubicin, daunorubicin, taxol, cisplatin, ccl065, ethidium bromide , mitomycin, etoposide, tenophoside, vincristine, vinblastine, colchicine, dihydroxy anthracenedione, actinomycin, diphtheria toxin, Pseudomonas (PE) exotoxin A, PE40, abrin, abrin A chain, modecin A chain, alpha- mitogelin, retstrictocin, phenomycin, enomycin, curicin, crotina, calicheamicin, Saponaria officinalis inhibitor, and glucocorticoids and other chemotherapeutic agents, as well as radioisotopes such as At211, I135, Y125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32 and radioactive isotopes of Lu including Lu177. The antibodies may also be conjugated to an anti-cancer prodrug activating enzyme capable of converting the prodrug into its active form.

As used herein, the term &quot; diabodies &quot; refers to small fragments of antibodies with two antigen binding sites, the fragments of which comprise a heavy chain variable domain (Vh) connected to a light chain variable domain (V1) in the same polypeptide chain (V1-V1). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen binding sites. The diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al. Proc. Nat1. Acad. Know. USA 90: 6444-48 (1993). &Quot; gene product &quot; is used herein to denote a peptide / protein or mRNA. For example, a &quot; gene product of the invention &quot; is sometimes referred to herein as a &quot; cancer amino acid sequence &quot;, &quot; cancer protein &quot; &quot; a cancer protein listed in Table I &quot;, a &quot; cancer mRNA &quot;, &quot; cancer listed in Table I &quot;, etc. In one embodiment, the cancer protein is encoded by a nucleic acid of Figure 1. The cancer protein may be a fragment, or alternatively, be a full length protein encoded by nucleic acids of Figure 1. In one embodiment, a cancer amino acid sequence is used to determine sequence identity or similarity. In another embodiment, the sequences are naturally occurring allelic variants of a protein encoded by a nucleic acid of Figure 1. In another embodiment, the sequences are sequence variants as further described herein.

Antibodies &quot; heteroconjugates &quot; are useful in the present methods and compositions. As used herein, the term &quot; heteroconjugate antibody &quot; refers to two covalently linked antibodies. Such antibodies may be prepared using methods known in synthetic protein chemistry, including the use of crosslinking agents. See, for example, U.S. Patent No. 4,676,980.

&Quot; High Throughput Screening &quot; for presence, absence, quantification, or other properties of particular nucleic acids or protein products are well known to those skilled in the art. Similarly, binding assays and reporter gene assays are equally well known. Thus, for example, U.S. Patent No. 5,559,410 discloses high throughput screening methods for proteins; U.S. Patent No. 5,585,639 discloses high throughput screening methods for nucleic acid binding (i.e., in matrices); while U.S. Patent Nos. 5,576,220 and 5,541,061 disclose high yield methods of ligand / antibody binding screening.

In addition, high throughput screening systems are commercially available (see, for example, Amersham Biosciences, Piscataway, NJ; Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, CA, Precision Systems, Inc., Natick, MA, etc.). These systems typically automate complete procedures, including all pipetting of samples and reagents, distribution of liquids, timed incubations, and final readings of the microplates in appropriate assay detectors. These configurable systems provide high throughput and fast start as well as a high degree of flexibility and customization. Manufacturers of such systems provide detailed protocols for various high performance systems. Thus, for example, Zymark Corp. provides technical bulletins describing screening systems for the detection of gene transcription modulation, ligand binding, and the like. The term &quot; homologue &quot; refers to a molecule which exhibits homology with another molecule, for example, having sequences of chemical residues that are the same or similar at corresponding positions.

In one embodiment, the antibody provided herein is a &quot; human antibody &quot;. As used herein, the term &quot; human antibody &quot; refers to an antibody in which essentially all sequences of the light chain and heavy chain sequences, including the complementarity determining regions (CDRs), are for human genes. In one embodiment, the human monoclonal antibodies are prepared by the trioma technique, human B cell technique (see, for example, Kozbor, et al., Immunol. Today 4: 72 (1983), EBV Transformation Technique (see, for example, Cole et al., MONOCLONAL ANTIBODIES AND CANCER THERAPY 77-96 (1985)), or using phage display (see, for example, Marks et al., J. Mol. Biol. : 581 (1991)) In a specific embodiment, the human antibody is generated in a transgenic mouse. particularly preferred, fully human antibody sequences are made in a transgenic mouse engineered to express human light and heavy chain antibody genes. An exemplary description of the preparation of transgenic mice that produce human antibodies those found in WO 02/43478 and in US Pat.

United States 6,657,103 (Abgenix) and its progeny. B cells from transgenic mice that produce the desired antibody can be fused to produce hybridoma cell lines for the continuous production of the antibody. See, for example, U.S. Patent Nos. 5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,545,806; and Jakobovits, Adv Drug Del. Rev. 31: 33-42 (1998); Green, et al., J. Exp. Med. 188: 483-95 (1998). &Quot; Human Leukocyte Antigen &quot; or &quot; HLA &quot; is a human class I or class II major Histocompatibility Complex (MHC) protein (see, for example, Stites, et al., IMMUNOLOGY, 8 to ED., Lange Publishing, Los Altos, CA (1994).

As used herein, the term &quot; humanized antibodies &quot; refers to antibody forms which contain non-human (e.g., murine) antibody sequences as well as human antibodies. Such antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody, optionally, will also comprise at least a portion of a constant region (Fc) of a

immunoglobulin, typically that of a human immunoglobulin. See, for example, U.S. Patent No. 4,816,567 to Cabilly; Queen et al. (1989) Proc. Nat'l Acad. Know. USA 86: 10029-10033; and ANTIBODY ENGINEERING: A PRACTICAL APPROACH (Oxford University Press 1996).

The terms &quot; hybridize &quot; &quot; &quot; &quot; hybridize &quot;, &quot; hybridize &quot; and the like, used in the context of polynucleotides, are intended to refer to conventional hybridization conditions, preferably such as hybridization in 50% formamide / 6xSSC / 0.1% SDS / 100 mg / ml ADSs, in which the temperatures for the hybridization are above 37 degrees C and temperatures for washing in 0.1xSSC / 0.1% SDS are above 55 degrees C.

The &quot; isolated &quot; and &quot; biologically pure &quot; refer to material which is substantially or essentially free of components which normally accompany it as found in its native state. Thus, isolated peptides according to the invention preferably do not contain materials normally associated with peptides in their in situ environment. For example, a polynucleotide is said to be &quot; isolated &quot; when it is substantially separated from contaminating polynucleotides that correspond to or are complementary to genes in addition to the PSCA genes or encoding polypeptides other than the PSCA gene product or fragments thereof. One skilled in the art can readily employ nucleic acid isolation procedures to obtain an isolated PSCA polynucleotide. A protein is said to be &quot; isolated &quot;, for example, when physical, mechanical or chemical methods are used to remove the PSCA proteins from the cellular constituents which are normally associated with the protein. One skilled in the art can readily employ standard purification methods to obtain an isolated PSCA protein. Alternatively, an isolated protein can be prepared by chemical means. &quot; Bookmarks &quot; Suitable materials include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescers, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such tags include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.

In addition, the antibodies provided herein may be useful as the antigen-binding component of fluorobodies. See, for example, Zeytun et al., Nat. Biotechnol. 21: 1473-79 (2003). The term &quot; mammal &quot; refers to organisms 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 &quot; metastatic prostate cancer &quot; and &quot; metastatic disease &quot; means prostate cancers that have spread to regional or distant lymph nodes and are intended to include stage D disease under the AUA system and TxNxM + stage under the TNM system. As is the case with locally advanced prostate cancer, surgery is generally not indicated for patients with metastatic disease, and hormone therapy (androgen ablation) is a preferred treatment modality. Patients with metastatic prostate cancer eventually develop an androgen-refractory state within 12 to 18 months of initiation of treatment. Approximately half of these androgen-refractory patients die within 6 months after developing this condition. The most common site for prostate cancer metastases is bone. Bone metastases from prostate cancer are often osteoblastic rather than osteolytic (ie, resulting in the formation of new bone). Bone metastases are most commonly found in the spine, followed by the femur, pelvis, rib cage, skull and humerus. Other common sites for metastasis include the lymph nodes, lung, liver and brain. Metastatic prostate cancer is typically diagnosed by laparoscopic or open pelvic lymphadenectomy, scanning of whole body radionuclides, skeletal radiography, and / or biopsy of bone lesions. The term &quot; modulator &quot; or &quot; test compound &quot; or &quot; drug candidate &quot; or grammatical equivalents as used herein describe any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., to be tested for the ability to directly or indirectly alter the cancer phenotype or expression of a cancer sequences, for example, protein or nucleic acid sequences, or effects of cancer sequences (e.g., signaling, gene expression, protein interaction, etc.). In one aspect, a modulator will counteract the effect of a cancer protein of the invention. By &quot; neutralizes &quot; it is understood that an activity of a protein is inhibited or blocked together with the consequent effect on the cell. In another aspect, a modulator will neutralize the effect of a gene, and its corresponding protein, of the invention by normalizing the levels of said protein. In preferred embodiments, the modulators alter the expression profiles, or nucleic acids, or expression profile proteins provided herein, or downstream effector pathways. In one embodiment, the modulator suppresses a cancer phenotype, for example, to a normal tissue fingerprint. In another embodiment, a modulator induced a cancer phenotype. Generally, a plurality of assay mixtures are run in parallel with different concentrations of agent to obtain a differential response at various concentrations. Typically, one of these concentrations serves as a negative control, i.e. at concentration zero or below the detection level.

Modulators, drug candidates or test compounds encompass numerous chemical classes, though typically they are organic molecules, preferably compounds of small organic molecules having a molecular mass of more than 100 and less than about 2,500 daltons. Preferred small molecules have less than 2000, or less than 1500 or less than 1000 or less than 500 D. Applicant agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least one amine group, carbonyl, hydroxyl or carboxyl, preferably at least two of the functional chemical groups. Candidate agents often comprise cyclic or heterocyclic carbon structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Modulators also comprise biomolecules such as peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Peptides are particularly preferred. One class of modulators are peptides, for example from about five to about 35 amino acids, with from about five to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. Preferably, the cancer modulating protein is soluble, includes a non-transmembrane region, and / or has an N-terminal Cys to aid in solubility. In one embodiment, the C-terminus of the fragment is maintained as a free acid and the N-terminus is a free amine to aid in coupling, i.e. to the cysteine. In one embodiment, a cancer protein of the invention is conjugated to an immunogenic agent as discussed herein. In one embodiment, the cancer protein is conjugated to BSA. The peptides of the invention, for example of preferred lengths, may be linked to one another or to other amino acids to create a longer peptide / protein. Modulatory peptides may be naturally occurring protein digestion products as outlined above, random peptides, or "deviated" random peptides. In a preferred embodiment, peptide / protein based modulators are antibodies, and fragments thereof, as defined herein.

Cancer modulators may also be nucleic acids. Nucleic acid modulating agents may be naturally occurring nucleic acids, random nucleic acids, or "&quot; random nucleic acids. &Quot; For example, products of prokaryotic or eukaryotic genome digestion may be used in an approach analogous to that outlined above for proteins. The term &quot; monoclonal antibody &quot; as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minimal quantities. Monoclonal antibodies are highly specific, directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of antibodies directed against (or specific for) different epitopes. In one embodiment, the polyclonal antibody contains a plurality of monoclonal antibodies with different specificities, affinities, or avidity for epitopes within a single antigen containing multiple antigenic epitopes. The &quot; monoclonal &quot; indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and should not be interpreted as necessitating the production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention may be made by the hybridoma method, first described by Kohler et al., Nature 256: 495 (1975), or may be made by recombinant DNA methods (see for example, U.S. Patent No. 4,816,567). &Quot; Monoclonal antibodies &quot; can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352: 624-628 (1991) and Marks et al., J. Mol. Biol. 222: 581-597 (1991), for example. These monoclonal antibodies will usually bind with at least one Kd of about 1 μΜ, more usually at least about 300 nM, typically at least about 30 nM, preferably at least about 10 nM, more preferably at least about 3 nM or more, usually determined by ELISA.

A &quot; motif &quot;, as in a biological motif of a PSCA-related protein, refers to any pattern of amino acids that forms part of the primary sequence of a protein, which is associated with a particular function (e.g., protein- protein, protein-DNA interaction, etc.) or modification (for example, that is phosphorylated, glycosylated or amidated), or localization (eg secretory sequence, nuclear localization sequence, etc.) or a sequence that is correlated with being immunogenic , either humorously or cellularly. A motif may contiguous or be able to be aligned in certain positions that are generally correlated with a particular function or property. In the context of HLA motifs, &quot; motif &quot; refers to the residue pattern in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a HLA class I motif and from about 6 to about 25 amino acids for a HLA motif of class II, 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 primary and secondary anchor residues. Reasons that occur frequently are set out in Table V.

A &quot; pharmaceutical excipient &quot; comprises a material such as an adjuvant, a carrier, buffering and pH adjusting agents, tonicity adjusting agents, wetting agents, preservative, and the like. &quot; Pharmaceutically acceptable &quot; refers to a composition which is non-toxic, inert, and / or which is physiologically compatible with humans or other animals. The term &quot; polynucleotide &quot; 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 any type of nucleotide, and is intended to include single and double stranded forms of DNA and / or RNA . In the art, this term is commonly used interchangeably with &quot; oligonucleotide &quot;. A polynucleotide may comprise a nucleotide sequence disclosed herein wherein thymidine (T), as shown for the example in Figure 1, may also be uracil (U); this definition refers 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 &quot; polypeptide &quot; means a polymer of at least about 4, 5, 6, 7, or 8 amino acids. Throughout the specification, one-letter or three-letter standard amino acid designations are used. In the art, this term is commonly used interchangeably with &quot; peptide &quot; or &quot; protein &quot;.

A &quot; primary anchor residue &quot; of HLA is an amino acid at a specific position throughout a peptide sequence which is intended to provide a point of contact between the immunogenic peptide and the HLA molecule. One to three, usually two, primary anchor residues within a peptide of defined length, usually defines a &quot; motif &quot; for an immunogenic peptide. These residues are understood to fit in close proximity to the peptide-binding well of an HLA molecule, with its sidechains hidden in specific pockets of the binding well. In one embodiment, for example, the primary anchoring residues for a class I HLA molecule are located at the 2-position (from the amino-terminal position) and at the carboxy-terminal position of a peptide epitope of 8,9 , 10, 11, or 12 residues according to the invention. Alternatively, in another embodiment, the primary anchoring residues of a peptide binding to an HLA class II molecule are spaced apart from each other, rather than to the ends of a peptide, wherein the peptide generally has at least 9 amino acids in length. The primary anchor positions for each motif and supermotive are set out in Table IV (a). For example, analog peptides may be created by altering the presence or absence of particular residues at the primary and / or secondary anchoring positions shown in Table IV. Such analogs are used to modulate the affinity and / or population coverage of a peptide comprising a particular HLA motif or supermotif. &quot; Radioisotopes &quot; include but are not limited to the following (non-limiting exemplary uses are also set out in Table IV (I)).

By &quot; randomized &quot; or grammatical equivalents as applied herein to nucleic acids and proteins means that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. These random peptides (or nucleic acids, discussed herein) may incorporate any nucleotide or amino acid at any position. The synthetic process may be designed to generate randomized proteins or nucleic acids to allow the formation of all or most of the possible combinations along the length of the sequence, thereby forming a library of randomized candidate bioactive proteinaceous agents.

In one embodiment, a library is &quot; fully randomized &quot; no sequence preferences or constants in any position. In another embodiment, the library is a &quot; diverted random library &quot;. That is, some positions within the sequence are held constant, or are selected from a limited number of possibilities. For example, nucleotides or amino acid residues are randomized into a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically detritated residues (whether small or large), toward the creation of nucleic acid binding domains, cysteine, for crosslinking, proline for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or for purines, etc.

A recombinant DNA or RNA molecule &quot; is a DNA or RNA molecule that has been subjected to molecular manipulation in vitro.

As used herein, the term &quot; single chain Fv &quot; antibody &quot; or &quot; scFv &quot; or &quot; single strand &quot; refers to antibody fragments comprising antibody Vh and V1 domains, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the Vh and V1 domains that enables sFV to form the desired structure for antigen binding. For a review of sFv, see Pluckthun, THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

Non-limiting examples of &quot; small molecules &quot; include compounds which bind or interact with PSCA, ligands including hormones, neuropeptides, chemokines, odorants, phospholipids, and functional equivalents thereof that bind and preferably inhibit the function of the PSCA protein. Such non-limiting small molecules preferably have a molecular mass of less than about 10 kDa, more preferably below 9, about 8, about 7, about 6, about 5, or about 4 kDa. In certain embodiments, the small molecules physically associate with, or bind to, the PSCA protein; are not found in naturally occurring metabolic pathways; and / or are more soluble in aqueous than non-aqueous solutions.

As used herein, the term &quot; specific &quot; refers to selective binding of the antibody to the target antigen epitope. Antibodies can be tested for binding specificity by comparing binding to an appropriate antigen with binding to an irrelevant antigen or antigen mixture under a given set of conditions. If the antibody binds to the appropriate antigen at least 2.5, 7, and preferably 10 times more than to an irrelevant antigen or mixture of antigens then it is considered to be specific. In one embodiment, a specific antibody is one that binds only to the PSCA antigen, but does not bind to an irrelevant antigen. In another embodiment, a specific antibody is one that binds to the human PSCA antigen but does not bind to non-human PSCA antigen with 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% or greater of amino acid homology with the PSCA antigen. In another embodiment, a specific antibody is one that binds to the human PSCA antigen and binds to the murine PSCA antigen, but with a greater degree of binding to the human antigen. In another embodiment, a specific antibody is one that binds to the human PSCA antigen and binds to the PSCA primate antigen, but with a greater degree of binding to the human antigen. In another embodiment, the specific antibody binds to the human PSCA antigen and any non-human PSCA antigen, but with a higher degree of binding to the human antigen or any combination thereof. The &quot; restraint &quot; of the hybridization reactions is readily determinable by one skilled in the art, and generally consists of an empirical calculation depending on the probe length, wash temperature, and salt concentration. In general, longer probes require higher temperatures for hybridization, while shorter probes require lower temperatures. Hybridization generally depends on the ability of the denatured nucleic acid sequences to rehybridize when complementary strands are present in an environment below their melting temperature. The higher the degree of homology desired between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, it follows that higher relative temperatures would tend to render the reaction conditions more restrictive, while lower temperatures less restrictive. For additional details and explanation of restrictiveness of hybridization reactions, see Ausubel et al. , Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995). &quot; Restriction conditions &quot; or &quot; high stringency conditions &quot; as defined herein are identified by, but not limited to, those which: (1) employ a low ionic strength and high wash temperature, for example 0.015 M sodium chloride / citrate 0.0015 M sodium / 0.1% sodium dodecyl sulfate at 50 ° C; (2) employ a denaturing agent during hybridization, 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 mM sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate , 5x Denhardt's solution, sonicated salmon sperm DNA (50æg / ml), 0.1% SDS, and 10% dextran sulfate at 42 ° C, with washes at 42 ° C in 0.2% SSC x (sodium chloride / sodium citrate) and 50% formamide at 55 ° C, followed by a high stringency wash consisting of SSC x 0.1 containing EDTA at 55 ° C. &quot; Moderately stringent conditions &quot; are described, 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 a wash solution and hybridization conditions (eg temperature, ionic strength and% SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 65øC in a solution comprising: 1% bovine serum albumin, 0.5 M sodium phosphate pH 7.5, 1.25 mM EDTA, and SDS a 7%, 5x SSC (150 mM NaCl, 15 mM trisodium citrate), followed by washing the filters in 2 x SSC / 1% SDS at about 50øC and 0.2 x SSC / 0.1 SDS % at 50 ° C. The skilled person will recognize how to adjust the temperature, ionic strength, etc., as needed to accommodate factors such as probe length and the like.

A supermotive of &quot; HLA &quot; is a peptide binding specificity shared by the HLA molecules encoded by two or more HLA alleles. The general phenotypic frequencies of HLA supertypes in different ethnic populations are set forth in Table IV (f). The non-limiting constituents of various supertypes are as follows: A2: A * 021, A * 0202, A * 0203, A * 0204, A * 0205, A * 0206, A * 6802, A * 6901, A * 0207 A3, All, A31, A * 3301, A * 6801, A * 0301, A * 1101, A * 3101 B7: B7, B * 3501-03, B * 51, B * 5301, B * 5401 , B * 5501, B * 5502, B * 5601, B * 6701, B * 7801, B * 0702, B * 5101, B * 5602 B44: B * 371, B * 4402, B * 4403, B * 60 (B * 4001), B61 (B * 4006)

A: A * 24, A * 30, A * 2403, A * 2404, A * 3002, A * 3003 B27: B * 1401-02, B * 1503, B * 1509, B * 1510, B * 1518, B * 3801- 02, B * 3903, B * 3903, B * B * 7301, B * 2701-8 B58: B * 1516, B * 1517, B * 5701, B * 5702, B58 B62: B * 4601, B52, B * 1501 (B62), B * 1502 (B75), B * 1513 (B77) Calculated population coverage achieved by different combinations of HLA supertypes is set forth in Table IV (g).

As used herein &quot; to treat &quot; or &quot; therapeutic &quot; and grammatically related terms refer to any improvement in any consequence of disease, such as prolonged survival, lower morbidity, and / or a decrease in side effects that are by-products of an alternative therapeutic modality; as is readily appreciated in the art, complete eradication of the disease is a preferred outcome although it is not a requirement for the treatment act.

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

As used herein, a &quot; vaccine &quot; of cellular or HLA immune response 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 of a polyepitopic peptide; or nucleic acids encoding such individual peptides or polypeptides, for example, a minigene encoding a polyepitopic peptide. &Quot; one or more peptides &quot; may include any complete unit integer from 1-150 or more, for example 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, 31,32,33,34,34,36,37,38,39,40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 135, 140, 145, or 150 or more peptides of the invention. The peptides or polypeptides may optionally be modified, such as by lipidation, addition of targeting sequences or the like. Class I HLA peptides of the invention may be admixed with, or ligated to, class II HLA peptides to facilitate the activation of both cytotoxic T lymphocytes and helper T lymphocytes. HLA vaccines may also comprise antigen presenting cells pulsed by peptides, for example, dendritic cells. The term &quot; variant &quot; refers to a molecule which exhibits a variation from a described type or standard, such as a protein having one or more different amino acid residues in the corresponding position (s) of a specifically described protein ( for example, the PSCA protein shown in Figure 1). An analog is an example of a variant protein. Splicing isoforms and single nucleotide polymorphisms (SNPs) are further examples of variants.

&Quot; PSCA-related proteins &quot; as disclosed herein include those specifically identified herein, as well as allelic variants, conservative substitution variants, analogs and homologs which may be isolated / generated and characterized without undue experimentation following the methods outlined herein or readily available in the art. Fusion proteins that combine portions of different PSCA proteins or fragments thereof, as well as fusion proteins of a PSCA protein and a heterologous polypeptide are also included. Such PSCA proteins are collectively referred to as the PSCA-related proteins, the proteins of the invention, or PSCA. The term &quot; PSCA-related protein &quot; refers to a polypeptide fragment or to a PSCA protein sequence of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or more than 25 amino acids; or at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 330, 335, 339 or more amino acids.

II.) PSCA polynucleotides Disclosed herein are polynucleotides corresponding or complementary to all or part of a PSCA gene, mRNA, and / or coding sequence, preferably in isolated form, including polynucleotides encoding a PSCA-related protein and fragments of DNA, RNA, RNA / DNA hybrid, and related molecules, polynucleotides and oligonucleotides complementary to a PSCA mRNA gene or sequence or a portion thereof, and polynucleotides and oligonucleotides that hybridize to a PSCA, mRNA, or with a polynucleotide encoding PSCA (collectively, &quot; PSCA polynucleotides &quot;). In all instances when referred to in this section, T can also be U in Figure 1.

PSCA polynucleotides include: a PSCA polynucleotide having the sequence shown in Figure 1, the PSCA nucleotide sequence as shown in Figure 1 wherein T is U; at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in Figure 1; or at least 10 contiguous nucleotides of a polynucleotide having the sequence as shown in Figure 1 where T is U.

Polynucleotides encoding relatively long portions of a PSCA protein are also within the scope of the invention. For example, polynucleotides that encode from amino acid 1 (or 20 or 30 or 40, etc.) to about amino acid 20, (or 30, or 40 or 50, etc.) of the PSCA protein &quot; or variant &quot; shown in Figure 1 or Figure 3 may be generated by a variety of techniques well known in the art. These polynucleotide fragments may include any portion of the PSCA sequence as shown in Figure 1. II.A.) PSCA II.A.1 Polynucleotide Uses. Monitoring of Genetic Abnormalities

The polynucleotides of the preceding paragraphs have a number of different specific uses. The human PSCA gene is mapped to the chromosomal location established in the Example titled &quot; Chromosomal Mapping of PSCA &quot;. For example, since the PSCA gene is mapped to this chromosome, the polynucleotides encoding distinct regions of the PSCA proteins are used to characterize cytogenetic abnormalities of this chromosomal site, 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, for example, Krajinovic et al., Mutat Res. 382 (3-4): 81-83 1998), Johansson et al., Blood 86 (10): 3905-3914 (1995) and Finger et al., PNAS 85 (23): 9158-9162 (1988)). Thus, polynucleotides encoding specific regions of the PSCA proteins provide new tools that can be used to delineate, more accurately than was previously possible, cytogenetic abnormalities in the chromosomal region encoding PSCA 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 to identify more subtle and less common chromosomal abnormalities (see for example Evans et al., Am. J. Obstet., Gynecol 171 (4) : 1055-1057 (1994)).

In addition, since PSCA has been shown to be highly expressed in prostate cancer and other cancers, PSCA polynucleotides are used in methods that assess the status of PSCA gene products in normal versus cancerous tissues. Typically, polynucleotides encoding specific regions of the PSCA proteins are used to assess the presence of disorders (such as deletions, insertions, point mutations, or changes resulting in a loss of an antigen, etc.) in specific regions of the PSCA gene , such regions containing one or more motifs. Exemplary assays include RT-PCR assays as well as single chain conformation polymorphism (SSCP) analysis (see, for example, Marrogi et al., J. Cutan, Pathol 26 (8): 369-378 (1999 ), both of which use polynucleotides that encode specific regions of a protein to examine these regions within the protein.

Also 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, are derived from natural or synthesized sources, and include molecules capable of inhibiting expression RNA or PSCA proteins. For example, antisense molecules may be RNAs or other molecules, including peptide nucleic acids (PNAs) or non-nucleic acid molecules such as phosphorothioate derivatives that specifically bind to DNA or RNA in a base pair-dependent manner. One skilled in the art can readily obtain these classes of nucleic acid molecules using the PSCA polynucleotides and polynucleotide sequences disclosed herein. Antisense technology involves the administration of exogenous oligonucleotides that bind to a target polynucleotide located within the cells. The term &quot; antisense &quot; refers to the fact that such oligonucleotides are complementary to their intracellular targets, for example, PSCA. See, for example, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRC Press, 1989; and Synthesis 1: 1-5 (1988). The PSCA antisense oligonucleotides of the present invention include derivatives such as S-oligonucleotides (phosphorothioate derivatives or S-oligos, see Jack Cohen, supra), which exhibit improved cancer cell growth inhibitory action. S-oligos (nucleoside phosphorothioates) are isoelectronic analogs of an oligonucleotide (O-oligo) in which a non-bridging oxygen atom of the phosphate group is replaced by a sulfur atom. S-oligos can be prepared by treating the corresponding S-oligos with 3 H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transfer reagent. See, for example, 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 PSCA antisense oligonucleotides include morpholino antisense oligonucleotides known in the art (see, for example, Partridge et al., 1996,

Antisense &amp; Nucleic Acid Drug Development 6: 169-175).

The PSCA antisense oligonucleotides disclosed herein may typically be RNA or DNA which is complementary to and which hybridizes stably with the first 100 codons to 5 'or last 100 3' codons of a PSCA or mRNA genomic sequence corresponding. Absolute complementarity is not necessary, although high degrees of complementarity are preferred. The use of a complementary oligonucleotide with this region allows selective hybridization with PSCA mRNA and not with mRNA specifying other protein kinase regulatory subunits. The PSCA antisense oligonucleotides disclosed herein may be 15 to 30 mer fragments of the antisense DNA molecule having a sequence that hybridizes to PSCA mRNA. Optionally, the PSCA antisense oligonucleotide is a 30 mer oligonucleotide which is complementary to a region in the first 10 codons at 5 'or last 10 codons at 3' of PSCA. Alternatively, the antisense molecules are modified to employ ribozymes in inhibiting PSCA expression, see, for example, L.A. Couture &amp; D. T. Stinchcomb; Trends Genet 12: 510-515 (1996). II.A.3. Initiators and Initiator Pairs

Other nucleotides disclosed herein include primers and primer pairs, which allow the specific amplification of polynucleotides of the invention or any specific parts thereof, and probes that selectively or specifically hybridize to nucleic acid molecules of the invention or to any part thereof . The probes may be labeled with a detectable label, such as, for example, a radioisotope, fluorescent compounds, bioluminescent compound, a chemiluminescent compound, metal chelator or enzyme. Such probes and primers are used to detect the presence of a PSCA polynucleotide in a sample and as a means to detect a cell expressing a PSCA protein.

Examples of such probes include polypeptides comprising all or part of the human PSCA cDNA sequence shown in Figure 1. Examples of primer pairs capable of specifically amplifying PSCA mRNAs are also described in the Examples. As will be understood by the skilled person, a large number of different primers and probes may be prepared based on the sequences provided herein and used effectively to amplify and / or detect a PSCA mRNA.

The PSCA polynucleotides disclosed herein are useful for a variety of purposes, including but not limited to their use as probes and primers for the amplification and detection of the PSCA 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 PSCA polypeptides; as tools for modulating or inhibiting the expression of the PSCA gene (s) and / or translation of the PSCA transcript (s); and as therapeutic agents. Disclosed herein is the use of any probe as described herein to identify and isolate a PSCA or PSCA-related nucleic acid sequence from a naturally occurring source such as humans and other mammals as well as the sequence nucleic acid alone, which could comprise all or most of the sequences found in the probe used.

II.A.4. Isolation of Nucleic Acid Molecules Encoding PSCA

The PSCA cDNA sequences described herein enable the isolation of other polynucleotides encoding the PSCA gene product (s). as well as the isolation of polynucleotides encoding homologs of PSCA gene products, alternative splicing isoforms, allelic variants, and mutant forms of a PSCA gene product as well as polynucleotides encoding PSCA-related protein analogues. Various molecular cloning methods that may be employed to isolate full length cDNAs encoding a PSCA gene are well known (see, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd 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 may conveniently be employed using commercially available cloning systems (e.g., Lambda ZAP Express, Stratagene). Phage clones containing DNAs from the PSCA gene may be identified by probing with a labeled PSCA cDNA or fragment thereof. For example, in one embodiment, a PSCA cDNA (e.g., Figure 1) or a portion thereof may be synthesized and used as a probe to recover full length and overlapping cDNAs corresponding to a PSCA gene. A PSCA gene on its own can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs), and the like, with probes or PSCA DNA primers. II.A.5. Recombinant Nucleic Acid Molecules and Vector-Host Systems There are disclosed herein DNA or RNA molecules containing a PSCA polynucleotide, fragment, analogue or homolog thereof, including but not limited to phage, 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).

Also disclosed herein is a host-vector system comprising a recombinant DNA molecule containing a PSCA polynucleotide, fragment, analogue 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 (for example, a cell susceptible to baculovirus infection such as a Sf9 or HighFive cell ). Examples of suitable mammalian cells include several prostate cancer cell lines such as DU145 and TsuPrl, other transfection or transduction prostate cancer cell lines, primary cells (PrEC), as well as a number of mammalian cells commonly used for the expression of recombinant proteins (e.g., COS, CHO, 293, 293T cells). More particularly, a polynucleotide comprising the PSCA coding sequence or a fragment, analog or homolog thereof may be used to generate PSCA proteins or fragments thereof using any number of host-vector systems routinely used and widely known in the art.

A wide variety of host-vector systems suitable for the expression of PSCA proteins or fragments thereof are available, see, for example, Sambrook et al., 1989, supra; Current Protocols in Molecular Biology, 1995, supra). Preferred vectors for expression in mammals include, but are not limited to, pcDNA 3.1 myc-His-tag (Invitrogen) and the retroviral vector pSROtkneo (Muller et al., 1991, MCB 11: 1785). Using these expression vectors, PSCA can be used in several prostate and non-prostate cancer cell lines, including for example 293, 293T, rat-1, NIH 3T3 and TsuPrl. Host-vector systems are useful for the production of a PSCA protein or fragment thereof. Such host-vector systems may be employed to study the functional properties of PSCA or PSCA mutations or analogs.

Recombinant human PSCA protein or an analog or homologue or fragment thereof can be produced by mammalian cells transfected with a construct encoding a PSCA-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding PSCA or a fragment, analog or homolog thereof thereof, a PSCA-related protein is expressed in the 293T cells, and the recombinant PSCA protein is isolated using standard purification methods (e.g., affinity purification using anti-PSCA antibodies). Alternatively, a PSCA coding sequence is subcloned into the retroviral vector pSRaMSVtkneo and used to infect several mammalian cell lines, such as NIH 3T3, TsuPrl, 293 and rat-1 in order to establish cell lines expressing PSCA. Various other expression systems well known in the art may also be employed. Expression constructs encoding a leader peptide bound in frame with a PSCA coding sequence may be used for the generation of a secreted form of recombinant PSCA protein.

As discussed herein, redundancy in the genetic code allows variation in the PSCA gene sequences. In particular, it is known in the art that specific host species usually have specific codon preferences, and thus the disclosed sequence may be adapted as preferred for a given host. For example, preferred analog codon sequences typically have rare codons (i.e., codons having a frequency of use of less than about 20% in known host sequences) substituted with higher frequency codons. Codon preferences for a specific species are calculated, for example, by using codon usage tables available on the INTERNET such as the URL dna.affrc.go.jp/~nakamura/codon.html.

Further sequence modifications are known to potentiate protein expression in a cellular host. These include the elimination of sequences encoding spurious polyadenylation signals, exon / intron splice site signals, transposon-like repeats, and / or such such well-characterized sequences that are deleterious to gene expression. The GC content of the sequence is adjusted to mean levels 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 secondary hairpin mRNA structures. Other useful modifications include the addition of a translation initiation consensus sequence at the beginning of an open reading frame as described in Kozak, Mol. Cell Biol., 9: 5073-5080 (1989). It is understood by those skilled in the art that the general rule that eukaryotic ribosomes initiate translation exclusively into the 5 'proximal AUG codon is abrogated only under rare conditions (see, for example, Kozak PNAS 92 (7): 2662-2666, 1995 ) and Kozak NAR 15 (20): 8125-8148 (1987)).

III.) PSCA-related proteins

Also disclosed herein are proteins related to PSCA. Specific examples of PSCA proteins comprise a polypeptide having all or part of the amino acid sequence of the human PSCA as shown in Figure 1, preferably Figure 1A. Alternatively, examples of PSCA proteins comprise variant, homologous or analog polypeptides having changes in the amino acid sequence of PSCA shown in Figure 1.

A PSCA polypeptide includes: a PSCA polypeptide having a sequence shown in Figure 1, a peptide sequence of a PSCA as shown in Figure 1 wherein T is U; at least 10 contiguous nucleotides of a polypeptide having the sequence as shown in Figure 1; or at least 10 contiguous peptides of a polypeptide having the sequence as shown in Figure 1 where T is U.

Amino acid abbreviations are provided in Table II. Conservative amino acid substitutions can often be performed on a protein by altering the conformation or function of the protein. The proteins may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, conservative substitutions.

The disclosures herein include a wide variety of variants or analogs of PSCA proteins accepted in the art such as polypeptides having amino acid insertions, deletions and substitutions. PSCA variants can be made using methods known in the art such as site-directed mutagenesis, alanine screening and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl Acids Res., 13: 4331 (1986), Zoller et al., Nucl Acids Res., 10: 6487 (1987)), cassette mutagenesis (Wells et al. , Gene 34: 315 (1985)), restriction selection mutagenesis (Wells et al., Philos., Trans. R. Soc. London SerA, 317: 415 (1986)) or other known techniques can be performed on the cloned DNA to produce the DNA of the PSCA variants. Amino acid screening assays may 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 screening amino acids are relatively small and neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred screening amino acid among this group because it eliminates the side chain in addition to the carbon-beta and is less likely to alter the conformation of the major strand of the variant. Alanine is also typically preferred because it is the most common amino acid. In addition, it is often found in both the occult and exposed positions (Creighton, The Proteins, (W.H. Freeman & Co., N.I.); Chothia, J. Mol. Biol., 150: 1 (1976)). If alanine substitution does not produce sufficient amounts of variant, an isosteric amino acid may be used.

As defined herein, variants, analogs or homologs of PSCA have the distinctive attribute of having at least one epitope that &quot; cross-reacts &quot; with a PSCA protein having an amino acid sequence of Figure 1. As used in this phrase, &quot; cross-reacts &quot; means that the antibody or T cell that specifically binds to a PSCA variant also specifically binds to a PSCA protein having the amino acid sequence set forth in Figure 1. A polypeptide ceases to be a variant of a protein shown in Figure 1, when it no longer contains an epitope capable of being recognized by an antibody or T cell that specifically binds to the starting PSCA protein. Those skilled in the art understand that antibodies that recognize proteins bind to variable size epitopes, and that a cluster of the order of about four or five contiguous or not, is a typical number of amino acids in a minimal epitope. See, for example, Nair et al. , J. Immunol 2000, 165 (12): 6949-6955; Hebbes et al., Mol Immunol (1989) 26 (9): 865-7;

Schwartz et al., J Immunol (1985) 135 (4): 2598-608.

Other classes of PSCA-related protein variants share 70%, 75%, 80%, 85% or 90% or more of similarity to an amino acid sequence of Figure 1, or a fragment thereof. Another specific class of PSCA protein variants or analogs comprises one or more of the biological motifs of PSCA described herein or presently known in the art. Thus, analogs of PSCA fragments (nucleic acids or amino acids) that have functional (e.g., immunogenic) properties altered relative to the starting fragment are encompassed by the disclosures herein. It should be appreciated that current motifs or those that become part of the art should be applied to the nucleic acid or amino acid sequences of Figure 1.

As discussed herein, the polypeptides may be smaller than the complete amino acid sequence of the PSCA protein shown in Figure 1. For example, the peptides / proteins may have any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of a PSCA protein shown in Figure 1.

PSCA-related proteins are generated using standard peptide synthesis technology or using standard chemical cleavage methods well known in the art. Alternatively, recombinant methods may be used to generate nucleic acid molecules encoding a PSCA-related protein. In one example, nucleic acid molecules provide a means for generating defined fragments of a PSCA protein (or variants, homologues or analogues thereof). III.A.) Forms of Protein Realization loading Reasons

Further illustrative disclosures herein include PSCA polypeptides comprising amino acid residues of one or more of the biological motifs contained within a PSCA polypeptide sequence set forth in Figure 1. Various motifs are known in the art, and a protein can be evaluated for the presence of such motifs through a number of Internet sites made available to the public (see, for example, the URL addresses: pfam.wustl.edu/; searchlauncher. bem.tme.edu/seq-search/struc-predict. epimatrix ™ and epimer ™, Brown University, brown .edu / Research / TB- HIV_Lab / epimatrix / epimatrix.html, and BIMAS, bimas.dcrt.nih.gov/.).

Subsequences that carry motifs of all variant PSCA proteins are established and identified in Tables V-XVIII and XXII-LI. Table IV (h) establishes several motifs that frequently occur based on pfam research (see URL address pfam.wustl.edu/). The columns in Table IV (h) list (1) abbreviation of the motif name, (2) percentage of identity found between different members of the motif family, (3) motif name or description and (4) most common function; the location information that is included in the reason is relevant to the location.

Polypeptides comprising one or more of the PSCA motifs discussed above are useful for elucidating the specific characteristics of a malignant phenotype, taking into account the observation that the PSCA motifs discussed above are associated with growth dysregulation and because the PSCA is about - expressed in certain cancers (see, for example, 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, for example, Chen et al., Lab Invest., 78 (2): 165-174 (1998), Gaiddon et al., Endocrinology 136 (10): 4331-4338 (1995), Hall et al., Nucleic Acids Research 24 (6): 1119-1126 (1996), Peterziel et al., Oncogene 18 (46): 6322-6329 (1999) and O'Brian, Oncol. Rep. 5 (2): 305-309 (1998)). In addition, both glycosylation and myristoylation are protein modifications also associated with cancer and cancer progression (see, for example, Dennis et al., Biochem Biophis, Acta 1473 (1): 21-34 (1999); Raju et al., Exp. Cell Res. 235 (1): 145-154 (1997)). Amidation is another protein modification also associated with cancer and cancer progression (see, for example, Treston et al., J. Natl. Cancer Inst. Monogr. (13): 169-175 (1992)).

In another disclosure, the proteins may comprise one or more of the immunoreactive epitopes identified according to methods accepted in the art, such as the peptides set forth in Tables V-XVIII and XXII-LI. CTL epitopes can be identified using specific algorithms to identify peptides within a PSCA protein that are capable of optimally binding to specified HLA alleles (e.g., Table IV, Epimatrix ™ and Epimer ™, Brown University, URL brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS, URL bimas.dcrt.nih.gov/.) In addition, methods for identifying peptides that have a sufficient binding affinity for HLA molecules and which are correlated with immunogenic epitopes, are well known in the art, and are carried out without undue experimentation. Additionally, methods 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 analogues of such epitopes in order to modulate immunogenicity. For example, it starts with an epitope that carries a CTL or HTL motif (see, for example, the HLA class I and HLA class II motifs / supermotifs of Table IV). The epitope is converted to analog by substituting one amino acid at one of the specified positions, and substituting it with another amino acid specified for that position. For example, based on residues defined in Table IV, a deleterious residue can be removed by substitution in favor of any other residue, such as a preferred residue; replacing a less preferred residue with a preferred residue; or substitute a preferred residue which occurs originally with another preferred residue. Substitutions may occur at primary anchoring positions or at other positions in a peptide; see, for example, Table IV.

A variety of references reflect the art concerning the identification and generation of epitopes in a protein of interest as well as analogues thereof, see, for example, WO 97/33602 to Chesnut et al .; Sette, Immunogenetics 1999 50 (3-4): 201-212; Sette et al., J. Immunol. 2001 166 (2): 1389-1397; Sidney et al. , Hum. Immunol. 1997 58 (1): 12-20; Kondo et al. , Immunogenetics 1997 45 (4): 249-258; Sidney et al., J. Immunol. 1996 157 (8): 3480-90; and Falk et al. , Nature 351: 290-6 (1991); Hunt et al. , Science 255: 1261-3 (1992); Parker et al. , J. Immunol. 149: 3580-7 (1992); Parker et al. , J. Immunol. 152: 163-75 (1994)), Kast et al., 1994 152 (8): 3904-12; Borras-Cuesta et al., Hum. Immunol. 2000 61 (3): 266-278; Alexander et al., J. Immunol. 2000 164 (3); 164 (3): 1625-1633; Alexander et al., PMID: 7895164, UI: 95202582; O'Sullivan et al., J. Immunol. 1991 147 (8): 2663-2669; Alexander et al., Immunity 1994 1 (9): 751-761 and Alexander et al., Immunol. Res. 1998 18 (2): 79-92.

Related disclosures include polypeptides which comprise combinations of different motifs set forth in the Table (s) IV (a), IV (b), IV (c), IV (d), and IV (h), and / or a or more of the predicted CTL epitopes of Tables V-XVIII and XXII-LI, and / or one or more of the predicted HTL epitopes of Tables XLVIII-LI, and / or one or more known T cell binding motifs in the art. Some polypeptides do not contain insertions, deletions or substitutions either within the motifs or within the intervening sequences of the polypeptides. In addition, polypeptides that include a number of N-terminal and / or C-terminal amino acid residues on either side of these motifs may be desirable (for example, to include a larger portion of the polypeptide architecture in which the subject is located). Typically, the number of N-terminal and / or C-terminal amino acid residues on each side of a motif is from 1 to about 100 amino acid residues, preferably 5 to about 50 amino acid residues.

PSCA-related proteins are carried out in many forms, preferably in isolated form. A PSCA protein molecule will be substantially free of other proteins or molecules that impair PSCA binding to antibodies, T cells or other ligands. The nature and degree of isolation and purification will depend upon the intended use. PSCA-related proteins may include purified PSCA-related proteins and functional and soluble PSCA-related proteins. In one disclosure, a functional and soluble PSCA protein or fragment thereof retains the ability to be bound by an antibody, T cell or other ligand.

The disclosures of the present document also provide PSCA proteins which comprise biologically active fragments of a PSCA amino acid sequence shown in Figure 1. Such proteins exhibit properties of the starting PSCA protein, such as the ability to elicit the generation of antibodies that are bind specifically to an epitope associated with the starting PSCA protein; to be bound by such antibodies; eliciting the activation of HTL and CTL; and / or, if recognized by HTL or CTL which also bind specifically to the starting protein.

PSCA-related polypeptides containing particularly interesting structures can be predicted and / or identified using various analytical techniques well known in the art, including, for example, the methods of analysis of Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus -Schultz or Jameson-Wolf, or based on immunogenicity. Fragments containing such structures are particularly useful in the generation of anti-PSCA antibodies or subunit-specific T cells or in the identification of factors that bind to PSCA. For example, hydrophilicity profiles can be generated, and immunogenic peptide fragments can be identified using the method of Hopp, T.P. and Woods, K.R., 1981, Proc. Natl. Acad. Know. USA 78: 3824-3828. Hydropatibility profiles can be generated, and immunogenic peptide fragments can be identified using the method of Kyte, J. and Doolittle, R.F., 1982, J. Mol. Biol. 157: 105-132. Percentage Profiles (%) of Accessible Residues can be generated, and immunogenic peptide fragments can be identified using the method of Janin J., 1979, Nature 277: 491-492. Medium Flexibility profiles can be generated, and immunogenic peptide fragments can be identified using the method of Bhaskaran R., Ponnuswamy P.K., 1988, Int. J. Pept. Protein Res. 32: 242-255. Binding profiles between beta sheets can be generated, and immunogenic peptide fragments can be identified using the method of Deleage, G., Roux B., 1987, Protein Engineering 1: 289-294.

CTL epitopes can be identified using specific algorithms to identify peptides within a PSCA protein that are capable of optimally binding to specified HLA alleles (e.g., using the SYFPEITHI website at the World Wide Web URL syfpeithi.bmi Epimatrix ™ and Epimer ™, Brown University, URL (brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html), and BIMAS, URL bimas.dcrt.nih.gov/). In this regard, epitopes of peptides from PSCA which are presented in the context of human class I MHC molecules, for example, HLA-A1, A2, A3, All, A24, B7 and B35 were predicted (see, for example, Tables V-XVIII, XXII-LI). Specifically, the complete amino acid sequence of the PSCA protein and relevant portions of other variants, i.e. for Class I HLA predictions, flanking residues on either side of a point mutation or exon junction, and for Class HLA predictions II 14 flanking residues on either side of a point mutation or junction of exons corresponding to that variant, where introduced into the Peptide Motif Search HLA algorithm found on the Bioinformatics and Molecular Analysis Section (BIMAS) website listed above; in addition to the SYFPEITHI website, at the syfpeithi.bmi-heidelberg.com/ URL. The HLA peptide motif search algorithm was developed by Dr. Ken Parker on the basis of binding specific peptide sequences in the well of Class I HLA molecules, in particular HLA-A2 (see, for example, 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 the localization and classification 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 Class I HLA molecules. Many HLA-binding peptides of Class I have 8, 9, 10 or 11 merits. 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, for example, Parker et al., J. Immunol., 149: 3580-7 (1992)). Selected results of precose binding peptides are shown in Tables V-XVIII and XXII-LI herein. In Tables V-XVIII and XXII-XLVIII, selected candidates of 9 mer and 10 mer for each family member are shown in conjunction with their location, the amino acid sequence of each specific peptide, and an estimated binding score. In Tables XLVIII-LI, selected candidates of 15 mer for each family member are shown in conjunction 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 peptide-containing complexes at 37 ° C at pH 6.5. Peptides with the highest binding score are predicted to be those which bind more strongly to Class I HLA on the cell surface for the longest period of time and thus represent the best immunogenic targets for T cell recognition. effective binding of peptides to an HLA allele can be assessed by stabilizing HLA expression in the defective T2 cell line in antigen processing (see, for example, Xue et al., Prostate 30: 73-8 (1997) and Peshwa et al., Prostate 36: 129-38 (1998)). The immunogenicity of specific peptides can be assessed in vitro by the stimulation of CD8 + cytotoxic T lymphocytes (CTL) in the presence of antigen presenting cells such as dendritic cells.

It should be appreciated that any epitope provided by the BIMAS website, Epimer ™ and Epimatrix ™ sites, or specified by the HLA class I or class II motifs available in the art or which became a part of the art such as those set forth in Table IV (or certain using the World Wide Web site URL syfpeithi.bmi-heidelberg.com/, or BIMAS, bimas.dcrt.nih.gov/) should be &quot; applied &quot; to a PSCA protein according to the present invention. As used in this context &quot; applied &quot; means that a PSCA protein is evaluated, for example, visually or by computer-based pattern searching methods, as appreciated by those skilled in the relevant art. Each subsequence of a PSCA protein of 8, 9, 10, or 11 amino acid residues harboring a Class I HLA motif, or a subsequence of 9 or more amino acid residues harboring a Class II HLA motif, within the scope of the disclosures in this document.

III.B.) Expression of PSCA-Related Proteins

In one of the following examples, the PSCA may conveniently be expressed in cells (such as 293T cells) transfected with a commercially available expression vector such as a CMV-derived expression vector encoding PSCA with a 6XHis and MYC label at the C-terminus (pcDNA3.1 / mycHIS, Invitrogen or Tag5, GenHunter Corporation, Nashville TN). The Tag5 vector provides an IgGK secretion signal which can be used to facilitate the production of a secreted PSCA protein in transfected cells. PSCA labeled HIS in the culture medium can be purified, for example, using a nickel column using standard techniques. III.C.) Protein Modifications Related to PSCA

Modifications of PSCA-related proteins such as covalent modifications are disclosed herein. One type of covalent modification includes reacting targeted amino acid residues of a PSCA polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or with the N or C terminal residues of a PSCA protein. Another type of covalent modification of a PSCA polypeptide comprises altering the native glycosylation pattern of a protein of the invention. Another type of covalent modification of PSCA comprises connecting a PSCA polypeptide with one of a variety of non-protein polymers, for example, polyethylene glycol (PEG), propylene glycol, or polyoxyalkylenes, as 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.

PSCA-related proteins may also be modified to form a chimeric molecule comprising PSCA fused to another polypeptide or heterologous amino acid sequence. Such a chimeric molecule may be synthesized chemically or recombinantly. A chimeric molecule may have a protein as disclosed herein fused to another tumor associated antigen or fragment thereof. Alternatively, a protein according to the disclosures herein may comprise a fusion of fragments of a PSCA sequence (amino acids or nucleic acids) so that a molecule is created which is not, through its length, directly homologous to the sequences amino acids or nucleic acids shown in Figure 1. Such a chimeric molecule may comprise multiples of the same subsequence of PSCA. A chimeric molecule may comprise a fusion of a PSCA-related protein with a polyhistidine epitope tag, which provides an epitope to which immobilized nickel may selectively bind, with cytokines or with growth factors. The epitope tag is generally placed at the amino or carboxyl terminus of a PSCA protein. In an alternative example, the chimeric molecule may comprise a fusion of a PSCA-related protein with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as &quot; immunoadhesin &quot;), such a fusion could be with the Fc region of an IgG molecule. Ig fusions preferably include substituting a soluble (transmembrane domain deleted or inactivated) form of a PSCA polypeptide in place of at least one variable region within an Ig molecule. The immunoglobulin fusion may include the hinge, CH2 and CH3 regions, or the hinge, CHI, CH2 and CH3 regions of an IgGI molecule. For the production of immunoglobulin fusions see, for example, U.S. Patent No. 5,428,130 issued June 27, 1995.

III.D.) Protein Uses Related to PSCA

The proteins disclosed herein have a number of different specific uses, as PSCA has been shown to be highly expressed in prostate cancer and other cancers, PSCA-related proteins are used in methods that assess the status of the PSCA gene products in normal to cancerous tissues, thus elucidating the malignant phenotype. Typically, polypeptides from specific regions of a PSCA protein are used to assess the presence of disorders (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 that target PSCA-related proteins which comprise amino acid residues from one or more of the biological motifs contained within a PSCA polypeptide sequence in order to evaluate the characteristics of this region in normal tissues against cancerous or eliciting an immune response against the epitope. Alternatively, PSCA-related proteins that contain the amino acid residues of one or more of the biological motifs in a PSCA protein are used to challenge factors that interact with that region of PSCA.

The PSCA protein fragments / subsequences are particularly useful for generating and characterizing domain-specific antibodies (for example, antibodies recognizing an extracellular or intracellular epitope of a PSCA protein) for the identification of PSCA-binding agents or cellular factors or a particular structural domain thereof, and in various diagnostic and therapeutic contexts, including, but not limited to, diagnostic assays, cancer vaccines, and methods of preparing such vaccines.

Proteins encoded by the PSCA genes, or by analogs, homologues or fragments thereof, have a variety of uses, including, but not limited to, the generation of antibodies and in methods of identifying ligands and other binding agents and cell constituents to the PSCA gene product. Antibodies raised against a PSCA protein or fragments thereof are useful in diagnostic and prognostic assays and imaging methodologies in the management of human cancers characterized by the expression of a PSCA protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods of treating patients with such cancers. PSCA-related nucleic acids or proteins are also used in the generation of HTL or CTL responses. Various immunological assays useful for the detection of PSCA proteins are used, including, but not limited to, various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunosorbent assay (ELIFA), immunocytochemical methods, and the like. Antibodies can be labeled and used as immunological imaging reagents capable of detecting cells expressing PSCA (for example, in radioclintigraphic imaging methods). PSCA proteins are also particularly useful in the generation of cancer vaccines, as further described herein.

IV.) Antibodies to PSCA

One aspect of the invention provides antibodies that bind to PSCA-related proteins. Preferred antibodies bind specifically to a PSCA-related protein and do not bind (or weakly bind) to peptides or proteins that are not PSCA-related proteins under physiological conditions. In this context, examples of physiological conditions include: 1) phosphate buffered saline; 2) Tris buffered saline containing 25 mM Tris and 150 mM NaCl; or normal saline (0.9% NaCl); 4) animal serum such as human serum; or, 5) a combination of any of 1) to 4); these reactions preferably taking place at a pH of 7.5, alternatively in a pH range of 7.0 to 8.0, or alternatively in a pH range of 6.5 to 8.5; furthermore, these reactions take place at a temperature between 4 ° C to 37 ° C. For example, antibodies that bind to PSCA may bind PSCA-related proteins such as homologues and analogs thereof.

The PSCA antibodies of the invention are particularly useful in cancer diagnostic and prognostic assays (see, for example, Table I) and imaging methodologies. Similarly, such antibodies are useful in the treatment, diagnosis, and or prognosis of prostate cancer and other cancers, insofar as PSCA is also expressed or overexpressed in these other cancers. In addition, intracellularly expressed antibodies (e.g., single chain antibodies) are therapeutically useful in the treatment of cancers in which expression of PSCA is involved, such as advanced or metastatic prostate cancers or other advanced or metastatic cancers.

Antibodies of the invention may also be used in various immunological assays useful for the detection and quantification of PSCA and mutant PSCA related proteins. Such assays may comprise one or more PSCA antibodies capable of recognition and binding to a PSCA-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 immunosorbent assays (ELIFA), and the like.

Non-antibody immunological assays also comprise T cell (inhibitory or stimulatory) immunogenicity assays as well as major histocompatibility complex (MHC) binding assays.

Additionally, methods of producing immunological images capable of detecting prostate cancer and other cancers that express PSCA are also disclosed herein, including, but not limited to, radioclintigraphic imaging methods using labeled PSCA antibodies. Such assays are clinically useful in the detection, monitoring, and prognosis of cancers that express PSCA such as prostate cancer.

Antibodies to PSCA are also used in methods for purifying a PSCA-related protein and for isolation of homologs of PSCA and related molecules. For example, a method of purifying a PSCA-related protein comprises incubating an antibody to PSCA which has been coupled to a solid matrix with a lysate or other solution containing a PSCA-related protein under conditions that allow the antibody to PSCA binds to PSCA-related protein; washing the solid matrix to remove impurities; and elution of the PSCA-related protein from the coupled antibody. Further uses of the antibodies for PSCA include generating anti-idiotypic antibodies that mimic a PSCA protein. Various methods for the preparation of antibodies are well known in the art. For example, antibodies may be prepared by immunizing a suitable mammalian host using a PSCA-related protein, peptide, or fragment, in isolated or immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press, Eds., Harlow, et al. Lane (1988); Harlow, Antibodies, Cold Spring Harbor Press, NI (1989)). In addition, PSCA fusion proteins may also be used, such as a GST PSCA fusion protein. In a particular embodiment, a GST fusion protein comprising all or most of the amino acid sequence of Figure 1 is produced, and then used as an immunogen to generate appropriate antibodies. In another embodiment, a PSCA-related protein is synthesized and used as an immunogen.

In addition, naked DNA immunization techniques known in the art are used (with or without PSCA-related protein or purified PSCA-expressing cells) to generate an immune response against the encoded immunogen (for review, see Donnelly et al., 1997 , Ann. Rev. Immunol 15: 617-648). The amino acid sequence of a PSCA protein as shown in Figure 1 can be analyzed to select specific regions of the PSCA protein to generate antibodies. For example, hydrophobicity or hydrophilicity analyzes of a PSCA amino acid sequence are used to identify hydrophilic regions in the PSCA framework. Regions of a PSCA protein having an immunogenic structure, as well as other regions and domains, can be readily identified using various other methods known in the art, such as Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus -Schultz or Jameson-Wolf. The hydrophilicity profiles can be generated using the method of Ηορρ, T.P. and Woods, K.R., 1981, Proc. Natl. Acad. Know. USA 78: 3824-3828. Hydropatibility profiles can be generated using the method of Kyte, J. and Doolittle, R.F., 1982, J. Mol. Biol. 157: 105-132. Percentage Profiles (%) of Accessible Residues can be generated using the method of Janin J., 1979, Nature 277: 491-492. The Average Flexibility profiles can be generated using the method of Bhaskaran R., Ponnuswamy P.K., 1988, Int. J. Pept. Protein Res. 32: 242-255. Binding profiles between beta sheets can be generated using the method of Deleage, G., Roux B., 1987, Protein Engineering 1: 289-294. Thus, each region identified by any of these programs or methods is within the scope of the present invention. Preferred methods for generating antibodies to PSCA are further illustrated by way of examples provided herein. Methods of 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 binding reagents such as those provided by Pierce Chemical Co., Rockford, IL, are effective. Administration of a PSCA immunogen is usually carried out by injection over a suitable period of time and with the use of a suitable adjuvant, as is understood in the art. During the immunization regimen, antibody titres may be taken to determine the suitability of the antibody formation.

PSCA monoclonal antibodies can be produced by various methods well known in the art. For example, immortalized cell lines secreting a desired monoclonal antibody are prepared using the standard Kohler and Milstein hybridoma technology or modifications that immortalize antibody-producing B cells, as is generally known. Immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is a PSCA-related protein. When the appropriate immortalized cell culture is identified, the cells can be expanded and the antibodies produced from cultures in vitro or from ascites fluid.

Antibodies or fragments of the invention may also be produced by recombinant means. Regions that specifically bind to desired regions of a PSCA protein may also be produced in the context of chimeric or grafted antibodies with complementarity determination regions (CDRs) originating from multiple species. Antibodies to human or humanized PSCAs can also be produced, and are preferred for use in therapeutic settings. Methods for humanizing murine and other non-human antibodies by substituting one or more of the CDRs of the non-human antibodies for corresponding human antibody sequences are well known (see, for example, Jones et al., 1986, Nature 32: 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. Know. USA 89: 4285 and Sims et al., 1993, J. Immunol. 151: 2296. Methods for the production of fully human monoclonal antibodies include transgenic and phage display methods (for review, see Vaughan et al., 1998, Nature Biotechnology 16: 535-539). Fully human PSCA monoclonal antibodies can be generated using cloning technologies that employ large combinatorial libraries of human Ig genes (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. -82). Completely human PSCA monoclonal antibodies can also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application WO 98/24893, Kucherlapati and Jakobovits et al., Published December 3, 1997 (see Also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7 (4): 607-614, U.S. Patent Nos. 6,162,963 issued December 19, 2000, 6,150,584 issued on November 12, 2000 and 6,114,598 issued September 5, 2000). This method avoids the in vitro manipulation required with the phage display technology and efficiently produces authentic human antibodies of high affinity. The reactivity of the antibodies to PSCA with a PSCA-related protein can be established through a number of well known means, including Western blot analysis, immunoprecipitation, ELISA, and FACS using, as appropriate, PSCA-related proteins, PSCA-expressing cells or extracts thereof. An antibody to PSCA or fragment thereof can be labeled with a label detectable or conjugated to a second molecule. Suitable detectable labels include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme. In addition, bispecific antibodies specific for two or more PSCA epitopes are generated using methods generally known in the art. Homodimeric antibodies may also be generated by crosslinking techniques known in the art (for example, Wolff et al., Cancer Res. 53: 2560-2565).

In one embodiment, the invention provides monoclonal antibodies identified as Hal-4.121, was sent (through Federal Express) to the American Type Culture Collection (ATCC), PO Box 1549, Manassas, VA 20108 on May 4, 2005 and assigned with the Access number PTA-6701. Antibodies identified as Hal-1.16, Hal-5.99, Hal-4.117, Hal-4.20, Hal-4.37 are also disclosed herein (through Federal Express) to the American Type Culture Collection (ATCC), PO Box 1549, Manassas , VA 20108 on May 4, 2005 and assigned with accession numbers PTA-6698 and PTA-6703 and PTA-6699 and PTA-6700 and PTA-6702 respectively.

V.) Cellular Immune Responses against PSCA The mechanism by which T cells recognize antigens was outlined. Effective peptide epitope vaccine compositions disclosed herein induce a therapeutic or prophylactic immune response in very broad segments of the world population. For an understanding of the value and efficacy of the compositions that induce cellular immune responses, a brief review of the technology related to immunology is provided.

A complex of an HLA molecule and a peptide antigen acts as the ligand recognized by HLA-restricted T cells (Buus, S. et al., Cell 47: 1071, 1986; Babbitt, BP et al., Nature 317: 359, 1985, Townsend, A. and Bodmer, H., Annu Rev. Immunol., 7: 601, 1989, Germain, RN, Annu Rev. Immunol 11: 403, 1993). Through the study of single amino acid substituted antigen analogues and the sequencing of endogenously linked, naturally processed peptides, critical residues corresponding to motifs required for specific binding to HLA antigen molecules have been identified and are set forth in Table IV (see, (eg, Southwood, et al., J. Immunol., 160: 3363, 1998), Rammensee et al., Immunogenetics 41: 178, 1995, Rammensee et al., SYFPEITHI, World Wide Web Access at URL (134.2.96.221/scripts.hlaserv-er.dll/home.htm), Sette, A. and Sidney, J. Curr Opin, Immunol 10: 478, 1998, Engelhard, VH Curr Curr Immunol 06 : Rigpert et al., J. Med. Chemol., 9, 9, 1994. Sette, A. and Gray, HM, Curr Opin, Immunol, 4:79, 1992, Sinigaglia, F. and Hammer, J. Curr. Biol. Cell 74: 929-937, 1993 Kondo et al., J. Immunol 155: 4307-4312, 1995 Sidney et al., J. Immunol 157: 3480-3490, 1996 Sidney et al., Human Immunol 45: 79-93, 1996. Sette, A. and Sidney, J. Immunogenetics Nov 1999; 50 (3-4): 201-12, Revision).

In addition, X-ray crystallographic analyzes of HLA-peptide complexes revealed pockets within the peptide-binding cleft / well of HLA molecules that accommodate, in an allele specific manner, residues supported by peptide ligands; these residues, in turn, determine the ability of HLA binding of the peptides in which they are present. (See, for example, Madden, DR Annu Rev. Immunol 13: 587, 1995, Smith, et al., Immunity 4: 203, 1996; Fremont et al., Immunity 8: 305, 1998; al., Structure 2: 245, 1994, Jones, EY Curr Opin, Immunol 9:75, 1997, Brown, JH et al., Nature 364: 33, 1993, Guo, HC et al., Proc. Natl. , Et al., Science 360: 367, 1992, Matsumura, M. et al., Science 257: The present invention relates to a method for the preparation of a compound of the formula (II) in the preparation of a compound of the general formulas (WO 92/99, 1992, Madden et al., Cell 70: 1035, 1992; Fremont, DH et al., Science 257: 919,1992, Saper, MA, Borker, PJ and Wiley, DC, J. Mol Biol. 219: 277, 1991.)

Accordingly, the definition of class I and class II allele-specific HLA binding motifs, class I and class II supermotives allows the identification of regions within a protein that are correlated with binding to particular antigen (s) of HLA.

Thus, through a process for identifying HLA motifs, candidates for epitope-based vaccines have been identified; such candidates may be further evaluated by HLA-peptide binding assays to determine the binding affinity and / or the time period of association of the epitope and its corresponding HLA molecule. Additional confirmatory work may be performed to select among these vaccine candidates epitopes having preferred characteristics in terms of population coverage and / or immunogenicity. A number of strategies may be used to evaluate cellular immunogenicity, including: 1) Evaluation of primary T cell cultures from normal individuals (see, for example, Wentworth, PA et al., Mol Immunol 32: 603, 1995; Celis, E. et al.

Proc. Natl. Acad. Know. USA 91: 2105, 1994; Tsai, V. et al., J. Immunol. 158: 1796, 1997; Kawashima, I. et al., Human Immunol. 59: 1, 1998). This procedure involves the stimulation of peripheral blood lymphocytes (PBLs) from normal individuals with a test peptide in the presence of antigen presenting cells in vitro over a period of several weeks. Peptide-specific T cells become activated during this time period and are detected using, for example, a lymphokine or 51 Cr release assay involving peptide-sensitized target cells. 2) Immunization of HLA transgenic mice (see, for example, Wentworth, PA et al., J. Immunol 26:97, 1996; Wentworth, PA et al., Int. Immunol., 8: 651, 1996 , Alexander, J. et al., J. Immunol., 159: 4753, 1997). For example, in such methods the peptides in incomplete Freund's adjuvant are administered subcutaneously to HLA transgenic mice. Several weeks after immunization, the splenocytes are removed and cultured in vitro in the presence of the test peptide for approximately one week. Specific peptide T cells are detected using, for example, a 51 Cr release assay involving peptide-sensitized target cells and target cells expressing the endogenously generated antigen. 3) Demonstration of memory T cell responses from immune subjects who have been vaccinated effectively and / or from chronically ill patients (see, for example, Rehermann, B. et al., J. Exp Med. , J. Immunol 159: 1047, 1995. Doolan, DL et al., Immunity 7:97, 1997, Bertoni, R. et al., J. Clin Invest 100: 503, 1997. Threlkeld SC et al., J. Immunol 159: 1648, 1997; Diepolder, HM et al., J. Virol 71: 6011, 1997). Accordingly, memory responses are detected by culturing PBL from individuals who have been exposed to the antigen due to disease and therefore have generated a &quot; naturally &quot; immune response, or from patients who have been vaccinated against the antigen . PBL from individuals are cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APC) to allow activation of &quot; memory &quot; T cells, compared to &quot; naive &quot; T cells. . At the end of the culturing period, T cell activity is detected using assays including 51Cr release involving peptide-sensitized targets, T-cell proliferation or lymphokine release.

VI.) Transgenic Animals for PSCA

Nucleic acids encoding a PSCA-related protein can also be used to generate transgenic animals or animals &quot; knock out &quot; which, in turn, are useful in the development and screening of therapeutically useful reagents. According to established techniques, the cDNA encoding PSCA can be used to clone genomic DNA encoding PSCA. The cloned genomic sequences can then be used to generate transgenic animals that contain cells expressing DNA encoding PSCA. 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 April 12, 1988 and 4,870,009 issued on September 26, 1989. Typically, particular cells would be targeted for incorporation of PSCA transgene with tissue-specific enhancers.

Transgenic animals which include a copy of a PSCA-encoding transgene can be used to examine the effect of increased expression of DNA encoding PSCA. Such animals may be used as test animals for reagents which are believed to provide protection against, for example, pathological conditions associated with their overexpression. According to the present disclosure, an animal is treated with a reagent and a reduced incidence of a pathological condition, compared to untreated animals carrying the transgene, would indicate a potential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of PSCA can be used to construct an animal &quot; knock out &quot; to PSCA having a gene encoding deficient or altered PSCA as a result of homologous recombination between the endogenous gene encoding PSCA and altered genomic DNA encoding PSCA introduced into an embryonic cell of the animal. For example, cDNA encoding PSCA can be used to clone genomic DNA encoding PSCA according to established techniques. A portion of the PSCA-encoding genomic DNA 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 5 'and 3' ends) are included in the vector (see, for example, Thomas and Capecchi, Cell, 51: 503 (1987) for a description of vectors of homologous recombination). The vector is introduced into an embryonic stem cell line (for example, by electroporation) and the cells into which the introduced DNA has homologously recombined with the endogenous DNA are selected (see, for example, Li et al., Cell, 69 : 915 (1992)). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or mouse) to form aggregation chimeras (see, for example, Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, EJ Robertson, ed. (IRL, Oxford, 1987), pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudo-pregnant female adoptive animal, and the embryo brought to term to create a &quot; knock out &quot; animal. The offspring carrying the homologously recombined DNA in their germ cells can be identified by standard techniques and used to create animals in which all the cells of the animal contain the homologously recombined DNA. Knock-out animals may be characterized, for example, as to their ability to defend themselves against certain pathological conditions or for their development of pathological conditions due to the absence of a PSCA polypeptide.

VII.) Methods for PSCA Detection

Another disclosure herein relates to methods for the detection of PSCA polynucleotides and PSCA-related proteins, as well as methods for identifying a cell expressing PSCA. The expression profile of PSCA makes it a diagnostic marker for metastatic disease. Accordingly, the PSCA gene product status provides useful information for predicting a variety of factors, including advanced disease susceptibility, rate of progression, and / or tumor aggressiveness. As discussed in detail herein, the state of PSCA gene products in patient samples can be analyzed by a variety of protocols which are well known in the art, including immunohistochemical analysis, the variety of Northern blotting techniques including hybridization in situ, RT-PCR analysis (for example in microdissected samples by laser capture), Western blot analysis and tissue matrix analysis.

More particularly, the disclosure provides assays for the detection of PSCA polynucleotides in a biological sample, such as serum, bone, prostate and other tissues, urine, semen, cell preparations, and the like. Detectable PSCA polynucleotides include, for example, a PSCA gene or fragment thereof, PSCA mRNA, PSCA mRNA, alternative splicing variants, and recombinant DNA or RNA molecules containing a PSCA polynucleotide. A number of methods for amplifying and / or detecting the presence of PSCA polynucleotides are well known in the art and can be employed in practicing this aspect of the invention.

A method for detecting a PSCA mRNA in a biological sample may comprise the production of cDNA from the sample by reverse transcription using at least one primer; amplification of the cDNA thus produced using PSCA polynucleotides as sense and antisense primers to amplify PSCA cDNA therein; and detecting the presence of the amplified PSCA cDNA. Optionally, the amplified PSCA cDNA sequence can be determined.

A method of detecting a PSCA gene in a biological sample may comprise first isolating genomic DNA from the sample; amplifying isolated genomic DNA using PSCA polynucleotides as sense and antisense primers; and detect the presence of the amplified PSCA gene. Any number of combinations of sense and antisense probes can be designed from a nucleotide sequence of PSCA (see, for example, Figure 1) and used for this purpose.

Also disclosed herein are assays for detecting the presence of a PSCA protein in a tissue or other biological sample, such as serum, semen, bone, prostate, urine, cell preparations and the like. Methods for the detection of a PSCA-related protein are also well known and include, for example, immunoprecipitation, immunohistochemical analysis, Western blot analysis, molecular binding assays, ELISA, ELIFA and the like. For example, a method for detecting the presence of a PSCA-related protein in a biological sample comprises first contacting the sample with a PSCA antibody, a PSCA fragment reactive thereof, or a recombinant protein containing a region of antigen binding of an antibody to PSCA; and then detecting the binding of the PSCA-related protein in the sample.

Methods for identifying a cell expressing PSCA are also within the scope of the disclosures. An assay to identify a cell expressing a PSCA gene may comprise detecting the presence of PSCA mRNA in the cell. Methods for detecting particular mRNAs in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled PSCA ribosounds, Northern blot and related techniques), and various amplification assays (such as RT-PCR using PSCA-specific complementary primers and other amplification type detection methods such as, for example, branched DNA, SISBA, TMA and the like).

Alternatively, an assay to identify a cell expressing a PSCA gene comprises detecting the presence of PSCA-related protein in the cell or secreted by the cell. Various methods for the detection of proteins are well known in the art and are employed for the detection of PSCA-related proteins and cells expressing PSCA-related proteins. Analysis of PSCA expression is also useful as a tool for identifying and evaluating agents that modulate expression of the PSCA gene. For example, PSCA expression is significantly upregulated in prostate cancer, and is expressed in cancers of the tissues listed in Table I. The identification of a molecule or biological agent that inhibits PSCA expression or overexpression in cancerous cells is of therapeutic value. For example, such an agent can be identified using a screen that quantifies PSCA expression by RT-PCR, nucleic acid hybridization or antibody binding. VIII.) Methods for Monitoring the Status of Genes Related to PSCA and Its Products Oncogenesis is known to be a multi-step process where cell growth becomes progressively unregulated and cells progress from a normal physiological state to precancerous and (see, for example, Alers et al., Lab Invest. 77 (5): 437-438 (1997) and Isaacs et al.

Cancer Surv. 23: 19-32 (1995)). In this context, examination of a biological sample for evidence of dysregulated cell growth (such as expression of aberrant PSCA in cancers) allows the early detection of such aberrant physiology, prior to a pathological state, such as cancer having progressed to a stage in which the therapeutic options are more limited and or the prognosis is worse. In such examinations, the PSCA status in a biological sample of interest can be compared, for example, to the PSCA status in a corresponding normal sample (e.g., a sample of that individual or, alternatively, another individual not affected by a pathology). A change in PSCA status in the biological sample (compared to the normal sample) provides evidence of unregulated cell growth. In addition to the use of a biological sample which is not affected by a pathology as a normal sample, a predetermined normative value may also be used such as a normal predetermined level of mRNA expression (see, for example, Grever et al. , J. Comp. Neurol. 1996 9 de dez; 376 (2): 306-14 and U.S. Patent No. 5,837,501) to compare the PSCA status in a sample. The term &quot; condition &quot; in this context is used in accordance with its accepted meaning in the art and refers to the condition or state of a gene and its products. Typically, those of skill in the art use a number of parameters to assess 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 localization of PSCA expressing cells), as well as the level, and biological activity of expressed gene products (such as PSCA mRNAs, polynucleotides, and polypeptides). Typically, a change in the PSCA state comprises a change in the localization of PSCA and / or PSCA expressing cells and / or an increase in PSCA mRNA and / or protein expression. The state of PSCA in a sample can be analyzed by a variety of means well known in the art, including, without limitation, immunohistochemical analysis, in situ hybridization, RT-PCR analysis on laser capture microdissected samples, Western blot analysis and analysis of tissue matrices. Typical protocols for assessing the state of a PSCA 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 state of PSCA in a biological sample is evaluated by various methods used by those skilled in the art including, but not limited to, Southern genomic analysis (for examining, for example, perturbations in a PSCA gene), Northern and / or PCR analysis of PSCA mRNA (for examining, for example, changes in polynucleotide sequences or levels of PSCA mRNA expression), and Western and / or immunohistochemical analysis (for examining, for example, changes in the sequences alterations in the localization of polypeptides in a sample, changes in the levels of protein expression and / or associations of PSCA proteins with polypeptide binding partners). Detectable PSCA polynucleotides include, for example, a PSCA gene or a fragment thereof, PSCA mRNA, alternative splicing variants, PSCA mRNA, and recombinant DNA or RNA molecules containing a PSCA polynucleotide. The PSCA expression profile makes it a diagnostic marker for local and / or metastatic disease, and provides information about the growth or oncogenic potential of a biological sample. In particular, the PSCA status provides useful information to predict susceptibility to particular stages of disease, tumor progression and / or aggressiveness. Methods and assays for the determination of PSCA status and diagnosis of PSCA-expressing cancers, such as cancers of the tissues listed in Table I, are disclosed herein. For example, since PSCA mRNA is so highly expressed in prostate cancers and others in relation to normal prostate tissue, assays that evaluate the levels of mRNA transcripts or PSCA proteins in a biological sample may be used to diagnose a disease associated with PSCA dysregulation, and may provide useful prognostic information to define the appropriate therapeutic options. The PSCA expression state provides information including the presence, location and stage of dysplastic, precancerous and cancer cells, predicting susceptibility at various stages of the disease, and / or for measuring tumor aggressiveness. In addition, the expression profile renders it useful as an imaging reagent for metastatic disease. Accordingly, various methods of molecular diagnosis and prognosis are being disclosed herein to examine the condition of PSCA in biological samples, such as those from individuals suffering from or suspected of suffering from a disorder characterized by unregulated cell growth, such as cancer.

As described above, the PSCA status in a biological sample can be examined by a number of procedures well known in the art. For example, the PSCA status 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 PSCA expressing cells (e.g., those expressing mRNA or PSCA proteins). This examination may provide evidence of dysregulated cell growth, for example, when cells expressing PSCA are found in a biological sample that does not normally contain these cells (such as a lymph node), since such changes in PSCA status in a biological sample are often associated with unregulated cell growth. Specifically, a deregulated cell growth indicator 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 cell growth is important, for example, because metastases hidden in the lymph nodes can be detected in a substantial proportion of patients with prostate cancer, and such metastases are associated with known predictors of disease progression (see, for example, Murphy et al., Prostate 42 (4): 315-317 (2000); Su et al., Semin Surg. Oncol 18 (1): 17-28 (2000) and Freeman et al. , J Urol 1995 Aug 154 (2 Pt 1): 474-8). There are disclosed herein methods for monitoring PSCA gene products by determining the state of PSCA gene products expressed by cells from an individual suspected of having a disease associated with unregulated cell growth (such as hyperplasia or cancer) and then comparing the condition thus determined with the state of PSCA gene products in a corresponding normal sample. The presence of aberrant PSCA gene products in the test sample relative to the normal sample provides an indication of the presence of unregulated cell growth between the cells of the subject. Assays useful in determining the presence of cancer in a subject are disclosed herein, comprising detecting a significant increase in PSCA mRNA or protein expression in a tissue sample or test cell relative to the expression levels in the corresponding normal cell or tissue. The presence of PSCA mRNA may, for example, be evaluated in tissues, including but not limited to those listed in Table I. The presence of significant PSCA expression in any of these tissues is useful to indicate appearance, presence and / or the severity of a cancer, since the corresponding normal tissues do not express PSCA mRNA or express it at lower levels.

In a related disclosure, the PSCA status is determined at the protein level rather than at the nucleic acid level. For example, such a method comprises determining the level of PSCA protein expressed by the cells in a test tissue sample and comparing the level thus determined with the level of PSCA expressed in a corresponding normal sample. The presence of the PSCA protein can be evaluated, for example, using immunohistochemistry methods. Antibodies against PSCA or binding partners capable of detecting PSCA protein expression are used in a variety of assay formats well known in the art for this purpose.

The state of nucleotides and amino acid sequences of PSCA can be evaluated in a biological sample in order to identify perturbations in the structure of these molecules. Such disorders may include insertions, deletions, substitutions and the like. These evaluations are useful because perturbations in nucleotide and amino acid sequences are observed in a large number of proteins associated with a deregulated growth phenotype (see, for example, Marrogi et al., 1999, J. Cutan. 8): 369-378). For example, a mutation in the PSCA sequence may be indicative of the presence or the promotion of a tumor. Therefore, such assays have diagnostic and predictive value where a mutation in PSCA indicates a potential loss of function or increase in tumor growth.

A wide variety of assays for the observation of nucleotide and amino acid sequence perturbations is well known in the art. For example, the size and structure of the nucleic acid or amino acid sequences of the PSCA gene products are observed by the Northern, Southern, Western, PCR and DNA sequencing protocols discussed herein. In addition, other methods for the observation of perturbations in nucleotide and amino acid sequences, such as single strand conformation polymorphism analysis are well known in the art (see, for example, U.S. Patent Nos. 5,382. 510 issued September 7, 1999 and 5,952,170 issued January 17, 1995).

In addition, the methylation state of a PSCA gene can be examined in a biological sample. Aberrant demethylation and / or hypermethylation of CpG islands in 5 'regulatory regions of the gene often occurs in immortalized and transformed cells, and may result in altered expression of several genes. For example, hypermethylation of the glutathione S-transferase pi-class promoter (a protein expressed in the normal prostate but not expressed in &gt; 90% of prostate carcinomas) appears to permanently silence the transcription of this gene and is the most frequent genomic change detected 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 the normal prostate, but is expressed in 25-50% of prostate cancers) is induced by deoxyazacytidine in lymphoblastoid cells, suggesting that tumor demethylation (Lethe et al., Int. J. Cancer 76 (6): 903-908 (1998)). A variety of assays to examine the methylation state of a gene is well known in the art. For example, in Southern hybridization approaches, methylation sensitive restriction enzymes that can not cleave sequences containing methylated CpG sites to assess the methylation status of CpG islands can be used. In addition, MSP (methylation specific PCR) can readily establish the methylation profile profile of all CpG sites present on a CpG island of a given gene. This procedure involves the initial modification of DNA through sodium bisulfite (which will convert all unmethylated cytosines to uracil) followed by amplification using specific primers for methylated DNA against unmethylated DNA. Protocols involving methylation interference may 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 PSCA status. Gene amplification is measured in a sample directly, for example, by conventional Southern blotting or Northern blotting to quantitate mRNA transcription (Thomas, 1980, Proc.Natl.A.A.A., 77: 5201 5205), dot blotting ( DNA analysis), or in situ hybridization using an appropriately labeled probe, based on the sequences provided herein. Alternatively, the antibodies that are employed recognize specific duplexes, including DNA duplexes, RNA duplexes, and hybrid duplexes of DNA and RNA or DNA and protein duplexes. Antibodies, in turn, are labeled and the assay is carried out where the duplex is bound to a surface so that, after surface duplex formation, the presence of antibody bound to the duplex can be detected.

Biopsy tissue or peripheral blood can be conveniently tested for the presence of cancer cells using, for example, Northern blot analysis, dot blot or RT-PCR to detect PSCA expression. The presence of PSCA mRNA amplifiable by RT-PCR provides an indication of the presence of cancer. RT-PCR assays are well known in the art. RT-PCR assays for peripheral blood tumor cells are currently being evaluated for use in the diagnosis and treatment of a number of human solid tumors. In the field of prostate cancer, these include RT-PCR assays for the detection of PSA and PSM-expressing cells (Verkaik et al., 1997, Urol Res 25: 373-384; Ghossein et al. Clin Oncol, 13: 1195-2000, Heston et al., 1995, Clin. Chem., 41: 1687-1688). An evaluation of an individual's susceptibility to developing cancer is disclosed herein. One method of predicting susceptibility to cancer may comprise the detection of PSCA mRNA or PSCA protein in a tissue sample, its presence indicating cancer susceptibility, wherein the degree of expression of PSCA mRNA correlates with the degree of susceptibility. In one method, the presence of PSCA in prostatic tissue or other is examined with the presence of PSCA in the sample providing an indication of susceptibility to prostate cancer (or the appearance or existence of a prostate tumor). Likewise, the integrity of the PSCA nucleotide and amino acid sequences can be assessed 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 the PSCA gene products in the sample is an indication of susceptibility to cancer (or the appearance or existence of a tumor).

Also disclosed herein are methods for measuring tumor aggressiveness. A method for measuring the aggressiveness of a tumor may comprise determining the level of PSCA mRNA or PSCA protein expressed by tumor cells by comparing the level thus determined with the level of PSCA mRNA or PSCA protein expressed in a corresponding normal tissue taken from the same subject or from a normal tissue reference sample, wherein the degree of PSCA mRNA or PSCA protein expression in the tumor sample relative to the normal sample indicates the degree of aggressiveness. In one method, the aggressiveness of a tumor is assessed by determining the extent to which PSCA is expressed in tumor cells, with higher expression levels indicating more aggressive tumors. Another method is to evaluate the integrity of nucleotide and amino acid sequences of PSCA 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 disorders indicates more aggressive tumors. Further described herein are methods for monitoring the progression of a malignant neoplasm in an individual over time. Methods for observing the progression of a malignancy in an individual over time may comprise determining the level of PSCA mRNA or PSCA protein expressed by cells in a tumor sample by comparing the level thus determined with the level of PSCA mRNA or PSCA protein expressed in an equivalent tissue sample taken from the same subject at a different time, wherein the degree of expression of PSCA mRNA or PSCA protein in the tumor sample over time provides information on the progression of cancer . In one method, the progression of a cancer is assessed by determining the expression of PSCA in tumor cells over time, where the increase in expression over time indicates a progression of the cancer. In addition, the integrity of the PSCA nucleotide and amino acid sequences in a biological sample can be assessed to identify perturbations in the structure of these molecules, such as insertions, deletions, substitutions and the like, wherein the presence of one or more disorders indicates a progression of cancer.

The above diagnostic approaches may be combined with any of a wide variety of prognostic and diagnostic protocols known in the art. Methods for observing a match between the expression of the PSCA gene and PSCA gene products (or disorders in the PSCA gene and PSCA gene products) and a factor that is associated with malignancy, as a means to diagnose and prognose the state of a tissue sample are also disclosed herein. A wide variety of factors associated with malignancy may be used, such as the expression of genes associated with malignancy (e.g., PSA, PSCA and PSM expression for prostate cancer, etc.), as well as crude cytological observations see, for example, Booking et al., 1984, Anal Quant. Cytol 6 (2): 74-88; Epstein, 1995, Hum. Pathol 26 (2): 223-9, Thorson et al. , 1998, Mod. Pathol. 11 (6): 543-51; Baisden et al., 1999, Am. J. Surg., Pathol 23 (8): 918-24). Methods for observing a match between the expression of the PSCA gene and PSCA gene products (or disorders in the PSCA gene and PSCA gene products) and another factor that is associated with malignancy are useful, for example, because presence of a set of specific factors that coincide with disease provides crucial information to diagnose and prognosticate the state of a tissue sample. Methods for observing a coincidence between the expression of the PSCA gene and PSCA gene products (or disorders in the PSCA gene and PSCA gene products) and another factor associated with malignancy may imply detecting over-expression of mRNA or PSCA 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 match of PSCA mRNA or protein and overexpression of mRNA or protein of PSA (or expression of PSCA or PSM). Expression of PSCA and PSA mRNA in prostate tissue can be examined where the overexpression of PSCA and PSA mRNA in the sample indicates the existence of prostate cancer, prostate cancer susceptibility or emergency or condition of the prostate. a prostate tumor. Methods for detecting and quantifying the expression of PSCA mRNA or protein are described herein, and standard protein and nucleic acid quantification and detection technologies are well known in the art. Standard methods for the detection and quantification of PSCA mRNAs include in situ hybridization using labeled PSCA ribosounds, Northern blot and related techniques using PSCA polynucleotide probes, RT-PCR analysis using PSCA-specific primers, and other type detection methods such as, for example, branched DNA, SISBA, TMA and the like. In a specific embodiment, semi-quantitative RT-PCR is used to detect and quantitate the expression of PSCA mRNA. Any number of primers capable of amplifying the PSCA may be used for this purpose including, but not limited to, the different sets of primers specifically described herein. Polyclonal or monoclonal antibodies specifically reactive with the PSCA wild type protein can be used in a biopsy tissue immunohistochemistry assay.

IX.) Identification of PSCA-Interacting Molecules The PSCA protein and nucleic acid sequences disclosed herein enable one skilled in the art to identify proteins, small molecules and other agents that interact with PSCA, as well as PSCA-activated pathways through any of a variety of protocols accepted in the art. For example, one of the so-called interaction trap systems (also referred to as &quot; double hybrid assay &quot;) may be used. In such systems, the molecules interact and reconstitute a transcription factor that drives the expression of a reporter gene, after which the expression of the reporter gene is tested. Other systems identify protein-protein interactions in vivo through the reconstitution of a eukaryotic transcriptional activator, see, for example, U.S. Patent 5,955,280 issued September 21, 1999, 5,925,523 issued on July 1999, 5,846,722 issued December 8, 1998 and 6,004,746 issued December 21, 1999. Algorithms are also available in the art for predictions based on the genome of protein function (see, for example, Marcotte, et al., Nature 402: 4 November 1999, 83-86).

Alternatively, peptide libraries can be screened to identify molecules that interact with PSCA protein sequences. In such methods, peptides that bind to PSCA are identified by screening libraries that encode a random or controlled collection of amino acids. The peptides encoded by the libraries are expressed as fusion proteins of bacteriophage coat proteins, the bacteriophage particles are then screened against the PSCA protein (s).

Accordingly, peptides having a wide variety of uses, such as therapeutic, diagnostic or prognostic reagents, are thus identified without any prior information about the structure of the expected ligand or receptor molecule. Typical peptide libraries and screening methods that can be used to identify molecules that interact with PSCA protein sequences are disclosed for example in U.S. Patent Nos. 5,723,286 issued March 3, 1998 and 5,733,731 issued on 31 of March 1998.

Alternatively, PSCA-expressing cell lines are used to identify protein-protein interactions mediated by PSCA. Such interactions can be examined using immunoprecipitation techniques (see, for example, Hamilton B.J., et al., Biochem Biophys Res Commun 1999, 261: 646-51). The PSCA protein can be immunoprecipitated from PSCA-expressing cell lines using anti-PSCA antibodies. Alternatively, antibodies against the His tag can be used in a cell line engineered to express fusions of PSCA and a His tag (vectors mentioned above). The immunoprecipitated complex can be examined for protein association through procedures such as Western blotting, 35S-methionine protein labeling, protein microsequencing, silver staining, and two-dimensional gel electrophoresis.

Ligands and small molecules that interact with the PSCA can be identified through related embodiments of such screening assays. For example, small molecules that interfere with protein function may be identified, including molecules that interfere with the ability of PSCA to mediate phosphorylation and dephosphorylation, interaction with the DNA or RNA molecules, as an indication of the regulation of the cell cycle , second messenger signaling or tumorigenesis. Likewise, small molecules that modulate ion channels, protein pumps, or communication functions of PSCA-related cells are identified and used to treat patients who have a PSCA-expressing cancer (see, for example, Hille, B., Ionic Channels of Excitable Membranes 2nd Ed., Sinauer Assoc., Sunderland, MA, 1992). In addition, ligands that regulate PSCA function can be identified based on their ability to bind to PSCA and activate a reporter construct. Typical methods are discussed for example in U.S. Patent No. 5,928,868 issued July 27, 1999 and include methods for the formation of hybrid ligands wherein at least one ligand is a small molecule. In an illustrative example, cells engineered to express a PSCA fusion protein and a DNA binding protein are used to co-express a ligand / small molecule hybrid fusion protein and a cDNA library transcriptional activator protein. The cells additionally contain a reporter gene, the expression of which is conditioned by the proximity of the first and second fusion proteins to each other, an event occurring only if the hybrid ligand binds to target sites in both hybrid proteins. Those cells expressing the reporter gene are selected and the unknown small molecule or unknown ligand is identified. This method provides a means of identifying modulators that activate or inhibit PSCA. Disclosed herein is a screening method for a molecule that interacts with a PSCA amino acid sequence shown in Figure 1, comprising the steps of placing a population of molecules in contact with a PSCA amino acid sequence, allowing the population of molecules and the amino acid sequence of PSCA interact under conditions facilitating an interaction, determining the presence of a molecule that interacts with the amino acid sequence of PSCA, and then separating the molecules that do not interact with the amino acid sequence of PSCA to molecules that do so. The method may further comprise the purification, characterization and identification of a molecule that interacts with the amino acid sequence of PSCA. The identified molecule can be used to modulate a function performed by a PSCA. The amino acid sequence of PSCA can be contacted with a library of peptides. X.) Therapeutic Methods and Compositions The identification of PSCA as a protein which is normally expressed in a restricted set of tissues, but is also expressed in cancers such as those listed in Table I, opens a series of therapeutic approaches for the treatment of such cancers. It is to be noted that targeted antitumor therapies have been useful even when the target protein is expressed in normal tissues, even in normal vital organ tissues. A vital organ is one that is needed to sustain life, such as the heart or the colon. A non-vital organ is one that can be removed after which the individual is still able to survive. Examples of non-vital organs are ovary, breast and prostate.

For example, Herceptin® is an FDA approved pharmaceutical agent consisting of an antibody that is immunoreactive with the protein also known as HER2, HER2 / neu and erb-b-2. It is marketed by Genentech and has been a commercially successful antitumor agent. Sales of Herceptin® reached almost $ 400 million in 2002. Herceptin® is a treatment for HER2-positive metastatic breast cancer. However, HER2 expression is not limited to these tumors. The same protein is expressed in several normal tissues. In particular, HER2 / neu is known to be present in the normal kidney and heart, so that these tissues are present in all human Herceptin receptors. The presence of HER2 / neu in the normal kidney is also confirmed by Latif, Z., et al., BJU International (2002) 89: 5-9. As shown in this article (which evaluated whether renal cell carcinoma should be a preferred indication for anti-HER2 antibodies, such as Herceptin), both mRNA and protein are produced in benign renal tissues. Notably, the HER2 / neu protein was strongly overexpressed in benign renal tissue.

Despite the fact that HER2 / neu is expressed in tissues such vital tissues as the heart and kidneys, Herceptin is a very useful and commercially successful FDA accepted drug. The effect of Herceptin on cardiac tissue, i.e. &quot; cardiotoxicity, &quot; has been merely a side effect of treatment. When patients were treated with Herceptin alone, significant cardiotoxicity occurred in a very low percentage of patients. To minimize cardiotoxicity there is a more stringent entry requirement for treatment with HER2 / neu. Factors such as predisposition to heart disease are assessed before treatment can occur.

Of particular interest, although kidney tissue is indicated to exhibit normal expression, possibly an even greater expression than cardiac tissue, the kidney does not suffer from any appreciable side effect of Herceptin. Furthermore, of the diversity of the normal set of tissues in which HER2 is expressed, there is very little occurrence of any side effect. Only cardiac tissue showed any significant side effects at all. A tissue such as kidney, where HER2 / neu expression is especially notable, was not the basis for any side effects.

In addition, favorable therapeutic effects have been found for antitumor therapies targeting the epidermal growth factor receptor (EGFR); Erbitux (ImClone). EGFR is also expressed in numerous normal tissues. There have been very limited side effects in normal tissues after the use of anti-EGFR therapies. A general side effect that occurs with EGFR treatment is a severe skin rash observed in 100% of patients undergoing treatment.

Thus, expression of a target protein in normal tissue, even in normal vital tissue, does not call into question the utility of a targeting agent for the protein as a therapeutic agent for certain tumors in which the protein is also overexpressed. For example, expression in vital organs is not in itself harmful. In addition, organs considered expendable, such as the prostate and ovary, can be removed without affecting mortality. Finally, some vital organs are not affected by normal organic expression due to an immunoprilege. Immunoprivileged organs are organs that are protected from blood by a blood-brain barrier and therefore are not accessible to immunotherapy. Examples of immunoprivileged organs are the brain and testicles.

Accordingly, therapeutic approaches that inhibit the activity of a PSCA protein are useful for patients suffering from a cancer that expresses PSCA. These therapeutic approaches generally fall into three classes. The first class modulates the function of PSCA with respect to the growth of tumor cells, leading to the inhibition or delay of the growth of tumor cells or inducing their death. The second class comprises various methods for inhibiting the binding or association of a PSCA protein with its binding partner or with other proteins. The third class comprises a variety of methods for inhibiting the transcription of a PSCA gene or translation of PSCA mRNA. X.A.) Anti-Cancer Vaccines Cancer vaccines comprising a PSCA-related protein or PSCA-related nucleic acid are disclosed herein. In view of the expression of PSCA, the cancer vaccines prevent and / or treat cancers expressing PSCA with minimal or absent effects on non-target tissues. The use of a tumor antigen in a vaccine that generates cell-mediated humoral immune responses as an 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 through the use of a PSCA-related protein, or a PSCA-encoding nucleic acid molecule, and recombinant vectors capable of expressing and displaying the PSCA immunogen (typically comprising a number of epitopes of T cell or antibody). Those skilled in the art understand that a wide variety of vaccine systems for delivery of immunoreactive epitopes are known in the art (see, for example, Heryln et al., Ann Med 1999 Feb 31 (1): 66-78; Maruyama et al. Cancer Immunol. Immunother 2000, 49 (3): 123-32). Briefly, such methods for generating an immune response (e.g., cell-mediated and / or humoral) in a mammal, comprise the steps of: exposing the mammalian immune system to an immunoreactive epitope (e.g., an epitope present on a mammalian PSCA shown in Figure 1 or an analogue or homologue thereof), such that the mammal produces an immune response that is specific for that epitope (e.g., generates antibodies that specifically recognize that epitope). The entire PSCA protein, immunogenic regions or epitopes thereof can be combined and distributed through various means. Such vaccine compositions may for example include lipopeptides (e.g., Vitiello, A. et al., J. Clin. Invest. 95: 341, 1995), peptide compositions encapsulated in poly (DL-lactide-co-glycolide ) (&quot; PLG &quot;) (see, for example, 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 immunostimulatory complexes (ISCOMS) (see, for example, Takahashi et al., Nature 344: 873-875, 1990; Hu et al., Clin Exp Immunol 113: 235-243, 1998), multiple antigen peptide systems (MAPs) (see, for example, Tam, JP, Proc Natl Acad Sci USA 85: 5409-5413, 1988 Tam, JP , 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, E. E. et al., In: Concepts in vaccine development, Kaufmann, S.E., ed., p. 379, 1996;

Chakrabarti, S. et al., Nature 320: 535, 1986; Hu, S. L. et al., Nature 320: 537, 1986; Kieny, M.-P. et al., AIDS Bio / Technology 4: 790, 1986; Top, F. H. et al. , J. Infect. Dis. 124: 148, 1971; Chanda, PK et al., Virology 175: 535, 1990), particles of viral or synthetic origin (for example, Kofler, N. et al., J. Immunol.Moves, 192: 25, 1996. Eldridge, JH et al. ), Adjuvants (Warren, HS, Vogel, FR and Chedid, LA Annu. Rev. Immunol, et al., Nature, 7: 649, 1995) 4: 369, 1986. Gupta, RK et al.

Vaccine 11: 293, 1993), liposomes (Reddy, R. et al., J.

Immunol. 148: 1585, 1992; Rock, K.L., Immunol. , U.S.A. 17: 131, 1996), or, cDNA absorbed in particles or naked (Ulmer, J.B. et al., Science 259: 1745, 1993; Robinson, H.L., Hunt, L.A. and Webster, R.G., Vaccine 11: 957, 1993;

Shiver, J. W. et al. , In: Concepts in vaccine development, Kaufmann, S. Η. 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 cancer associated with PSCA, the vaccine compositions may also be used in conjunction with other treatments used for cancer, for example, surgery, chemotherapy, drug therapeutics, radiation therapeutics, etc., including use in combination with adjuvants such as IL-2, IL-12, GM-CSF, and the like.

Cellular Vaccines:

CTL epitopes can be determined using specific algorithms to identify peptides within the PSCA protein that bind to corresponding HLA alleles (see, for example, Table IV; Epimer ™ and Epimatrix ™, Brown University (URL brown.edu/ (Bimas.dcrt.nih.gov/ SYFPEITHI at URL syfpeithi.bmi-heidelberg.com/) A PSCA immunogen may contain one or more of the following: more amino acid sequences identified using techniques well known in the art, such as the sequences shown in Tables V-XVIII and XXII-LI or a peptide of 8, 9, 10 or 11 amino acids specified by a HLA class I motif / supermotif for example, Table IV (A), Table IV (D) or Table IV (E)) and / or a peptide of at least 9 amino acids which comprises a Class II HLA motif / supermotif (e.g. ) or Table IV (C).) As is appreciated in the art, the Class I HLA binding well is essentially closed-end peptides such that peptides of only a particular size range can fit into the well and are ligated, generally Class I HLA epitopes are 8, 9, 10, or 11 amino acids in length. In contrast, the Class II HLA binding well is essentially open ended; therefore, a peptide of about 9 or more amino acids may be attached by a Class II HLA molecule. Due to the differences in the binding cavity between the Class I and II HLA motifs, the Class I HLA motifs are specific for length, i.e. the two position of a Class I motif is the second amino acid in an amino to carboxyl of the peptide. Amino acid positions in a Class II motif are relative only to each other, not to the entire peptide, i.e., additional amino acids may be attached to the amino and / or carboxyl terminus of a motif carrier sequence. Class II HLA epitopes are often 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length, or more than 25 amino acids.

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 hybridoma generation). Methods of generating an immune response in a mammal comprise exposing the mammalian immune system to an immunogenic epitope in a protein (e.g., a PSCA protein), so that an immune response is generated. A typical embodiment is a method for generating an immune response against PSCA in a host by contacting the host with a sufficient amount of at least one B cell or PSCA cytotoxic T cell epitope or an analog thereof; and at least a periodic interval, subsequently re-contacting the host with the B cell or PSCA cytotoxic T cell epitope or an analog thereof. A specific embodiment is a method of generating an immune response against a PSCA-related protein or a man-made multiepitopic peptide comprising: administering a PSCA immunogen (e.g., a PSCA protein or a peptide fragment thereof, a PSCA fusion protein or the like, etc.) in a vaccine preparation to a human or other mammal. Typically, such vaccine preparations additionally contain a suitable adjuvant (see, for example, U.S. Patent No. 6,146,635) or a universal auxiliary epitope such as a PADRE ™ peptide (Epimmune Inc., San Diego, CA; see Alexander et al., Immunity 1994 1 (9): 751-761 and Alexander et al., J. Immunol, 2000, 164 (3): 164 (3): 1625-1633. , Immunol Res 1998 18 (2): 79-92). An alternative method comprises generating an immune response in a subject against a PSCA immunogen through: in vivo administration to the muscle or skin of the subject's body of a DNA molecule comprising a DNA sequence encoding a PSCA immunogen, DNA sequence operably linked to regulatory sequences that control the expression of the DNA sequence; wherein the DNA molecule is taken up by the cells, the DNA sequence is expressed in the cells and an immune response is generated against the immunogen (see, for example, U.S. Patent No. 5,962,428). Optionally, a genetic vaccine facilitator such as anionic lipids; saponins; lectins; estrogenic compounds; hydroxylated lower alkyls; dimethylsulfoxide; and urea is also administered. In addition, an anti-idiotypic antibody mimicking PSCA may be administered so as to generate a response against the target antigen.

Nucleic Acid Vaccines:

The vaccine compositions disclosed herein include nucleic acid mediated modalities. DNA or RNA encoding the protein (s) of the invention may be administered to a patient. Methods of genetic immunization may be employed to generate humoral and prophylactic or therapeutic cellular immune responses directed against cancer cells expressing PSCA. Constructs comprising DNA encoding a PSCA-related protein / immunogen and suitable regulatory sequences may be injected directly into the muscle or skin of an individual such that the muscle or skin cells absorb the construct and express the protein / immunogen of PSCAs encoded. Alternatively, a vaccine comprises a PSCA-related protein. Expression of the PSCA-related protein immunogen results in the generation of prophylactic or therapeutic cellular or humoral immunity and against cells bearing a PSCA protein. Various techniques of prophylactic and therapeutic genetic immunization known in the art may be used (for review, see the information and references published on the internet address genweb.com). Nucleic acid-based delivery is described, for example, in Wolff et al., Science 247: 1465 (1990), as well as U.S. Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720. Examples of DNA-based delivery technologies include &quot; DNA &quot;, facilitated distribution (mediated by bupivicaine, polymers, peptides), cationic lipid complexes, and particle mediated (&quot; biobalistic &quot;) or pressure- for example, US Patent No. 5,922,687).

For therapeutic or prophylactic immunization purposes, the proteins described herein may be expressed through viral or bacterial vectors. Various viral gene delivery systems which may be used include, but are not limited to, vaccinia virus, avian pox, canary pox, adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus and sindbis virus (see Resens, 1996, Curr. Opin Immunol, 8: 658-663, Tsang et al., J. Natl Cancer Inst 87: 982-990 (1995)). Non-viral delivery systems may also be employed by introducing naked DNA encoding a PSCA-related protein into the patient (e.g., intramuscularly or intradermally) to induce an antitumor response. Vaccinia virus is used, for example, as a vector for expressing nucleotide sequences encoding the peptides described herein. Upon introduction into a host, the recombinant vaccinia virus expresses the immunogenic peptide of the protein, and thereby elicits an immune response from the host. Vaccinia vectors and methods useful in immunization protocols are described in, for example, U.S. Patent No. 4,722,848. Another vector is BCG (Bacillus Calmette-Guérin). Vectors of BCG are described in Stover et al., Nature 351: 456-460 (1991). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides, for example, adenovirus or 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 of this document.

Thus, gene delivery systems are used to deliver a PSCA-related nucleic acid molecule. Full length human PSCA cDNA can be employed. In another method, PSCA nucleic acid molecules encoding cytotoxic T lymphocytes (CTL) and / or specific antibody epitopes are employed.

Ex Vivo Vaccines Several strategies ex vivo can also be used to generate an immune response. One approach involves the use of antigen presenting cells (APCs), such as dendritic cells (DC), to present the PSCA antigen to a patient's immune system. Dendritic cells express Class I and II MHC molecules, costimulator B7 and IL-12, and are therefore highly specialized antigen presenting cells. In prostate cancer, autologous dendritic cells pulsed with prostate-specific membrane antigen (PSMA) peptides are being used in a Phase I clinical trial to stimulate the immune systems of prostate cancer patients (Tjoa et al., 1996, Prostate 28: 65-69; Murphy et al., 1996, Prostate 29: 371-380). Thus, dendritic cells can be used to present PSCA peptides to T cells in the context of Class I or II MHC molecules. In one method, autologous dendritic cells pulsed with PSCA peptides are capable of binding to MHC class I and / or class II molecules. In another method, the dendritic cells are pulsed with the full PSCA protein. Yet another strategy involves manipulating the overexpression of a PSCA gene in dendritic cells using various delivery 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 transfection of RNA derived from tumor (Ashley et al., 1997, J. Exp Med 186: 1177-1182). Cells expressing PSCA may also be engineered to express immune modulators, such as GM-CSF, and be used as immunizing agents. X.B.) PSCA as a Target for Antibody-Based Therapy PSCA is an attractive target for antibody-based therapeutic strategies. A number of antibody strategies are known in the art for targeting both extracellular and intracellular molecules (see, for example, complement mediated killing and ADCC as well as the use of intrabodies). Since PSCA is expressed by cancer cells of different lineages relative to corresponding normal cells, the systemic administration of prepared PSCA immunoreactive compositions which exhibit excellent sensitivity without toxic effects, non-specific and / or non-target effects caused by binding of the composition immunoreactive to non-target organs and tissues. Antibodies specifically reactive with PSCA domains are useful for the treatment of cancers that express PSCA systemically, either as conjugated with a toxin or a therapeutic agent, or as naked antibodies capable of inhibiting cell proliferation or function.

Antibodies against PSCA can be introduced into a patient so that the antibody binds to PSCA and modulates a function, such as an interaction with a binding partner, and thereby mediates the destruction of tumoral cells and / or inhibits the growth of tumor cells . The mechanisms by which such antibodies exert a therapeutic effect may include complement mediated cytolysis, antibody dependent cellular cytotoxicity, modulation of the physiological function of PSCA, inhibition of ligand binding or signal transduction pathways, modulation of tumor cell differentiation, alteration of tumor angiogenesis factor profiles, and / or apoptosis. Examples include Rituxan® for non-Hodgkins lymphoma, Herceptin® for metastatic breast cancer, and Erbitux® for colorectal cancer.

Those skilled in the art will appreciate that antibodies can be used to target and specifically bind to immunogenic molecules such as an immunogenic region of a PSCA sequence shown in Figure 1. In addition, those skilled in the art will comprise a routine combining antibodies with cytotoxic agents (see, for example, Slevers et al., Blood 93:11, 3678-3684 (June 1, 1999)). When cytotoxic and / or therapeutic agents are delivered directly to the cells, such as through their conjugation with antibodies specific for a molecule expressed by that cell (e.g., PSCA), the cytotoxic agent will exert its known biological effect (e.g., cytotoxicity) on these cells.

A wide variety of compositions and methods for the use of antibody-cytotoxic agent conjugates to kill the cells are known in the art. In the context of cancers, typical methods involve administering to an animal having a tumor a biologically effective amount of a conjugate comprising a cytotoxic agent and / or selected therapeutic agent linked to a targeting agent (for example, an antibody anti-PSCA) which binds to a label (e.g., PSCA) expressed, accessible to binding or located on cell surfaces. A typical embodiment is a method of administering a cytotoxic agent and / or therapeutic agent to a PSCA-expressing cell, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a PSCA epitope and exposes the cell to the conjugate of antibody-agent. Another illustrative embodiment is a method of treating a subject suspected of suffering from metastatic cancer, comprising a step of parenterally administering to said subject a pharmaceutical composition comprising a therapeutically effective amount of an antibody conjugated to a cytotoxic agent and / or therapeutic. Cancer immunotherapy using anti-PSCA antibodies can be made according to 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. 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 ), Colorectal cancer (Moun et al., 1994, Cancer Res. 54: 6160-6166, Velders et al., 1995, Cancer Res. 55: 4398-4403) and breast cancer ( Shepard et al., 1991, J. Clin Immunol., 11: 117-127). Some therapeutic approaches involve conjugation of naked antibody to a toxin or radioisotope, such as conjugation of Y91 or I131 with anti-CD20 antibodies (e.g., Zevalin ™, IDEC Pharmaceuticals Corp. or Bexxar ™, Coulter Pharmaceuticals), respectively, while others involve the co-administration of antibodies and other therapeutic agents, such as Herceptin ™ (trastuzuMAb) with paclitaxel (Genentech, Inc.). The antibodies may be conjugated to a therapeutic agent. To treat prostate cancer, for example, antibodies against PSCA may be administered in conjunction with radiation, chemotherapy, or hormonal ablation. In addition, the antibodies may be conjugated to a toxin, such as calicheamicin (for example, Mylotarg ™, Wyeth-Ayerst, Madison, NJ, a recombinant humanized IgG4 antibody conjugated with antitumor antibiotic calicheamicin) or a maytansinoid , taxane-based tumor-activated prodrug, TAP, platform, ImmunoGen, Cambridge, MA, see also, for example, U.S. Patent 5,416,064) or Auristatin E (Nat Biotechnol, July 2003; ): 778-84 (Seattle Genetics)).

While antibody therapy for PSCA is useful for all stages of cancer, antibody therapy may 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 cycles of chemotherapy. Alternatively, therapy with the antibodies of the invention is combined with a regimen of chemotherapy or radiation for patients who have not received chemotherapeutic treatment. In addition, antibody therapy may allow the use of reduced dosages of concomitant chemotherapy, particularly for patients who do not tolerate very well the toxicity of the chemotherapeutic agent.

Fan et al. (Cancer Res. 53: 4637-4642, 1993), Prewett et al. (International J. of Onco, 9: 217-224, 1996) and Hancock et al. (Cancer Res. 51: 4575-4580, 1991) describe the use of various antibodies together with chemotherapeutic agents.

Cancer patients may be evaluated for the presence and level of PSCA expression, preferably through tumor tissue immunohistochemistry evaluations, quantitative PSCA imaging, or other techniques that reliably indicate the presence and degree of PSCA expression. Immunohistochemical analysis of tumor biopsies or surgical specimens is preferred for this purpose. Methods for the immunohistochemical analysis of tumor tissues are well known in the art.

Anti-PSCA 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-PSCA monoclonal antibodies (MAbs) can induce lysis of tumor cells either by complement mediated mechanisms or antibody dependent cellular cytotoxicity (ADCC) mechanisms, both of which require an intact Fc portion of the molecule of immunoglobulin for interaction with the Fc receptor sites of effector cells on complement proteins. In addition, anti-PSCA mAbs that exert a direct biological effect on tumor growth are useful in the treatment of cancers that express PSCA. The mechanisms by which the directly cytotoxic MAbs act include: inhibition of cell growth, modulation of cell differentiation, modulation of tumor angiogenesis factor profiles and induction of apoptosis. The mechanism (s) by which a particular anti-PSCA MAb exerts an antitumor effect is assessed using any number of in vitro assays that assess cell death, such such as ADCC, ADMMC, cellular complement mediated lysis, and so on, as is generally known in the art.

In some patients, the use of murine or other non-human monoclonal antibodies or chimeric human / mouse MAbs can induce moderate to strong immune responses against non-human antibody. This may result in the elimination of the antibody from circulation and reduced efficacy. In more severe cases, such an immune response may lead to the extensive formation of immune complexes that may potentially cause renal failure. Accordingly, preferred monoclonal antibodies used in the therapeutic methods of the invention are those which are either fully human or humanized and which bind specifically to the target PSCA antigen with high affinity but exhibit low or no antigenicity in the patient.

Therapeutic uses of the present invention contemplate the administration of anti-PSCA MAbs alone, while other uses disclosed herein contemplate combinations, or cocktails, of different MAbs. Such MAbs cocktails may have certain advantages in that they contain MAbs that target different epitopes, exploit different effector mechanisms or combine directly cytotoxic MAbs with MAbs that are based on immune effector functionality. Such MAbs in combination may exhibit synergistic therapeutic effects. In addition, anti-PSCA 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 therapy . The anti-PSCA MAbs are administered in their &quot; naked &quot; form &quot; or unconjugated, or may have one or more therapeutics conjugated therewith.

Anti-PSCA antibody formulations are administered via any route capable of delivering the antibodies to a tumor cell. The routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumoral, intradermal and the like. The treatment usually involves repeated administration of the anti-PSCA antibody preparation, via an acceptable route of administration, such as intravenous (IV) injection, typically at a dose in the range of about 0.1, 0.2, 0.3 , 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg / kg body weight. In general, doses in the range of 10-1000 mg MAb per week are effective and well tolerated.

Based on clinical experience with MAb Herceptin ™ in the treatment of metastatic breast cancer, an initial loading dose of approximately 4 mg / kg body weight of patient IV, followed by weekly doses of about 2 mg / kg IV of the preparation of anti-PSCA MAb represents an acceptable dosage regimen. Preferably, the initial loading dose is administered as an infusion of 90 minutes or more. The periodic maintenance dose is administered as an infusion of 30 minutes or longer provided the initial dose is well tolerated. As understood by those skilled in the art, various factors may influence the optimal dosage regimen in a particular case. Such factors include, for example, the binding affinity and half-life of the antibody or MAbs used, the degree of expression of PSCA in the patient, the extent of circulating PSCA antigen released, the level of desired constant state concentration, frequency of treatment and the influence of chemotherapeutic or other agents used in combination with the method of treatment, as well as the health status of a particular patient.

Optionally, patients should be evaluated for PSCA levels in a given sample (for example, circulating levels of PSCA antigen and / or PSCA expressing cells) in order to assist in determining the most effective dosage regimen, etc. Such evaluations are also used for control purposes during therapy, and are useful for measuring therapeutic success in combination with evaluation of other parameters (e.g., urine cytology and / or ImmunoCyt levels in bladder cancer therapy, or by analogy, serum PSA levels in prostate cancer therapy).

Anti-idiotypic anti-PSCA antibodies may also be used anti-cancer therapeutics as a vaccine to induce an immune response in cells expressing a PSCA-related protein. In particular, the generation of anti-idiotypic antibodies is well known in the art; this methodology can be readily adapted to generate anti-idiotypic anti-PSCA antibodies mimicking an epitope in a PSCA-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 may be used in cancer vaccine strategies.

It is an object of the present invention to provide antibodies against PSCA, which inhibit or retard the growth of PSCA-expressing tumor cells. It is a further object of the present invention to provide methods for inhibiting angiogenesis and other biological functions and thereby reducing the growth of tumors in mammals, preferably humans, using such antibodies against PSCA, and in particular using such antibodies against combined PSCAs with radiation and chemo or both.

In one embodiment, there is synergy when tumors, including human tumors, are treated with antibodies against PSCA in conjunction with chemotherapeutic agents or radiation or combinations thereof. In other words, inhibition of tumor growth by a PSCA antibody is potentiated more than expected, when combined with chemotherapeutic agents or radiation or combinations thereof. Synergy may be demonstrated, for example, by a greater inhibition of tumor growth with combined treatment than would be expected from a treatment with only antibodies against PSCA or by the additive effect of treatment with a PSCA antibody and a chemotherapeutic agent or radiation. Preferably, synergy is demonstrated by remission of cancer where remission of cancer is not expected by treatment, either with a naked PSCA antibody or with treatment using an additive combination of a PSCA antibody and a chemotherapeutic agent or radiation. The method of inhibiting the growth of tumor cells using an antibody to PSCA and a combination of chemotherapy or radiation, or both comprises administering the antibody to PSCA prior to, during, or after the initiation of chemotherapy or radiation therapy, as well as any combination of (ie before and during, before and after, during and after, or before, during and after the onset of chemotherapy and / or radiation therapy). For example, the antibody to PSCA is typically administered between 1 and 60 days, preferably between 3 and 40 days, more preferably between 5 and 12 days prior to commencement of chemotherapy and / or radiation therapy. However, depending on the treatment protocol and the patient's specific needs, the method is performed in a way that will provide the most effective treatment and ultimately extend the life of the patient. Administration of chemotherapeutic agents can be achieved in a variety of ways, including systemically the parenteral and enteral routes. In one embodiment, the antibody to PSCA and the chemotherapeutic agent are administered as separate molecules. In another embodiment, the antibody to PSCA is linked, for example, by conjugation to a chemotherapeutic agent. (See the Example titled &quot; Clinical trials in humans for the treatment and diagnosis of human carcinomas through the use of anti-PSCA antibodies in vivo &quot;) and (see the section entitled &quot; PSCA as a target for therapy antibody based &quot;). Particular examples of chemotherapeutic or chemotherapeutic agents include cisplatin, dacarbazine (DTIC), dactinomycin, mecloretamine (nitrogen mustard), streptozocin, cyclophosphamide, carmustine (BC-TG), lomustine (CCNU), doxorubicin (adriamycin), daunorubicin, procarbazine , mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere), aldesleucine, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon alpha, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobromane, plicamycin, streptozocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil, taxol and combinations thereof. The radiation source, used in combination with an antibody to PSCA, may be external or internal to the patient being treated. When the source is external to the patient, the therapy is known as external beam radiation therapy (EBRT). When the source of radiation is internal to the patient, the treatment is called brachytherapy (BT). The radiation is administered according to well known standard techniques using standard equipment manufactured for this purpose, such as AECL Theratron and

Varian Clinac. The radiation dose depends upon numerous factors as is well known in the art. Such factors include the organ being treated, healthy organs in the path of radiation that may be inadvertently adversely affected, the patient's tolerance for radiation therapy, and the area of the body that needs treatment. The dose will typically be between 1 and 100 Gy, and more particularly between 2 and 80 Gy. Some doses that have been reported include 35 Gy for the spinal cord, 15 Gy for the kidneys, 20 Gy for the liver and 65-80 Gy for the prostate. It should be emphasized, however, that the invention is not limited to any particular dosage. The dose will be determined by the attending physician according to the particular factors of a given situation, including the factors mentioned above. The distance between the external radiation source and the point of entry into the patient may be any distance representing an acceptable balance between killing target cells and minimizing side effects. Typically, the source of external radiation is between 70 and 100 cm from the point of entry into the patient. Brachytherapy is usually performed by placing the source of radiation on the patient. Typically, the radiation source is placed approximately 0-3 cm from the tissue to be treated. Known techniques include interstitial, interstitial and surface brachytherapy. Radioactive seeds can be implanted permanently or temporarily. Some typical radioactive atoms that have been used in permanent implants include iodine-125 and radon. Some typical radioactive atoms that have been used in temporary implants include radio, cesium-137 and iridium-192. Some additional radioactive atoms that have been used in brachytherapy include americium-241 and gold-198. The radiation dose for brachytherapy may be the same as that mentioned above for external beam radiation therapy. In addition to the above-mentioned factors to determine the dose of external beam radiotherapy, the nature of the radioactive atom used is also taken into account in determining the dose of brachytherapy. X.C.) PSCA as a Target for Immune Cell Responses

Vaccines and methods of preparing vaccines which contain an immunogenically effective amount of one or more HLA-binding peptides as described herein are further described herein. In addition, the vaccines according to the disclosures herein encompass compositions of one or more of the disclosed peptides. A peptide may be present in a vaccine individually. Alternatively, the peptide may exist as a homopolymer comprising multiple copies of the same peptide, or as a heteropolymer of various peptides. Polymers have the advantage of a greater immunological reaction and, when different peptide epitopes are used to constitute the polymer, the additional ability to induce antibodies and / or CTLs which react with different antigenic determinants of the pathogenic organism or tumor related peptide directed to an immune response. The composition may be a region of a naturally occurring antigen or may be prepared, for example, recombinantly or by chemical synthesis.

Carriers that can be used with vaccines disclosed herein are well known in the art, and include, for example, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, poly amino acids such as poly-L-lysine, poly -glutamic acid, influenza, hepatitis B virus core protein, and the like. The vaccines may contain a physiologically tolerable (i.e., acceptable) diluent such as water, or saline, preferably phosphate buffered saline. 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 may be prepared by conjugating peptides of the invention to lipids, such as tripalmitoyl-S-glycerylcysteine-lyserine-serine (P3CSS). In addition, an adjuvant such as an oligonucleotide containing synthetic cytosine-phosphorothioate-guanine (CpG) has been found to increase CTL responses by 10 to 100-fold (see, for example, Davila and Celis, J. Immunol., 165: 539 -547 (2000)).

Following immunization with a peptide composition, by injection, aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal, or other suitable routes, the host immune system responds to the vaccine by producing large amounts of CTLS and / or HTLs the desired antigen. Accordingly, the host becomes at least partially immune to the further development of cells expressing or overexpressing the PSCA antigen, or derives at least some therapeutic benefit when the antigen was tumor associated.

In some examples, it may be desirable to combine the class I peptide components with components that induce or facilitate neutralizing antibody responses or helper T cells directed to the target antigen. There is disclosed herein a composition comprising class I and class II epitopes. An alternative example of such a composition comprises a class I and / or class II epitope, together with a cross-reacted HTL epitope such as a PADRE ™ molecule (Epimmune, San Diego, CA) (described for example in the document U.S. Patent No. 5,736,142).

A vaccine may also include antigen presenting cells (APCs), such as dendritic cells (DC), as a vehicle to present peptides of the invention. The vaccine compositions may be raised in vitro after mobilization and collection of dendritic cells, whereby loading of dendritic cells occurs in vitro. For example, the dendritic cells are transfected, for example, with a minigene according to the invention, or are pulsed with peptides. The dendritic cell can then be administered to a patient to elicit immune responses in vivo. The vaccine compositions, whether based on DNA or peptides, may also be administered in vivo in combination with the mobilization of the dendritic cells, whereby loading of the dendritic cells occurs in vivo.

Preferably the following principles are used when selecting a set 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. Multiple epitopes to be incorporated into a given vaccine composition may be, but need not be, contiguous following the native antigen from which the epitopes are derived. 1.) Epitopes are selected which, upon administration, mimic immune responses that have been found to correlate with tumor clearance. For Class I HLA this includes 3-4 epitopes from at least one tumor associated antigen (TAA). For Class II HLA a similar logic is employed; again 3-4 epitopes are selected from at least one TAA (see, for example, Rosenberg et al., Science 278: 1447-1450). Epitopes from a TAA can be used in combination with epitopes of one or more additional TAAs to produce a vaccine that targets tumors with different expression patterns of TAAs frequently expressed. 2.) Epitopes are selected which have the required binding affinity established as being correlated with immunogenicity: for class I HLA an IC50 of 500 nM or less, often 200 nM or less; and for class II an IC 50 value of 1000 nM or less. 3.) Peptide-bearing supermotifs, or a sufficient set of peptides bearing specific allele motifs, are selected to provide broad population coverage. For example, it is preferable to have a population coverage of at least 80%. A Monte Carlo analysis, a statistical evaluation known in the art, can be used to assess the amplitude or redundancy of population coverage. 4.) In selecting epitopes from cancer-related antigens it is often useful to select analogs because the patient may develop tolerance to the native epitope. 5.) Epitopes designated &quot; internal epitopes (nested) &quot; are of particular relevance. Internal epitopes occur when at least two epitopes overlap in a given peptide sequence. An internal peptidic sequence may comprise B cell epitopes, HLA class I and / or HLA class II epitopes. In providing internal epitopes, a general aim is to provide the largest number of epitopes per sequence. Thus, one aspect is to avoid providing a peptide that is longer than the amino terminus of the amino terminal epitope and than the carboxyl terminus of the carboxy terminal epitope on the peptide. In providing a multiepitopic sequence, such as a sequence comprising internal epitopes, it is generally important to trace the sequence so as to ensure that it has no pathological or other deleterious biological properties. 6.) Whether a polyepitopic protein is created, or in creating a minigene, one goal is to generate the smallest possible peptide spanning the epitopes of interest. This principle is similar, if not the same as that used when selecting a peptide comprising internal epitopes. However, with an artificial polyepitopic peptide, the goal of size minimization is balanced against the need to integrate any spacer sequences between epitopes in the polyepitopic protein. Spacer amino acid residues may, for example, be introduced to prevent junction epitopes (an epitope recognized by the immune system, not present on the target antigen, and only created by man-made epitope juxtaposition), or to facilitate cleavage between epitopes and , thus improving the presentation of epitopes. Junction epitopes should generally be avoided because the receptor can generate an immune response against that non-native epitope. A junction epitope that is a &quot; dominant epitope &quot; is of particular concern. A dominant epitope may lead to such a zealous response that immune responses to other epitopes are diminished or suppressed. 7.) When sequences of multiple variants of the same target protein are present, potential peptidic epitopes may also be selected based on their conservation. For example, a criterion for preservation may define that the entire sequence of an HLA class I-binding peptide or that the entire 9-mer nucleus of a binding class II binding peptide is conserved in a designated percentage of the sequences evaluated by a specific protein antigen. X.C.l. Minigenes Vaccines

A number of different approaches are available that allow the simultaneous distribution of multiple epitopes. Nucleic acids encoding the peptides disclosed herein are a particularly useful embodiment of the invention. Epitopes for inclusion in a minigene are preferably selected according to the guidelines established in the previous section. A preferred means for administering nucleic acids encoding the disclosed peptides utilizes minigene constructs encoding a peptide comprising one or more epitopes of the invention. The use of multi-epitopic 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 supercarrier and / or PSCA derived motif epitopes, the PADRE ™ universal helper T cell epitope, or various HTL epitopes from PSCA (see, for example, Tables V-XVIII and XXII to L1), and an endoplasmic reticulum translocation signal sequence can be manipulated. A vaccine may also comprise epitopes that are derived from other TAAs. The immunogenicity of a multi-epitope minigene can be confirmed in transgenic mice to assess the magnitude of CTL induction responses against the tested epitopes. In addition, 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 may show that the minigene serves to: 1.) generate a CTL response and 2.) that the induced CTLs recognize 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 can be translated inversely. A framework for the use of human codons can be used to guide the choice of codons for each amino acid. These DNA sequences encoding epitopes may be directly contiguous so that, when translated, a continuous polypeptide sequence is created. To optimize expression and / or immunogenicity, additional elements may be incorporated into the design of the minigene. Examples of amino acid sequences that may 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 a targeting signal from the endoplasmic reticulum. In addition, the HLA presentation of the CTL and HTL epitopes can be improved by including synthetic (e.g., poly-alanine) or naturally occurring flanking sequences adjacent to the CTL or HTL epitopes; these larger peptides comprising the epitope (s) are within the scope of the invention. The minigene sequence can be converted into DNA by joining oligonucleotides encoding the negative and positive strands of the minigene. Overlapping oligonucleotides (30-100 bases in length) can be synthesized, phosphorylated, purified and hybridized under suitable conditions using well known techniques. The ends of the oligonucleotides may be joined, for example, using T4 DNA ligase. This synthetic minigene, encoding the epitope of the polypeptide, can then be cloned into a desired expression vector.

Normal regulatory sequences well known to those skilled in the art are preferably included in the vector to ensure expression in the target cells. Various elements of the vector are desirable: a promoter with a downstream cloning site for the insertion of the minigene; a polyadenylation signal for efficient transcription termination; an origin of E. coli replication; and a selectable E. coli marker (e.g., ampicillin or kanamycin resistance). Numerous promoters may be used for this purpose, for example, the human cytomegalovirus (hCMV) promoter. See, for example, U.S. Patent Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

Further modifications of the vector may be desired to optimize the expression and immunogenicity of the minigene. In some cases, introns are required for efficient gene expression, and one or more naturally occurring or synthetic introns may 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 increased expression of the minigene.

Once the 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 the DNA is prepared using standard techniques. The DNA sequence and orientation of the minigene, as well as all other elements included in the vector, are confirmed by restriction mapping and DNA sequence analysis. Bacterial cells carrying the correct plasmid can be stored as a main cell bank and a working cell bank.

In addition, the immunostimulatory sequences (ISSs or CpGs) appear to play a role in the immunogenicity of the DNA vaccines. These sequences may be included in the vector, outside the coding sequence of the minigene, if desired to increase immunogenicity.

In some embodiments, a bi-cistronic expression vector allowing the production of both epitopes encoded by minigene and a second protein (included to increase or decrease immunogenicity) may be used. Examples of proteins or polypeptides that could beneficially enhance the immune response if coexpressed include cytokines (e.g., IL-2, IL-12, GM-CSF), cytokine induction molecules (e.g., LeIF), molecules co-stimulatory, or HTL responses, Pan-DR binding proteins (PADRE ™, Epimmune, San Diego, CA). Helper epitopes (HTLs) may be linked to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows the targeting of the HTL epitopes to a cell compartment different from that of the CTL epitopes. If necessary, this could facilitate more effective entry of the HTL epitopes into the HLA class II pathway, thereby improving the induction of HTL. In contrast to the induction of CTL or HTL, specifically decreasing the immune response through coexpression of immunosuppressive molecules (e.g., TGF-β) may be beneficial in certain diseases.

Therapeutic quantities of plasmid DNA may 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 shake flasks or in a bioreactor according to well-known techniques. Plasmid DNA can be purified using standard biosparation technologies such as solid phase anion exchange resins provided by QIAGEN, Inc. (Valencia, California). If necessary, the supercoiled DNA can be isolated from the open linear and circular 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 the reconstitution of lyophilized DNA into sterile phosphate buffered saline (PBS). This approach, known as &quot; DNA nu &quot;, is currently being used for intramuscular (IM) administration in clinical trials. To maximize the immunotherapeutic effects of minigene DNA vaccines, an alternative method for the formulation of 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 may also be used in the formulation (see, for example, as described by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6 (7): 682 (1988); In addition, peptides and compounds collectively referred to as protective, interactive compounds, are disclosed in US Pat. No. 5,279,833, WO 91/06309, and Feigner, et al., Proc Natl Acad Sci USA 84: 7413 (1987) non-condensing (PINC) could also be complexed with purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organ or cell types. Target cell sensitization may be used as a functional assay for expression and presentation of HLA class I CTL epitopes encoded by minigenes. For example, plasmid DNA is introduced into a mammalian cell line which is suitable as a target for CTL assays of and standard chromium release. The method of transfection used will be dependent on the final formulation. Electroporation can be used for &quot; naked &quot; DNA, whereas cationic lipids allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to enable enrichment of transfected cells using fluorescence activated cell sorting (FACS). These cells are then labeled with chromium-51 (51 Cr) and used as target cells for epitope-specific CTL lines; the cytolysis, detected by 51Cr release, indicates both production of, and presentation of, HLA from, CTL epitopes encoded by minigene. Expression of HTL epitopes can be assessed in an analogous manner using assays to evaluate HTL activity. In vivo immunogenicity is a second approach to 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 (for example, IM to DNA in PBS, intraperitoneal (i.p.) for lipid complexed DNA). Twenty-one days after immunization, splenocytes are harvested and re-stimulated for one week in the presence of peptides encoding each epitope to be tested. Thereafter, for CTL effector cells, assays are conducted for cytolysis of 51Cr-labeled, peptide-loaded target cells using standard techniques. Lysis of target cells which were sensitized by HLA-loaded peptide epitopes, corresponding to the minigene-encoded epitopes, demonstrates the function of the DNA vaccine for the in vivo induction of CTLs. The immunogenicity of the HTL epitopes is confirmed in transgenic mice in an analogous manner.

Alternatively, the nucleic acids can be administered using the ballistic distribution, as described, for example, in U.S. Patent No. 5,204,253. By using this technique, particles consisting exclusively of DNA are administered. In a further alternative embodiment, the DNA may be adhered to the particles, such as gold particles.

Minigenes may also be delivered using other bacterial or viral delivery systems well known in the art, for example, an expression construct encoding epitopes as disclosed herein may be incorporated into a viral vector such as vaccinia. X.C.2. Combinations of CTL Peptides with Helper Peptides

Vaccine compositions comprising the CTL peptides as described herein may be modified, for example, analogously, to provide desired attributes, such as improved serum half-life, extended population coverage or improved immunogenicity.

For example, the ability of a peptide to induce CTL activity can be improved by binding the peptide to a sequence that contains at least one epitope that is capable of inducing a helper T cell response. Although a CTL peptide can be directly attached to a helper T peptide, the CTL / HTL epitope epitope conjugates are often linked by a spacer molecule. The spacer is typically composed of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions. Spacers are typically selected from, for example, Ala, Gly or other neutral spacers of non-polar amino acids or neutral polar amino acids. It will be understood that the optionally present spacer does not need to consist of the same residues and thus may be a homo or hetero oligomer. When present, the spacer will generally have at least one or two residues, more usually three to six residues and sometimes 10 or more residues. The CTL peptide epitope may be attached to the epitope of the T helper peptide either directly or through a spacer, either at the amino or carboxyl terminus of the CTL peptide. The amino terminus of either the immunogenic peptide or the helper T peptide can be acylated.

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

X.C.3. Combinations of CTL Peptides with T-Cell Priming Agents

It may be desirable to include in the pharmaceutical compositions as disclosed herein at least one component that priming B or T lymphocytes. Lipids have been identified as agents capable of priming CTL in vivo. For example, palmitic acid residues may be attached to the ε-amino and α-amino groups of a lysine residue and then attached, for example, by means of 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, for example Freund's incomplete adjuvant. A particularly effective immunogenic composition may comprise palmitic acid linked to ε-amino and α-amino groups of Lys, which is linked by binding, for example, 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-glyceryl cysteinylserylserine (P3CSS) can be used to priming virus-specific CTLs when covalently attached to a peptide (see, for example, Deres, et al., Nature 342: 561, 1989). The peptides may be coupled to P3CSS, for example, and the lipopeptide administered to a subject to specifically priming an immune response to the target antigen. Furthermore, since the induction of neutralizing antibodies can also be primed with P3CSS-conjugated epitopes, two such compositions can be combined to more effectively trigger both humoral and cell-mediated responses.

X.C.4. Vaccine Compositions Comprising DC Pulsed with CTL and / or HTL Peptides

An example of a vaccine composition according to the disclosures herein comprises ex vivo administration of a cocktail of epitopes bearing peptides to PBMC, or DCs therefrom, from the patient's blood. A pharmaceutical agent for facilitating collection of DC can be used, such as Progenipoietin ™ (Pharmacia-Monsanto, St. Louis, MO) or GM-CSF / IL-4. After pulsating DC with peptides and prior to reinfusion in the patients, DCs are washed to remove unbound peptides. A vaccine may comprise peptide-pulsed DCs, which exhibit pulsed peptidic epitopes complexed with HLA molecules on their surfaces.

DCs can be pulsed ex vivo with a cocktail of peptides, some of which stimulate CTL responses to PSCA. Optionally, a helper T-cell peptide (HTL), such as a vaguely restricted natural or artificial class II HLA peptide, may be included to facilitate the CTL response. Thus, a vaccine can be used to treat a cancer that expresses or overexpresses PSCA. X.D.) Immunotherapy Adoptive PSCA-related antigenic peptides are also used to elicit an ex vivo CTL and / or HTL response. The resulting CTL or HTL cells can be used to treat tumors in patients who do not respond to other conventional forms of therapy or who will not respond to a therapeutic vaccine peptide or nucleic acid in accordance with the disclosures of the present invention. Ex vivo CTL or HTL responses against a particular antigen are induced by tissue culture incubation of the patient's CTL and HTL precursor cells, or genetically compatible, together with a source of antigen-presenting cells (APCs), such as 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 the destruction (HTL) of its specific target cell (e.g., a tumor cell). Transfected dendritic cells can also be used as antigen presenting cells. X.E.) Administration of Vaccines for Therapeutic or Prophylactic Purposes

Pharmaceutical and vaccine compositions as described herein may be used to treat and / or prevent a cancer that expresses or overexpresses PSCA. In therapeutic applications, peptide and / or nucleic acid compositions are administered to a patient in an amount sufficient to elicit an effective antigen B, CTL and / or HTL cell response and to cure or at least partially halt or slow down the antigen. symptoms and / or complications. An amount suitable to achieve this is defined as &quot; therapeutically effective dose &quot;. Amounts effective for this use will depend, for example, on the particular composition administered, the mode of administration, the stage and severity of the disease to be treated, the weight and general condition 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 a subject already harboring a PSCA-expressing tumor. The peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences. Patients may 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 cancer associated with PSCA. This is followed by booster doses until at least symptoms are substantially decreased and during a later period. 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) administered to the patient may vary according to the stage of the disease. illness or health condition of the patient. For example, in a patient with a PSCA-expressing tumor, a vaccine comprising a specific PSCA CTL may be more effective at killing tumor cells in a patient with an advanced disease than alternative embodiments. It is generally important to provide an amount of the epitope of the administered peptide through a means of administration sufficient to efficiently stimulate a cytotoxic T cell response; compositions that stimulate helper T cell responses may also be provided in accordance with the present disclosure. The dosage for an initial therapeutic immunization generally occurs in a unit dose range where the lowest value is about 1, 5, 50, 500, or 1,000 pg, and the highest value is about 10,000; 20,000; 30,000; or 50,000 mg. Dosage values for a human typically range from about 500æg to about 50,000æg per patient of 70 kilograms. Strength dosages of from about 1.0 pg to about 50,000 pg of peptide according to a regimen of reinforcement over weeks to months may be administered, depending on the response and condition of the patient, as determined by measurement of 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 neoplasm has been eliminated or reduced and for a later period. The dosages, routes of administration, and dosage regimens are adjusted according to the methodologies known in the art.

The peptides and compositions disclosed herein may be employed in severe disease states, i.e., fatal or life-threatening conditions. In such cases, as a result of the minimal amounts of foreign substances and the relatively non-toxic nature of the peptides in preferred compositions disclosed herein, it is possible, and may be desirable on the part of the attending physician, to administer substantial excesses of such peptide compositions in dose quantities indicated.

Vaccine compositions may also be used purely as prophylactic agents. Generally, the dosage for an initial prophylactic immunization generally occurs in a unit dose range where the lowest value is about 1, 5, 50, 500, or 1000 pg, and the highest value is about 10,000; 20,000; 30,000; or 50,000 pg. Dosage values for a human typically range from about 500æg to about 50,000æg per patient of 70 kilograms. This is followed by booster dosages of from about 1.0 pg to about 50,000 pg of peptide administered at set intervals from about four weeks to six months after the initial administration of the vaccine. 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.

Pharmaceutical compositions for therapeutic treatment are for parenteral, topical, oral, nasal, intrathecal, or local administration (for example as a topical cream or ointment). Preferably, the pharmaceutical compositions are administered parenterally, for example, intravenously, subcutaneously, intradermally, or intramuscularly. Thus, disclosed herein are compositions for parenteral administration which comprise a solution of the immunogenic peptides dissolved or suspended in an acceptable carrier, preferably in an aqueous carrier.

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

The compositions may contain pharmaceutically acceptable auxiliary substances as necessary to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like, for example, sodium acetate, lactate sodium chloride, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. The concentration of the peptides disclosed herein in the pharmaceutical formulations may vary widely, i.e., from less than about 0.1%, usually at or at least 2% to as much as 20% to 50% or more by weight , and will be selected primarily by fluid volumes, viscosities, etc., according to the particular mode of administration selected.

A unit dosage form for the human of a composition is typically included in a pharmaceutical composition comprising a human unit dosage form of an acceptable carrier, in one embodiment an aqueous carrier, and is administered in a volume / quantity which is known to those of skill in the art. (see, for example, Remington's Pharmaceutical Sciences, 17th Edition, A. Gennaro, Editor, Mack Publishing Co., Easton, Pennsylvania, 1985). For example a peptide dose for initial immunization may be from about 1 to about 50,000 pg, generally 100-5,000 pg, for a 70 kg patient. For example, for nucleic acids, an initial immunization can be performed using a nucleic acid expression vector as administered IM (or SC or ID) in amounts of 0.5-5 mg at various sites. Nucleic acid (0.1 to 1000 pg) can also be administered using biobalistics. After an incubation period of 3-4 weeks, a booster dose is then administered. The boost may be recombinant avian pox virus administered in a dose of 5-107 to 5x109 pfu.

For antibodies, a treatment generally involves repeated administration of the anti-PSCA antibody preparation, via an acceptable route of administration such as intravenous (IV) injection, 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. In addition, 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-PSCA MAb preparation represents an acceptable dosage regimen. As appreciated by those skilled in the art, a number of factors may influence the optimal dose in a particular case. Such factors include, for example, half-life of a composition, the binding affinity of an Ac, the immunogenicity of a substance, the degree of PSCA expression in the patient, the extent of PSCA antigen released in circulation, the concentration level of the desired constant state, frequency of treatment, and the influence of chemotherapeutic or other agents used in combination with the use of the invention, as well as the health condition of a particular patient. Preferred and non-limiting unit dose rates for humans are, for example, 500 mg-1 mg, 1 mg-50 mg, 50 mg-100 mg, 100 mg-200 mg, 200 mg-300 mg, 400 mg-500 mg , 500 mg-600 mg, 600 mg-700 mg, 700 mg-800 mg, 800 mg-900 mg, 900 mg-1 g, or 1 mg-700 mg. In certain embodiments, the dose is in the range of 2-5 mg / kg body weight, for example, with weekly monitoring doses of 1-3 mg / kg; 0.5 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg / kg body weight followed, for example, in two, three or four weeks per weekly doses; 0.5-10 mg / kg body weight, for example, followed in two, three or four weeks per weekly doses; 225, 250, 275, 300, 325, 350, 375, 400 mg per m2 of body area, weekly; 1-600 mg per m2 of body area weekly; 225-400mg per m2 of body area weekly; these doses may be followed by weekly doses for 2, 3, 4, 5, 6, 7, 8, 9, 19, 11, 12 or more weeks.

In one embodiment, unitary human dosage forms of polynucleotides comprise a suitable dosage range or effective amount that provides any therapeutic effect. As appreciated by one skilled in the art, a therapeutic effect depends on a number of factors, including the polynucleotide sequence, polynucleotide molecular mass and route of administration. Dosages are generally selected by the physician or other healthcare professional according to a variety of parameters known in the art, such as severity of symptoms, patient history, and the like. Generally, for a polynucleotide of about 20 bases, a dosage range may be selected from, for example, an independently selected lower limit such as about 0.1, 0.25, 0.5, 50, 100, 200, 300, 400 or 500 mg / kg to an independently selected upper limit of the lower limit of about 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg / kg. For example, a dose may be any of the following: 0.1 to 100 mg / kg, 0.1 to 50 mg / kg, 0.1 to 25 mg / kg, 0.1 to 10 mg / kg, 1 500 to 400 mg / kg, 200 to 300 mg / kg, 1 to 100 mg / kg, 100 to 200 mg / kg, 300 to 400 mg / kg, 400 to 500 mg / kg, 500 to 500 mg / kg. 1000 mg / kg, 500 to 5000 mg / kg, or 500 to 10,000 mg / kg. Generally, parenteral routes of administration may require higher doses of polynucleotides as compared to more direct application of the nucleotide to diseased tissue, as may be required by increasing polynucleotides.

Unit dosage forms for the human T cell may comprise a suitable dosage range or effective amount which provides any therapeutic effect. As appreciated by one skilled in the art, a therapeutic effect depends on a number of factors. Dosages are generally selected by the physician or other healthcare professional according to a variety of parameters known in the art, such as severity of symptoms, patient history, and the like. A dose may be from about 104 cells to about 106 cells, about 106 cells to about 108 cells, about 108 to about 1011 cells, or about 108 to about 5 × 10 10 cells. A dose may also be from about 106 cells / m 2 to about 10 10 cells / m 2, or about 106 cells / m 2 to about 108 cells / m 2.

Protein (s) and / or nucleic acids encoding the protein (s) may also be administered through liposomes, which may also serve to: 1) target the protein (s) to a particular tissue, such as lymphoid tissue; 2) to selectively target disease cells; or, 3) to increase the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In such preparations the peptide to be administered is incorporated as part of a liposome, alone or together with a molecule that binds to a prevailing receptor between lymphoid cells, such as monoclonal antibodies that bind to the CD45 antigen, or other immunogenic compositions or therapeutic. Thus, filled or decorated liposomes with a desired peptide can be targeted to the lymphoid cell site, where the liposomes then distribute the peptide compositions. Liposomes can be formed from standard vesicle-forming lipids, which generally include neutral or negative charge phospholipids and a sterol, such as cholesterol. Lipid selection is generally oriented by consideration of, for example, liposome size, acid instability and stability of liposomes in the bloodstream. A variety of methods are available for the preparation of liposomes, as described in, for example, 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 immune system cells, a ligand to be incorporated into the liposome may include, for example, antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A suspension of liposomes containing a peptide can be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the form of administration, the peptide to be administered, and the stage of the disease to be treated.

For solid compositions, solid, non-toxic, conventional carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate and similar. For oral administration, a non-toxic pharmaceutically acceptable composition is formed by incorporating any of the commonly employed excipients, such as those carriers previously listed, and generally 10-95% of the active ingredient, i.e., one or more peptides of the invention, and more preferably at a concentration of 25% -75%.

For aerosol administration, the immunogenic peptides are preferably provided in finely divided form with a surfactant or propellant. Typical percentages of the peptides are about 0.01% -20% by weight, preferably about 1% -10%. The surfactant should, of course, be non-toxic, and preferably soluble in the propellant. Representative of such agents are esters or partial esters of fatty acids containing from about 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters such as mixed or natural glycerides may be employed. The surfactant may constitute about 0.1% -20% by weight of the composition, preferably about 0.25-5%. The remainder of the composition is commonly propellant. A carrier may also be included, as desired, such as, for example, lecithin for intranasal administration.

XI.) Ways to Perform Diagnosis and Prognosis of PS CA

As disclosed herein, polynucleotides, polypeptides, reactive cytotoxic T-cells (CTL), reactive T helper (HTL) cells and anti-PSCA polypeptide antibodies are used in well-known diagnostic, prognostic and therapeutic assays which examine conditions associated with the deregulated cell growth such as cancer, in particular cancers listed in Table I (see, for example, both its specific tissue expression pattern as well as its overexpression in certain types of cancer, as described by Example in the Example entitled &quot; Analysis of PSCA expression in normal tissues and patient specimens &quot;).

Analogous to PSCA with a prostate-associated antigen, PSA, the archetypal marker that has been used by physicians for years to identify and monitor the presence of prostate cancer (see, for example, 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, for example, Tulchinsky et al., Int J Mol Med 1999 Jul 4 (1): 99-102 and Minimoto et al. (I.e.

Cancer Detect Prev 2000; 24 (1): 1-12). Therefore, the present disclosure of polypeptides and PSCA polynucleotides (as well as PSCA polynucleotide probes and anti-PSCA antibodies used to identify the presence of these molecules) and their properties enable those skilled in the art to utilize these molecules in methods which are analogous to used, for example, in a variety of diagnostic assays directed to the examination of conditions associated with cancer.

Typical examples of diagnostic methods utilizing polynucleotides, polypeptides, reactive T cells and PSCA antibodies are analogous to those methods from well established diagnostic assays employing, for example, polynucleotides, polypeptides, reactive T cells or PSA antibodies . For example, as the PSA polynucleotides are used as probes (for example in Northern analysis, see, for example, Sharief et al., Biochem. Mol. Biol. Int. 33 (3): 567-74 1994)) and primers (for example, in PCR analysis, see for example Okegawa et al., J. Urol. 163 (4): 1189-1190 (2000)) to observe the presence and / or level of PSA mRNA in methods of monitoring PSA overexpression or prostate cancer metastases, the PSCA polynucleotides described herein may be used in the same manner to detect overexpression of PSCA or cancers metastases of the prostate and other cancers that express this gene. Alternatively, just as the PSA polypeptides are used to generate PSA specific antibodies which can then be used to observe the presence and / or level of PSA proteins in methods for monitoring the overexpression of the PSA protein (see , for example, Stephan et al., Urology 55 (4): 560-3 (2000)) or prostate cell metastases (see, for example, Alanen et al., Pathol. Res. Pract., 192 (3): 233-7 (1996)), the PS PS polypeptides described herein can be used to generate antibodies for use in the detection of PSCA overexpression or metastases of prostate cells and cells from other cancers that express this gene.

Specifically, since metastases involve 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), biological sample for the presence of polynucleotide-expressing cells and / or PSCA polypeptides can be used to provide evidence of metastases. For example, when it is found that a biological sample from a tissue which does not normally contain PSCA (lymph node) expressing cells contains PSCA expressing cells such as PSCA expression observed in LAPC4 and LAPC9 xenografts isolated from metastases of lymph nodes and bone, respectively, this finding is indicative of metastases.

Alternatively the PSCA polynucleotides and / or polypeptides may be used to provide evidence of cancer, for example, when cells in a biological sample that do not normally express PSCA or express PSCA on a different level are expressed, express PSCA or have increased expression of PSCA (see, for example, the expression of PSCA in the cancers listed in Table I and in patient samples shown in the attached Figures). In such assays, the skilled persons may wish to, in addition, generate additional evidence of metastasis by testing the biological sample for the presence of a second tissue restricted marker (in addition to PSCA) such as PSA, PSCA etc. (see, for example, Alanen et al., Pathol. Res. Pract., 192 (3): 233-237 (1996)). The use of immunohistochemistry to identify the presence of a PSCA polypeptide within a tissue section may indicate an altered state in certain cells within that tissue. It is well understood in the art that the ability of an antibody to localize to a polypeptide that is expressed in cancer cells is a way of diagnosing the presence of disease, stage and progression of disease and / or aggressiveness of the tumor. Such an antibody may also detect an altered distribution of the polypeptide within the cancer cells, as compared to corresponding non-malignant tissue. The PSCA polypeptide and immunogenic compositions are also useful in view of the localization phenomena of altered subcellular protein in disease states. Altering cells from normal to a disease state causes changes in cell morphology and is usually associated with changes in the location / distribution of subcellular protein. For example, cell membrane proteins that are expressed in a polarized form in normal cells may be altered in the disease, resulting in a distribution of the protein in a non-polar manner along the entire cell surface. The phenomenon of localization of altered subcellular protein in a disease state has been demonstrated with expression of MUC1 and Her2 protein through the use of immunohistochemical means. Normal epithelial cells have a typical apical distribution of MUC1, in addition to some supranuclear localization of the glycoprotein, while malignant lesions often demonstrate a supporting staining pattern (Diaz et al., The Breast Journal, 7, 40-45 (2001 ), Zhang et al, Clinical Cancer Research, 4: 2669-2676 (1998): Cao, et al., The Journal of Histochemistry and Cytochemistry, 45: 1547-1557 (1997)). In addition, normal mammary epithelium is negative for Her2 protein or exhibits only a basolateral distribution whereas malignant cells can express the protein along the entire cell surface (De Potter, et al., International Journal of Cancer, 44, 969- 974 (1989): McCormick, et al., 117: 935-943 (2002)). Alternatively, the protein distribution may be altered from a solely surface location to include diffuse cytoplasmic expression in the disease state. Such an example can be seen with MUC1 (Diaz, et al., The Breast Journal, 7: 40-45 (2001)). Alternation in the location / distribution of a protein in the cell, as detected by immunohistochemistry methods, may also provide valuable information regarding the favorability of certain treatment modalities. This last point is illustrated by a situation where a protein may be intracellular in normal tissue but be on the cell surface in malignant cells; the location on the surface of the cell makes the cells favorably amenable to the application of antibody-based treatment and diagnosis regimens. When such a change in the location of the protein occurs for PSCA, the PSCA protein and related immune responses are very useful. Accordingly, the ability to determine whether the subcellular protein localization has changed to 24P4C12 makes the PSCA protein and related immune responses very useful. The use of the PSCA compositions allows those skilled in the art to make important therapeutic and diagnostic decisions.

PSCA-specific immunohistochemistry reagents are also useful for detecting metastases of PSCA-expressing tumors when the polypeptide appears in tissues where PSCA is not normally produced.

Thus, polypeptides and antibodies to PSCA that result from immune responses thereto are useful in a variety of important contexts such as diagnostic, prognostic, preventive and / or therapeutic purposes known to those skilled in the art.

As the polynucleotide fragments and PSA polynucleotide variants are employed by experts for use in PSA monitoring methods, polynucleotide fragments and PSCA polynucleotide variants are used in an analogous manner. In particular, typical PSA polynucleotides used in PSA monitoring methods are probes or primers consisting of fragments of the PSA cDNA sequence. In this aspect, primers used to amplify a PSA polynucleotide must include less than the complete PSA sequence to function in the polymerase chain reaction. In the context of such PCR reactions, the skilled person generally creates a variety of different polynucleotide fragments which can be used as primers in order to amplify different portions of a polynucleotide of interest or to optimize the amplification reactions (see, for example , Caetano-Anolles, G. Biotechniques 25 (3): 472-476, 478-480 (1998); Robertson et al., Methods Mol. Biol. 98: 121-154 (1998)). A further illustration of the use of such fragments is provided in the Example entitled &quot; Analysis of PSCA expression in normal tissues and patient specimens &quot;, where a PSCA polynucleotide fragment is used as a probe to show expression of PSCA RNAs in cancer cells. In addition, variant polynucleotide sequences are typically used as primers and probes for the corresponding mRNAs in PCR and Northern analyzes (see, for example, Sawai et al., Fetal Diagn. Ther. 1996, Dec-Dec 11 (6) : 407-13 and Current Protocols In Molecular Biology, Volume 2, Unit 2, Frederick, A. Ausubel et al., 1995)). Fragments and variants of polynucleotides are useful in this context where they are capable of binding to a target polynucleotide sequence (e.g., a PSCA polynucleotide shown in Figure 3 or variant thereof) under high stringency conditions.

In addition, PSA polypeptides that contain an epitope that can be recognized by an antibody or T cell that specifically binds to that epitope is used in PSA monitoring methods. The polypeptide fragments and PSCA polypeptide analogs or variants may also be used in an analogous manner. This practice of using polypeptide fragments or polypeptide variants to generate antibodies (such as anti-PSCA or T cell antibodies) is typical in the art with a wide variety of systems, such as fusion proteins, being used by practitioners (see, for example, Current Protocols In Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubel et al., 1995). In this context, each epitope (s) functions in order to provide the architecture with which an antibody or T cell is reactive. Typically, experts create a variety of different polypeptide fragments that can be used in order to generate specific immune responses to different portions of a polypeptide of interest (see, for example, U.S. Patent No. 5,840,501 and US Pat. U.S. Patent No. 5,939,533). For example, it may be preferable to use a polypeptide comprising one of the bio motifs of PSCA discussed herein or a subsequence bearing a motif that is readily identifiable by one of skill in the art based on motifs available in the art. Fragments, variants or analogs of polypeptides 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 (for example, a PSCA polypeptide shown in Figure 1).

As shown herein, PSCA polypeptides and polynucleotides (as well as PSCA polynucleotide probes and anti-PSCA antibodies or T cells used to identify the presence of these molecules) exhibit specific properties that make them useful in the diagnosis of cancers such as those listed in Table I. Diagnostic tests that measure the presence of PSCA genomic products in order to assess the presence or onset of a disease condition described herein, such as prostate cancer, are used to identify patients for measures preventive or additional monitoring, and were successfully used with PSA. In addition, these materials satisfy a need in the art for molecules having characteristics similar to or complementary to PSA in situations where, for example, a definitive diagnosis of prostate-origin metastases can not be performed on the basis of a PSA test alone (see, for example, Alanen et al., Pathol. Res. Pract., 192 (3): 233-237 (1996)), and consequently, materials such as polypeptides and PSCA polynucleotides (as well as PSCA polynucleotide probes and anti-PSCA antibodies used to identify the presence of these molecules) should be employed to confirm metastasis of prostatic origin.

Finally, in addition to their use in diagnostic assays, the PSCA polynucleotides disclosed herein have a number of other utilities such as their use in identifying chromosomal abnormalities associated with oncogenetics in the chromosomal region in which the PSCA gene is mapped (see the Example titled &quot; Chromosomal Mapping of PSCA &quot; below). In addition, in addition to their use in diagnostic assays, the PSCA-related proteins and polynucleotides disclosed herein have other utilities such as their use in forensic analysis of tissues of unknown origin (see, for example,

Takahama K Forensic Sci Int June 28, 1996; 80 (1-2): 63-9).

In addition, the PSCA-related proteins and polynucleotides as disclosed herein may be used to treat a pathological condition characterized by overexpression of PSCA. For example, the amino acid or nucleic acid sequence of Figure 1, or fragments of any, can be used to generate an immune response against a PSCA antigen. Antibodies and other molecules that react with PSCA can be used to modulate the function of this molecule, and thus provide a therapeutic benefit.

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

In one approach, a recombinant vector encoding single chain antibodies that bind specifically to PSCA is introduced into PSCA expressing cells via gene transfer technologies. Accordingly, the encoded single-chain anti-PSCA antibody is expressed intracellularly, binds to the PSCA protein, and thus inhibits its function. Methods for manipulating such intracellular single chain antibodies are well known. Such intracellular antibodies, also known as &quot; intrabodies &quot;, are specifically targeted to a specific compartment within the cell, providing control over where the inhibitory activity of the treatment is focused. This technology has been successfully applied in the art (for review, see Richardson and Marasco, 1995, TIBTECH vol.13). Intrabodies have been shown to virtually eliminate the expression of otherwise abundant cell surface receptors, for example, Richardson et al. , 1995, Proc. Natl. Acad. Know. 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 heavy and light chain variable domains 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 attached to the light chain constant region. Well known intracellular trafficking signals are manipulated into recombinant polynucleotide vectors encoding such single chain antibodies so as to accurately direct the Intrabody to the desired intracellular compartment. For example, intrabodies directed to the endoplasmic reticulum (ER) are manipulated 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 manipulated to include a nuclear localization signal. Lipid fractions are attached to intrabodies in order to attach the intrabody to the cytosolic side of the plasma membrane. Intrabodies can also be targeted to exert cytosolic function. 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, the intrabodies are used to capture PSCA in the nucleus, thereby preventing its activity within the nucleus. Nuclear targeting signals can be manipulated in such PSCA intrabodies in order to achieve the desired targeting. Such PSCA intrabodies are designed to specifically bind to a particular PSCA domain.

In another embodiment, cytosolic intrabodies that specifically bind to a PSCA protein are used to prevent PSCA from gaining access to the nucleus, thereby preventing it from exerting any biological activity within the nucleus (for example, by preventing PSCA from forming complexes of transcription with other factors).

In order to specifically direct the expression of such intrabodies to particular cells, intrabody transcription is placed under the regulatory control of an appropriate tumor specific promoter and / or enhancer. In order to direct expression of the intrabody specifically for the prostate, for example, the PSA promoter and / or promoter / enhancer can be used (see, for example, US Patent No. 5,919,652 issued July 6 of 1999). XII.B.) Inhibition of PSCA with Recombinant Proteins

In another approach, recombinant molecules bind to PSCA and thus inhibit PSCA function. For example, these recombinant molecules prevent or inhibit PSCA from binding to its binding partner (s) or associating with other protein (s). These recombinant molecules may, for example, contain the reactive part (s) of a PSCA-specific antibody molecule. The PSCA binding domain of a PSCA binding partner can be manipulated into a dimeric fusion protein, whereby the fusion protein comprises two PSCA ligand binding domains attached to the Fc portion of a human IgG, such as human IgG1 . Such an IgG moiety may contain, for example, the CH2 and CH3 domains and the hinge region, but not the CH1 domain. Such dimeric fusion proteins are administered in soluble form to patients suffering from a cancer associated with PSCA expression, wherein the dimeric fusion protein binds specifically to PSCA and blocks the interaction of PSCA with a binding partner. Such dimeric fusion proteins are further combined in multimeric proteins using known antibody binding technologies.

XII.C.) Inhibition of Transcription or PSCA Translation Various methods and compositions for inhibiting transcription of the PSCA gene are described herein. Likewise, methods and compositions for inhibiting the translation of PSCA mRNA into protein are disclosed herein.

In one approach, a method of inhibiting transcription of the PSCA gene comprises contacting the PSCA gene with an antisense PSCA polynucleotide. In another approach, a method of inhibiting the translation of PSCA mRNA comprises contacting a PSCA mRNA with a

antisense polynucleotide. In another approach, a PSCA specific ribozyme is used to cleave a PSCA message, thereby inhibiting translation. Such antisense and ribozyme-based methods may also be directed to the regulatory regions of the PSCA gene, such as PSCA promoter and / or enhancers. Likewise, proteins capable of inhibiting a transcription factor of the PSCA gene are used to inhibit transcription of PSCA mRNA. The various polynucleotides and compositions useful in the above-mentioned 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 PSCA transcription through interference with transcriptional activation of PSCA are also useful for the treatment of cancers that express PSCA. Likewise, factors that interfere with PSCA processing are useful for treating cancers that express PSCA. Methods of treating cancer using such factors are also within the scope of the invention. XII.D.) General Considerations for Therapeutic Strategies

Gene transfer and gene therapy technologies may be used to deliver therapeutic polynucleotide molecules to PSCA (i.e., antisense, ribozyme, polynucleotide encoding intrabodies and other PSCA inhibitor molecules) synthetic tumor cells. A number of gene therapy approaches are known in the art. Recombinant vectors encoding antisense polynucleotides, PSCA, ribozymes, factors capable of interfering with PSCA transcription, and so on, can be delivered to target tumor cells using such gene therapy approaches.

The above therapeutic approaches may be combined with any of a wide variety of surgery, chemotherapy or radiotherapy regimens. The therapeutic approaches of the invention may allow the use of reduced dosages of chemotherapy (or other therapies) and / or less frequent administration, an advantage for all patients, and in particular for those who do not tolerate well the toxicity of the chemotherapeutic agent. The antitumor activity of a particular composition (for example, antisense, ribozyme, intrabody), or a combination of such compositions, may be evaluated using various in vitro and in vivo assay systems. In vitro assays that assess therapeutic activity include cell growth assays, soft agar assays and other assays indicative of tumor promoter activity, binding assays capable of determining the extent to which a therapeutic composition will inhibit PSCA binding to a connection partner, etc.

In vivo, the effect of a therapeutic composition of PSCA can be assessed in an appropriate animal model. For example, xenogenic prostate cancer models may be used, wherein explants or xenograft tissues passed from human prostate cancer are introduced into immunocompromised animals, such as nude mice or SCIDs (Klein et al., 1997, Nature Medicine 3: 402-408). For example PCT Patent Application WO98 / 16628 and U.S. Patent 6,107,540 disclose various models of human prostate cancer xenograft capable of recapitulating the development of primary tumors, micrometastases, and the formation of osteoblastic metastases characteristic of phase disease late Efficacy can be predicted using assays that measure inhibition of tumor formation, tumor regression or metastases, and the like.

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

Therapeutic compositions used in the practice of the foregoing methods may be formulated into pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carriers include any material which when combined with the therapeutic composition retains the antitumor function of the therapeutic composition and is generally unreactive 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, bacteriostatic water and the like (see, generally, Remington's Pharmaceutical Sciences 16th Edition, A. Osal. Ed., 1980).

The therapeutic formulations may 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, intratumoral, intradermal, intraorganic, orthotopic and the like. A preferred formulation for intravenous injection comprises the therapeutic composition in a solution of preserved bacteriostatic water, sterile water not preserved and / or diluted in polyvinyl chloride or polyethylene bags containing sterile 0.9% sodium chloride for injection, USP. Therapeutic protein preparations may 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.

The dosages and administration protocols for the treatment of cancers using the above methods will vary with the target method and cancer, and will generally depend on a number of other factors appreciated in the art. XIII.) Identification, Characterization and Use of

PSCA Modulators Methods for Identifying and Using Modulators Screening can be performed to identify modulators that induce or suppress a particular expression profile, suppress or induce specific pathways, preferably thereby generating the associated phenotype. In another example, after major differentially expressed genes have been identified in a given state; screenings are performed to identify modulators that alter the expression of individual genes, either increasing or decreasing. Screening can be performed to identify modulators that alter a biological function of the expression product of a differentially expressed gene. Once again, once the importance of a gene in a particular state has been identified, screenings are performed to identify agents that bind and / or modulate the biological activity of the gene product.

In addition, screenings are made for the genes that are induced in response to a candidate agent. Upon identification of a modulator (which suppresses a cancer expression pattern leading to a normal expression pattern, or a modulator of a cancer gene leading to expression of the gene such as in normal tissue) a screening is performed to identify genes which are specifically modulated in response to the agent. Comparison of expression profiles between normal tissue and agent treated cancer tissue reveals genes that are not expressed in normal tissue or cancer tissue, but are expressed in agent treated tissue, and vice versa. These specific agent sequences are identified and used by methods described herein for genes or cancer proteins. In particular, these sequences and the proteins they encode are used in the labeling or identification of agent treated cells. In addition, the antibodies are raised against the agent-induced proteins and used to direct new therapeutics to the sample of treated cancer tissue.

Modulator Related Identification and Screening Assays:

Gene Expression Assays

The proteins, nucleic acids and antibodies of the invention and, as otherwise described herein, may be used in screening assays. Cancer associated proteins, antibodies, nucleic acids, modified proteins and cells containing these sequences are used in screening assays, such as evaluating the effect of drug candidates on a &quot; gene expression profile, &quot; profile of polypeptide expression or alteration of biological function. In one example, expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring of expression profile genes following treatment with a candidate agent (e.g., Davis, GF, et al., J. Immunol. Biol Screen 7:69 (2002), Zlokarnik, et al., Science 279: 84-8 (1998); Heid, Genome Res 6: 986-94, 1996).

Cancer proteins, antibodies, nucleic acids, modified proteins and cells containing the native or modified cancer proteins or genes are used in screening assays. That is, disclosed herein are methods for screening compositions that modulate the cancer phenotype or a physiological function of a cancer protein of the invention. This is done on a gene per se or by evaluating the effect of drug candidates on a &quot; gene expression profile &quot; or biological function. Expression profiles may preferably be used in conjunction with high throughput screening techniques to enable monitoring after treatment with a candidate agent, see Zlokarnik, supra.

A variety of assays are performed targeting the genes and proteins of the invention. Assays are performed at an individual nucleic acid or protein level. That is, after a particular gene has been identified as being upregulated in cancer, the test compounds are screened for the ability to modulate gene expression or for binding to the cancer protein, as disclosed herein. &quot; Modulation &quot; in this context includes an increase or a decrease in gene expression. The preferred amount of modulation will depend upon the original alteration of gene expression in normal tissue tissues submitted to cancer, with changes of at least 10%, preferably 50%, more preferably 100-300%, and in some embodiments 300-1000 % or superior. Thus, if a gene exhibits a 4-fold increase in cancer tissue compared to normal tissue, a decrease of about four times is often desired; likewise a 10-fold decrease in cancer tissue as compared to normal tissue, a target value of a 10-fold increase in expression by the test compound is often desired. Modulators that exacerbate the type of gene expression observed in cancer are also useful, for example, as a positively regulated target in further analyzes. The amount of gene expression is monitored using nucleic acid probes and quantification of levels of gene expression, or, alternatively, a gene product is itself controlled, for example, through the use of antibodies to the cancer protein and immunoassays conventional. Proteomics and separation techniques also allow expression quantification.

Monitoring Expression for Identification of Compounds Modifying Gene Expression Monitoring of gene expression, i.e. an expression profile, can be monitored simultaneously for a number of entities. Such profiles will typically involve one or more of the genes of Figure 1. In this example, for example, cancer nucleic acid probes are linked to biochips to detect and quantify cancer sequences in a particular cell. Alternatively, PCR may be used. Thus, a series, for example, wells of a microtiter plate, may be used with primers dispensed into desired wells. A PCR reaction can then be performed and analyzed for each well. Expression monitoring is performed to identify compounds that modify the expression of one or more cancer associated sequences, for example a polynucleotide sequence set forth in Figure 1. Generally, a test modulator is added to the cells prior to analysis. In addition, screening is also provided to identify agents that modulate cancer, modulate cancer proteins of the invention, bind to a cancer protein as disclosed herein, or interfere with the binding of a cancer protein and an antibody or another connection partner. High throughput screening methods may involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such &quot; combinatorial chemical libraries &quot; are then screened in one or more assays to identify library members (particular chemical species or subclasses) that exhibit a desired characteristic activity. The compounds thus identified may serve as &quot; guiding compounds &quot; as screening compounds, or as therapeutic agents.

Combinatorial libraries of potential modulators can be screened for ability to bind to a cancer polypeptide or modulate the activity. Conventionally, novel chemical entities with useful properties are generated by identifying a chemical compound (called a &quot; guiding compound &quot;) with some desirable property or activity, e.g., inhibitory activity, creating variants of the guiding compound, and evaluating the property and activity of these variant compounds. Often, high throughput screening (HTS) methods are employed for this type of analysis.

As mentioned above, monitoring of gene expression is conveniently used to test candidate modulators (e.g., protein, nucleic acid or small molecule). After the candidate agent is added and the cells allowed to incubate for a period, the sample containing a target sequence to be analyzed is, for example, added to a biochip.

If necessary, the target sequence is prepared using known techniques. For example, a sample is treated to lyse the cells, using known lysis buffers, electroporation, etc., with purification and / or amplification as performed by PCR, as appropriate. For example, an in vitro transcription with tags covalently attached to the nucleotides is performed. Generally, the nucleic acids are labeled with biotin-FITC or PE, or with cy3 or cy5. The target sequence may be labeled with, for example, a fluorescent, chemiluminescent, chemical or radioactive signal, to provide a means of detecting specific binding of the target sequence to a probe. The label may also be an enzyme, such as alkaline phosphatase or horseradish peroxidase, which when supplied with a suitable substrate produces a product which is detected. Alternatively, the label is a labeled compound or small molecule, such as an enzyme inhibitor, that binds but is not altered or catalyzed by the enzyme. The label may also be a fraction or compound, such as an epitope tag or biotin, which specifically binds streptavidin. For the biotin example, streptavidin is labeled as described above thereby providing a detectable signal for the bound target sequence. The unbound labeled streptavidin is typically removed prior to analysis.

As will be appreciated by those skilled in the art, such assays may be direct hybridization assays or may comprise &quot; sandwich assays &quot;, which include the use of multiple probes, as is generally described in US Patent Nos. 5,681,702. ; 5,597,909; 5,545,730; 5,594,117; 5,591,584; 5,571,670; 5,580,731; 5,571,670; 5,591,584; 5,624,802; 5,635,352; 5,594,118; 5,359,100; 5124, 246 and 5,681,697. In this method, in general, the target nucleic acid is prepared as described above, and is then added to the biochip comprising a plurality of nucleic acid probes, under conditions that allow the formation of a hybridization complex.

A variety of hybridization conditions may be used, including high, medium and low stringency conditions as described above. The assays are generally performed under stringency conditions that allow formation of the probe and marker hybridization complex only in the presence of target. Restriction can be controlled by changing a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration, pH, organic solvent concentration, etc. These parameters may also be used to control non-specific binding, as is generally described in U.S. Patent No. 5,681,697. Thus, it may be desirable to effect certain steps under higher stringency conditions to reduce non-specific binding.

The reactions outlined herein can be achieved in a variety of ways. Reaction components may be added simultaneously, or sequentially, in different orders, with preferred embodiments described below. In addition, the reaction may include a variety of other reagents. These include salts, buffers, neutral proteins, for example, albumin, detergents, etc., which can be used to facilitate optimum hybridization and detection and / or to reduce nonspecific or background interactions. Reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc., may also be used as appropriate, depending on the methods of sample preparation and the purity of the target. Assay data are analyzed to determine individual gene expression levels, and changes in expression levels as between states, forming a gene expression profile.

Assays Related to Biological Activity Methods disclosed herein are disclosed for identifying or screening a compound that modulates the activity of a cancer related gene or protein, as disclosed herein. The methods comprise the addition of a test compound, as defined above, to a cell comprising a cancer protein of the invention. The cells contain a recombinant nucleic acid encoding a cancer protein. A library of candidate agents can be tested on a plurality of cells.

In one method, assays are evaluated in the presence or absence or prior or subsequent exposure of physiological signals, for example hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogens, or other cells (i.e., cell-cell contacts). In another example, the determinations are made at different stages of the cell cycle process. Accordingly, the compounds that modulate the genes or proteins of the present invention are identified. Compounds with pharmacological activity are capable of potentiating or interfering with the activity of the cancer protein of the invention. Once identified, similar structures are evaluated to identify the critical structural characteristics of the compound.

A method of modulating (e.g., inhibiting) the cancer cell division is disclosed; the method comprises administering a cancer modulator. A method of modulating (e.g., inhibiting) cancer is also disclosed; the method comprises administering a cancer modulator. In a further example, methods are provided for treating cancer cells or individuals; the method comprises administering a cancer modulator.

A method for modulating the state of a cell expressing a gene of the invention is disclosed. As used herein, the term comprises such accepted parameters in the art, such as growth, proliferation, survival, function, apoptosis, senescence, localization, enzymatic activity, signal transduction, etc., of a cell. In one method, a cancer inhibitor is an antibody, as discussed above. In another method, the cancer inhibitor is an antisense molecule. A variety of cell growth, proliferation and metastasis assays are known to those skilled in the art, as described herein.

High Performance Screening for Identification of

Modulators

Assays to identify suitable modulators are capable of high throughput screening. Preferred assays thus detect a potentiation or inhibition of cancer gene transcription, inhibition or potentiation of polypeptide expression, and inhibition or potentiation of polypeptide activity.

In one example, modulators evaluated in high throughput screening methods are proteins, normally proteins or naturally occurring fragments of naturally occurring proteins. Thus, for example, cell extracts containing proteins or random or directed digestion products of protein cell extracts are used. In this way, the protein libraries are made for screening in the methods of the invention. Particularly preferred in this method are libraries of bacterial, fungal, viral and mammalian proteins, the latter being preferred, and human proteins being especially preferred. Particularly, a useful test compound will be directed to the class of proteins to which the target belongs, for example substrates for enzymes, or ligands and receptors.

Use of Growth in Soft Agar and Formation of Colonies to Identify and Characterize Modulators

Normal cells require a solid substrate to set and grow. When the cells are transformed, they lose this phenotype and grow detached from the substrate. For example, transformed cells can be grown in a stirred suspension culture or suspended in semi-solid medium, such as semi-solid or mole agar. Transformed cells, when transfected with tumor suppressor genes, can regenerate the normal phenotype and, once again, require a solid substrate to set and grow. Growth on soft agar or colony formation in the assays are used to identify cancer sequence modulators, which when expressed in host cells, inhibit abnormal cell proliferation and transformation. A modulator reduces or eliminates the ability of host cells to grow in suspension in solid or semi-solid media, such as agar.

Techniques for growth in soft agar or colony formation in suspension assays are described in Freshney, Culture of Animal Cells, Manual of Basic Technique (3rd ed., 1994). See also, the methods section of Garkavtsev et al. (1996), supra.

Evaluation of Contact Inhibition and Growth Density Limitation to Identify and Characterize Modulators

Normal cells usually grow in a flat pattern and organized into cell culture until they touch other cells. When the cells touch, they are inhibited by the contact and stop growing. The transformed cells, however, are not inhibited by contact and continue to grow to high densities in disorganized foci. Thus, the transformed cells grow to a higher saturation density than the corresponding normal cells. This is detected morphologically by the formation of a disoriented cell monolayer or cells in foci. Alternatively, the (3H) -thymidine labeling index at saturation density is used to measure growth limitation by density, similarly an MTT or Alamar blue assay will reveal the proliferation capacity of cells and the ability of modulators to affect it. See Freshney (1994), supra. Transformed cells, when transfected with tumor suppressor genes, can regenerate a normal phenotype and become inhibited by contact and could grow to a lower density.

In this assay, the (3 H) -thymidine labeling index on saturation density is a preferred method for measuring density limitation of growth. The transformed host cells are transfected with a cancer associated sequence and allowed to grow for 24 hours at a saturation density under non-limiting medium conditions. The percentage labeling of the cells with (3H) -thymidine is determined by incorporated cpm. Contact independent growth is used to identify modulators of cancer sequences, which led to abnormal cell proliferation and transformation. A modulator reduces or eliminates contact-independent growth, and returns the cells to a normal phenotype. Evaluation of Growth Factor Dependence or Serum to Identify and Characterize Modulators

Transformed cells have less serum dependence than their normal counterparts (see, for example, Temin, J. Natl Cancer Inst. 37: 167-175 (1966);

Eagle et al. , J. Exp. Med. 131: 836-879 (1970)); Freshney, supra. This is in part due to the release of various growth factors by the transformed cells. The degree of growth factor or serum dependence of transformed host cells can be compared with that of the control. For example, the growth factor or serum dependence of a cell is monitored in methods for identifying and characterizing compounds that modulate cancer associated sequences disclosed herein.

Use of Specific Tumor Marker Levels to Identify and Characterize Modulators

Tumor cells release a higher amount of certain factors (hereinafter &quot; tumor specific markers &quot;) than their normal counterparts. For example, plasminogen activator (PA) is released from human glioma at a level higher than normal brain cells (see, for example, Gullino, Angiogenesis, Tumor Vascularization, and Potential Interference with Tumor Growth, in Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985)). Likewise, the Tumor Angiogenesis Factor (TAF) is released at a higher level in tumor cells than in its normal counterparts. See, for example, Folkman, Angiogenesis and Cancer, Sem. Cancer Biol. (1992)), while bFGF is released from endothelial tumors (Ensoli, B. et al.). Various techniques that measure the release of these factors are described in Freshney (1994), supra. See also Unkless et al., J. Biol. Chem. 249: 4295-4305 (1974); Strickland &amp; Beers, J. Biol. Chem. 251: 5694-5702 (1976); WHUR et al., Br. J. Cancer 42: 305-312 (1980); Gullino, Angiogenesis, Tumor Vascularization, and Potential Interference with Tumor Growth, in Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985); Freshney, Anticancer Res. 5: 111-130 (1985). For example, levels of tumor-specific markers are monitored in methods for identifying and characterizing compounds that modulate cancer associated sequences as disclosed herein.

Intrusion Capacity in Matrigel for Identifying and Characterizing Modulators The degree of invasiveness in Matrigel or in an extracellular matrix constituent can be used as an assay to identify and characterize compounds that modulate cancer associated sequences. Tumor cells exhibit a positive correlation between malignancy and cell invasiveness in Matrigel or any other constituent of the extracellular matrix. In this assay, tumorigenic cells are typically used as host cells. Expression of a tumor suppressor gene in these host cells would decrease the host cells' ability to invade. Techniques described in Cancer Res. 1999; 59: 6010; Freshney (1994), supra, may be used. Briefly, the level of host cell invasion is measured using filters coated with Matrigel or any other constituent of the extracellular matrix. Penetration on the gel, or even to the distal side of the filter, is classified as invasion capability and histologically classified by the number of cells and distance traveled, or by pre-labeling the 125 I cells and counting the radioactivity on the distal side of the filter or plate background. See, for example, Freshney (1984), supra.

Evaluation of In Vivo Tumor Growth to Identify and Characterize Modulators

The effects of cancer-associated sequences on cell growth are tested on transgenic or immunodepressed organisms. Transgenic organisms are prepared in a variety of ways accepted in the art. For example, knock-out transgenic organisms, for example mammals such as mice, are made, in which a cancer gene is disrupted or in which a cancer gene is inserted. Knock-out transgenic mice are made by inserting a marker gene or other heterologous gene into the site of the endogenous cancer gene in the mouse genome through homologous recombination. Such mice can also be produced by replacing the endogenous cancer gene with a mutated version of the cancer gene, or by mutating the endogenous cancer gene, for example, by exposure to carcinogens.

To prepare transgenic chimeric animals, for example mice, a DNA construct is introduced into the nucleus of the embryonic stem cells. Cells containing the newly manipulated genetic lesion are injected into a host mouse embryo, which is reimplanted into a recipient female. Some of these embryos are grown in chimeric mice that have germ cells, some of which are derived from the mutant cell line. Therefore, by reproduction of the chimeric mice it is possible to obtain a new line of mice containing the introduced genetic lesion (see, for example, Capecchi et al., Science 244: 1288 (1989)). Chimeric mice may be derived according to U.S. Patent 6,365,797, issued April 2, 2002; U.S. Patent 6,107,540 issued August 22, 2000; Hogan et al., Manipulating the Mouse Embryo: A laboratory

Manual, Cold Spring Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem Cells: A Practical

Approach, Robertson, ed., IRL Press, Washington, D.C., (1987).

Alternatively, several immunosuppressed or immunodeficient host animals may be used. For example, a mouse &quot; naked &quot; (see, for example, Giovanella et al., J. Natl Cancer Inst 52: 921 (1974)), a SCID mouse, a thymectomized mouse, or an irradiated mouse (see, for example, Bradley et al. al., Br. J. Cancer 38: 263 (1978); Selby et al., Br. J. Cancer 41:52 (1980)) can be used as a host. Transplantable tumor cells (typically about 106 cells) injected into isogenic hosts produce invasive tumors in a high proportion of cases, while normal cells of similar origin do not. In hosts that have developed invasive tumors, cells expressing the cancer associated sequences are injected subcutaneously or orthopedically. The mice are then separated into groups, including control groups and treated experimental groups (for example, treated with a modulator). After a suitable period of time, preferably 4-8 weeks, tumor growth is measured (for example, by volume or by its two largest dimensions, or by weight) and compared to the control. Tumors that have a statistically significant reduction (using, for example, Student's t-test) are considered to have inhibited growth.

In Vitro Assays to Identify and Characterize Modulators

Assays for identifying compounds with modulating activity can be performed in vitro. For example, a cancer polypeptide is first contacted with a potential modulator and incubated for a suitable period of time, for example, from 0.5 to 48 hours. In one example, cancer polypeptide levels are determined in vitro by measuring the level of protein or mRNA. The level of protein is measured using immunoassays, such as Western blotting, ELISA and the like with an antibody that selectively binds to the cancer polypeptide or a fragment thereof. For the measurement of mRNA, amplification, for example using PCR, CSF or hybridization assays, for example, Northern blotting, RNase protection, dot blotting, are preferred. The level of protein or mRNA is detected using directly or indirectly labeled detection agents, for example, fluorescently or radioactively labeled nucleic acids, radioactively or enzymatically labeled antibodies, and the like, as described herein.

Alternatively, a reporter gene system may be designed using a cancer protein promoter operably linked to a reporter gene, such as luciferase, green fluorescent protein, CAT, or P-gal. The reporter construct is typically transfected into a cell. After treatment with a potential modulator, the amount of transcription, translation or activity of the reporter gene is measured according to standard techniques known to those skilled in the art (Davis GF, supra; Gonzalez, J. & Negulescu, P. Curr. Opin Biotechnol 1998: 9: 624).

As described above, in vitro screenings are performed on individual genes and gene products. That is, upon identification of a particular gene differentially expressed as important in a particular state, screening for modulators of gene expression or the gene product itself is performed. Screening of modulators of specific gene (s) expression can be performed. Typically, the expression of only one or a few genes is evaluated. The screenings may be designed to, first, find compounds that bind to differentially expressed proteins. These compounds are then evaluated for the ability to modulate differentially expressed activity. In addition, once the initial candidate compounds are identified, the variants can be further screened to better evaluate the activity ratios of the framework.

Binding Assays to Identify and Characterize Modulators

In binding assays, according to the disclosures described herein, an isolated or purified gene product of the invention is generally used. For example, antibodies are generated for a protein of the invention, and immunoassays are performed to determine the amount and / or location of the protein. Alternatively, cells comprising the cancer proteins are used in the assays.

Thus, the methods comprise combining a cancer protein as disclosed herein and a candidate compound as a ligand, and determining the binding of the compound to the cancer protein of the invention. Some examples utilize the human cancer protein; animal models of human disease can also be developed and used. In addition, other analogous mammalian proteins may also be used as appreciated by those skilled in the art. In addition, in some embodiments variant or derived cancer proteins are used.

Generally, the cancer protein as disclosed herein, or the ligand, is non-diffusibly bound to an insoluble support. The carrier may, for example, be one having isolated sample receiving areas (a microtiter plate, a matrix, etc.). The insoluble supports may be made from any composition to which the compositions may be attached, which is readily separated from the soluble material, and is otherwise compatible with the general screening method. The surface of such supports may be solid or porous and in any convenient way.

Examples of suitable insoluble supports include microtiter plates, matrices, membranes and beads. These are typically made from glass, plastic (e.g., polystyrene), polysaccharides, nylon, nitrocellulose or Teflon ™, etc. Microtiter plates and matrices are especially convenient because a large number of assays can be performed simultaneously using small amounts of reagents and samples. The particular manner of binding of the composition to the support is not crucial, as long as it is compatible with the reactants and the general methods described herein, maintains the activity of the composition and is non-diffusible. Preferred methods of binding include the use of antibodies that do not sterically block either the ligand binding site or the activation sequence upon attachment of the protein to the support, direct binding to &quot; cohesive &quot; or ionic, chemical crosslinking, protein or surface synthesis, etc. After binding of the protein or ligand / carrier to the carrier, excess unbonded material is removed by washing. The sample receiving areas can then be blocked by incubation with bovine serum albumin (BSA), casein or other innocuous protein or other fraction.

Once a cancer protein is attached to the support and a test compound is added to the assay. Alternatively, the candidate binding agent is attached to the support and the cancer protein of the invention is then added. Binding agents include specific antibodies, unnatural binding agents identified in screenings of chemical libraries, peptide analogues, etc.

Of particular interest are assays to identify agents that have a low toxicity to human cells. A wide variety of assays may be used for this purpose, including proliferation assays, cAMP assays, in vitro labeled protein-protein binding assays, electrophoretic mobility shift assays, protein binding immunoassays, functional assays phosphorylation, etc.) and the like.

A determination of the binding of the test compound (ligand, binding agent, modulator, etc.) to a cancer protein of the invention can be made in a number of ways. The test compound may be labeled and the binding determined directly, for example, by binding all or a portion of the cancer protein of the invention to a solid support, addition of a labeled candidate compound (e.g., a fluorescent label) , washing off excess reagent, and determining whether the label is present on the solid support. Various blocking and washing steps may be used, as appropriate.

In certain embodiments, only one of the components is labeled, for example, a protein of the invention or labeled ligands. Alternatively, more than one component is labeled with different labels, for example I 125, for proteins and a fluorophore for the compound. Proximity reagents, for example, quenching or energy transfer reagents are also useful.

Competitive Link to Identify and Characterize Modulators

In one embodiment, the binding of the &quot; test compound &quot; is determined by the competitive binding assay with a &quot; competitor &quot;. The competitor is a binding moiety that binds to the target molecule (e.g., a cancer protein of the invention). Competitors include compounds, such as antibodies, peptides, binding partners, ligands, etc. Under certain circumstances, the competitive binding between the test compound and the competitor displaces the test compound. In one embodiment, the test compound is labeled. Either the test compound, the competitor, or both, is added to the protein for a period of time sufficient to allow binding. Incubations are performed at a temperature which facilitates optimal activity, typically between four and 40 ° C. Incubation periods are typically optimized, for example, to facilitate rapid high throughput screening; typically between zero and one hour will suffice. Excess reagent is generally removed or washed. The second component is then added and the presence or absence of the labeled component is followed to indicate binding.

In one method, the competitor is first added, followed by the test compound. Competitor shift is an indication that the test compound is binding to the cancer protein and is therefore capable of binding to, and potentially modulating, the activity of the cancer protein. In this embodiment, any component can be labeled. Thus, for example, if the competitor is labeled, the presence of label in the wash solution of the post-test compound indicates displacement by the test compound. Alternatively, if the test compound is labeled, the presence of the label on the carrier indicates displacement.

In an alternative method, the test compound is added first, with incubation and washing, followed by the competitor. Lack of binding by the competitor indicates that the test compound binds to the cancer protein with higher affinity than the competitor. Thus, if the test compound is labeled, the presence of the label on the carrier, together with a lack of competitor binding, indicates that the test compound binds and thus potentially modulates the cancer protein of the invention.

Accordingly, competitive binding methods comprise differential screening to identify agents that are capable of modulating the activity of the cancer proteins of the invention. The methods may comprise the combination of a cancer protein and a competitor in a first sample. A second sample comprises a test compound, the cancer protein and a competitor. Competitor binding is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the cancer protein and potentially modulating its activity. That is, if the competitor binding is different in the second sample relative to the first sample, the agent is capable of binding to the cancer protein.

Alternatively, differential screening is used to identify drug candidates that bind to the native cancer protein, but which can not bind to modified cancer proteins. For example, the structure of the cancer protein is modeled and used in the rational design of drugs to synthesize agents that interact with that site, agents that do not generally bind to modified site proteins. In addition, such drug candidates that affect the activity of a native cancer protein are also identified through drug screening for the ability to potentiate or reduce the activity of such proteins.

Positive controls and negative controls may be used in the assays. Preferably, control and test samples are run in at least triplicate, to obtain statistically significant results. Incubation of all samples occurs for a period of time sufficient to allow binding of the agent to the protein. After incubation, samples are washed from non-specifically bound material and the amount of generally labeled bound agent is determined. For example, where a radioactive label is employed, the samples may be counted in a scintillation counter to determine the amount of bound compound.

A variety of other reagents may be included in the screening assays. These include reagents such as salts, neutral proteins, for example, albumin, detergents, etc. which are used to facilitate optimal protein-protein binding and / or to reduce nonspecific or background interactions. Reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc., may also be used. The mixture of components is added in an order which provides the necessary binding.

Use of Polynucleotides to Negatively Regulate or Inhibit a Protein as Disclosed in the Present Document.

The cancer polynucleotide modulators can be introduced into a cell containing the target nucleotide sequence by forming a conjugate with a ligand binding molecule as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines or other ligands that bind to cell surface receptors. Preferably, the conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block the entry of the sense or antisense oligonucleotide, or conjugate thereof, in the cell. Alternatively, a cancer polynucleotide modulator may be introduced into a cell containing the target nucleic acid sequence, for example, by forming a polynucleotide-lipid complex, as described in WO 90/10448. It is understood that the use of antisense or knock out and knock-in molecules in templates can also be used in screening assays as discussed above in addition to the methods of treatment.

Inhibitory and Antisense Nucleotides The activity of a cancer associated protein can be down-regulated, or totally inhibited, by the use of antisense polynucleotide or small nuclear RNA (snRNA) inhibitory, i.e., a nucleic acid complementary to and which may preferably hybridize specifically with a nucleic acid sequence of coding mRNA, for example, a cancer protein as disclosed herein, mRNA, or a subsequence thereof. Binding of the antisense polynucleotide to the mRNA reduces the translation and / or stability of the mRNA.

In the context of the present specification, the antisense polynucleotides may comprise naturally occurring nucleotides or synthetic species formed from naturally occurring subunits or their close homologues. The antisense polynucleotides may also have altered sugar fractions or inter-sugar bonds. Exemplary among these are phosphorothioate and other sulfur-containing species which are known for use in the art. Analogs are understood by the present invention as long as they function effectively to hybridize to nucleotides as disclosed herein. See, for example, Isis Pharmaceuticals, Carlsbad, CA; Sequitor, Inc., Natick, MA.

Such antisense polynucleotides may be readily synthesized using recombinant means, or may be synthesized in vitro. Equipment for such synthesis is sold by several suppliers, including Applied Biosystems. The preparation of other oligonucleotides, such as phosphorothioates and alkylated derivatives is also well known to those skilled in the art.

Antisense molecules as used herein include antisense or sense oligonucleotides. The sense oligonucleotides can, for example, be used to block transcription by binding to the antisense strand. Antisense and sense oligonucleotides comprise a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to mRNA (sense) or DNA (anti-sense) target sequences for cancer molecules. The antisense or sense oligonucleotides according to the present invention comprise a fragment generally of at least about 12 nucleotides, preferably of from about 12 to 30 nucleotides. The ability to derive an antisense or sense oligonucleotide based on a cDNA sequence encoding a given protein is described in, for example, Stein and Cohen (Cancer Res. 48: 2659 (1988) and van der Krol et al. (BioTechniques 6: 958 (1988)).

Ribozymes

In addition to antisense polynucleotides, the ribozymes may be used to direct and inhibit the transcription of nucleotide sequences associated with cancer. A ribozyme is an RNA molecule that catalytically cleaves other RNA molecules. Different types of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, ARNase P, and axehead ribozymes (see, for example, Castanotto et al., Adv. In Pharmacology 25: 289-317 ( 1994) for a general review of the properties of different ribozymes).

General characteristics of hairpin ribozymes are described, for example, in Hampel et al., Nucl. Acids Res. 18: 299-304 (1990); European Patent Publication No. 0360257; U.S. Patent No. 5,254,678. Methods of preparation are well known to those skilled in the art (see, for example, WO 94/26877; Ojwang et al., Proc Natl Acad Sci USA 90: 6340-6344 (1993); Yamada et al., Human Gene Therapy 1: 39-45 (1994) Leavitt et al., Proc Natl Acad Sci USA 92: 699-703 (1995) Leavitt et al., Human Gene Therapy 5: 1151- 120 (1994); and Yamada et al., Virology 205: 121-126 (1994)).

Use of Modulators in Phenotypic Screening

A test compound can be administered to a population of cancer cells, which have an associated cancer expression profile. By &quot; administration &quot; or &quot; contact &quot; it is understood herein that the modulator is added to the cells so as to allow the modulator to act on the cell, either by absorption and intracellular action, or by action on the cell surface. A nucleic acid encoding a proteinaceous agent (i.e., a peptide) may be placed in a viral construct such as an adenoviral or retroviral construct, and added to the cell such that expression of the peptidic agent is achieved, for example, document PCT US97 / 01019. Adjustable gene therapy systems can also be used. Once the modulator has been administered to the cells, the cells are washed if desired and allowed to incubate under preferably physiological conditions for a certain period. The cells are then harvested and a new gene expression profile is generated. Thus, for example, the cancer tissue is screened for agents that modulate, for example, induce or suppress the cancer phenotype. A change in at least one gene, preferably several, of the expression profile indicates that the agent has an effect on cancer activity. Likewise, alteration of a biological function or signaling pathway is indicative of modulatory activity. By defining such a signature for the cancer phenotype, screenings for novel drugs that alter the phenotype are designed. With this approach, the drug target does not have to be known and need not be represented on the original gene / protein expression screening platform, nor does the level of transcription for the target protein need to change. The inhibitory function of the modulator will serve as a substitute marker.

As outlined above, screenings are performed to evaluate genes or gene products. That is, after a particular gene differentially expressed as important in a particular state has been identified, the screening of modulators of gene expression or of the gene product itself is performed.

Use of Modulators to Affect Peptides

Measurements of cancer polypeptide activity, or cancer phenotype are performed using a variety of assays. For example, the effects of the modulators on the function of one or more cancer polypeptides are measured by analyzing the parameters described above. A physiological change affecting physiological activity is used to evaluate the influence of a test compound on the polypeptides as disclosed herein. When functional results are determined using intact or animal cells, a variety of effects can be evaluated, such as in the case of cancer associated with solid tumors, tumor growth, tumor metastasis, neovascularization, hormone release, transcription changes for both markers (e.g., Northern blots), changes in cellular metabolism, such as changes in cell growth or pH, and changes in intracellular second messengers such as cGNIP. Methods of Identification Characterizing Sequences Associated with Cancer Expression of different gene sequences is correlated with cancer. Accordingly, disorders based on mutant or variant cancer genes are determined. In one example, there are methods for identifying cells containing variant cancer genes, for example, by determining the presence of all or part of the sequence of at least one endogenous cancer gene in a cell. This is accomplished using any number of sequencing techniques. Methods of identifying the cancer genotype of an individual are disclosed herein, for example, by determining all or part of the sequence of at least one gene of the invention in the subject. This is generally done in at least one tissue of the subject, for example a tissue set forth in Table I, and may include evaluation of a number of different tissues or samples of the same tissue. The method may include comparing the sequenced gene sequence with a known cancer gene, i.e., a wild type gene for determining the presence of family members, homologies, mutations or variants. The sequence of all or part of the gene may then be compared to the sequence of a known cancer gene to determine if there are any differences. This is done using any number of known homology programs, such as BLAST, Bestfit, etc. The presence of a difference in sequence between the patient's cancer gene and the known cancer gene correlates with a disease state or a disease state propensity, as described herein.

The cancer genes can be used as probes to determine the number of copies of the cancer gene in the genome. The cancer genes are used as probes to determine the chromosomal location of the cancer genes. Such information as chromosomal location finds use in providing a diagnosis or prognosis, in particular, when chromosomal abnormalities such as translocations and the like are identified at the locus of the cancer gene. XIV.) IRNA and therapeutic use of small interfering RNAs (siRNAs)

Also disclosed herein are siRNA oligonucleotides, particularly double stranded RNAs encompassing at least one fragment of the PSCA coding region or 5 ', or complement, regions, or any antisense oligonucleotides specific for the PSCA sequence. Such oligonucleotides may be used to elicit a PSCA function, or may be used to screen or evaluate modulators of PSCA function or expression. In another method, the expression of the PSCA gene is reduced using siRNA transfection and results in a significantly decreased proliferative capacity of transformed cancer cells that endogenously express the antigen; cells treated with PSCA-specific siRNAs show reduced survival as measured, for example, by a metabolic readout of cell viability, correlating with reduced proliferative capacity. Thus, the PSCA siRNA compositions comprise siRNA (double stranded RNA) corresponding to the nucleic acid sequence ORF of the PSCA protein or subsequences thereof; these subsequences are generally 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35 or more than 35 contiguous RNA nucleotides in length and contain sequences which are complementary and not complementary to at least a portion of the mRNA coding sequence. Subsequences may be 19-25 nucleotides in length, more preferably 21-23 nucleotides in length. RNA interference is a novel approach to in vitro and in vivo gene silencing, so small double stranded RNAs (siRNAs) are valuable therapeutic agents. The power of siRNAs to silence specific gene activities has now been brought to animal models of disease and is also used in humans. For example, it has been shown that the hydrodynamic infusion of a solution of siRNA in a siRNA against a particular target is therapeutically effective. The pioneering work by Song et al. indicates that a type of fully natural nucleic acid, small interfering RNAs (siRNAs), served as therapeutic agents without further chemical modification (Song, E., et al. &quot; RNA interference targeting Fas protects mice from fulminant hepatitis &quot; Nat. Med. 9 (3): 347-51 (2003)). This work provided the first in vivo evidence that infusion of siRNA into an animal could alleviate the disease. In that case, the authors gave siRNA injections to mice designed to silence the FAS protein (a cell death receptor that, when overactivated during the inflammatory response induces the death of hepatocytes and other cells). The next day, the animals received an antibody specific for Fas. Control mice died of acute liver failure within a few days, while more than 80% of the siRNA-treated mice remained free of severe disease and survived. About 80% to 90% of their liver cells incorporated the naked siRNA oligonucleotides. In addition, the RNA molecules functioned for 10 days before losing their effect after 3 weeks.

For use in human therapy, siRNA is delivered by efficient systems that induce the long-term activity of iRNA. An important caveat for clinical use is to deliver siRNAs to the appropriate cells. The hepatocytes appear to be particularly receptive to exogenous RNA. Nowadays, targets located in the liver are attractive because the liver is an organ that can be readily established as the target of nucleic acid molecules and viral vectors. However, other target tissues and organs are also preferred.

Formulations of siRNAs with compounds that promote passage through cell membranes are used to improve administration of siRNA therapy. Synthetic and chemically modified siRNAs which are nucleoside resistant and have serum stability have the duration of the effects of improved iRNA are also disclosed herein.

Thus, siRNA technology is a therapy against human malignant disease by delivery of PSCA targeting siRNA molecules to individuals with cancers, such as those listed in Table 1. Such administration of siRNAs leads to decreased growth of cancerous cells express PSCA, and provides an antitumor therapy, decreasing the morbidity and / or mortality associated with disease malignancy. The effectiveness of this modality of gene product knockdown is significant when measured in vitro or in vivo. In vitro efficacy is readily demonstrable by application of siRNAs to cultured cells (as described above) or to biopsy aliquots of cancer patients when in vitro methods are used to detect reduced PSCA protein expression. XV.) Kits / Articles of Manufacture

For use in laboratory, prognostic, prophylactic, diagnostic and therapeutic applications described herein, kits are within the scope of disclosures. Such kits may comprise a carrier, package or container that is compartmentalized to receive one or more containers such as vials, tubes and the like, each container, comprising one of the separate elements to be used in the method, together with a label or package insert comprising instructions for use, such as a use described herein. For example, the container (s) may comprise a probe that is or can be detectably labeled. Such a probe may be an antibody or a polynucleotide specific to a protein or a gene or message of the invention, respectively. Where the method utilizes nucleic acid hybridization to detect the target nucleic acid, the kit may also have containers containing nucleotide (s) for amplification of the target nucleic acid sequence. The kits may comprise a container comprising a reporter, such as a biotin binding protein, such as avidin or streptavidin, attached to a reporter molecule, such as an enzymatic, fluorescent or radioisotope label; such a reporter may be used with, for example, a nucleic acid or antibody. The kit may include all or part of the amino acid sequences in Figure 1, Figure 2 or Figure 3 or analogues thereof, or a nucleic acid molecule encoding such amino acid sequences. The kit may comprise the above-described container and one or more other containers associated therewith which comprise commercially and user-desirable materials, including buffers, diluents, filters, needles, syringes; labels of conveyors, packaging, containers, vials and / or tubes listing contents and / or instructions for use, and leaflets with instructions for use.

A label may be present on or with the container to indicate that the composition is used for specific therapy or non-therapeutic application, such as prognostic, prophylactic, diagnostic or laboratory application and may also indicate guidelines for in vivo or in vitro, such as those described herein. Instructions or other information may also be included in one or more information leaflets or labels that are included on or with the kit. The label may be on or associated with the container. A label may be on a container when letters, numbers or other characters that form the label are molded or engraved on the container itself; a label may be associated with a container when it is present in a receptacle or conveyor which also carries the container, for example as an information booklet. The label may indicate that the composition is used for the diagnosis, treatment, prophylaxis or prognosis of a condition, such as a tissue neoplasm set forth in Table I.

The terms &quot; kit &quot; and &quot; article of manufacture &quot; can be used as synonyms.

In another example disclosed herein, one or more articles of manufacture containing compositions, such as amino acid sequence (s), small molecule (s), nucleic acid sequence (s) and / or antibody (s), for example, materials useful for the diagnosis, prognosis, prophylaxis and / or treatment of tissue neoplasms, such as those set out in Table I are provided. The article of manufacture typically comprises at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes and test tubes. The containers may be formed from a variety of materials such as glass, metal or plastic. The container may contain amino acid sequence (s), small molecule (s), nucleic acid sequence (s), cell population (s) and / or antibody (s). In one embodiment, the container contains a polynucleotide for use in analyzing the mRNA expression profile of a cell, together with reagents used for this purpose. A container may comprise an antibody, binding fragment thereof, or specific binding protein for use in evaluating the expression of the PSCA protein in cells and tissues, or for relevant laboratory, prognostic, diagnostic, prophylactic and therapeutic purposes; indications and / or orientations for such uses may be included on or with such a container, such as reagents and other compositions or tools used for such purposes. A container may comprise materials for eliciting a cellular or humoral immune response, together with indications and / or associated orientations. A container may comprise materials for adoptive immunotherapy, such as cytotoxic T cells (CTLs) or helper T cells (HTL), together with indications and / or associated orientations; reagents and other compositions or tools used for such purpose may also be included. The container may alternatively contain a composition which is effective for the treatment, diagnosis, prognosis or prophylaxis of a condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper punctured by a hypodermic injection needle). The active agents in the composition may be an antibody capable of specifically binding to PSCA and modulating the CA PS function. The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution and / or dextrose solution. It may also include other desirable materials from a commercial and user's point of view, including other tampons, diluents, filters, needles, agitators, syringes, and / or information leaflets with indications and / or instructions for use. EXAMPLES: Various aspects of the invention are further described and illustrated by the following various examples, none of which are intended to limit the scope of the invention.

Example 1

Expression Analysis of PSCA Variants in Tissues

Norms and Patient Specimens

Previously, the PSCA, referred to herein as PSCA v.l, has been identified as an antigen expressed in prostate cancer. Its expression has been detected in more than 80% of primary prostate cancers and in most prostate metastases.

It has also been shown to be expressed in bladder cancer, ovarian cancer, and pancreatic cancer; these cancers are listed in Table I. Through immunohistochemical analysis, PSCA has been shown to be overexpressed on the cell surface of most transitional urothelial carcinomas and in 60% of primary pancreatic adenocarcinomas. Foral PSCA expression data reported in patent publications (PCT / US98 / 04664, PCT / US / 28883, PCT / US00 / 19967) and peer reviewed scientific articles (Saffran et al., Proc Natl Acad Sci USA. 27 Feb 2001; 98 (5): 2658-2663; Amara et al., Cancer Res. 15 Jun 2001; 61 (12): 4660-65; Reiter et al., Proc Natl Acad Sci USA 17 Feb 1998; 95 (4): 1735-40; Argani et al., Cancer Res., 1 Jun 2001; 61 (11): 4320-24). Specific expression of different variants of PSCA is studied in specimens from normal and cancer patients. The primers were designed to differentiate between PSCA v.l / v.2 / v.4, PSCA v.3 and PSCA v.5. PSCA v1 / v.2 / v.4 leads to a 425 bp PCR product, PSCA v.3 leads to a 300 bp PCR product, while PSCA v.5 leads to a 910 bp PCR product of size (Figure II (a)) First-strand cDNA was prepared from the bladder, brain, heart, kidney, liver, lung, prostate, spleen, skeletal muscle, testis, pancreas, colon, normal stomach, prostate cancer clusters, bladder cancer, renal cancer, colon cancer, lung cancer, ovarian cancer, breast cancer, cancer metastases, and pancreatic cancer (Figure II (b).) Normalization was performed by PCR using actin primers, using the specific primers of variants was performed in 30 cycles of amplification.

The results show expression of PSCA v.5 primarily in mammary tissue, cancer metastasis, and pancreatic cancer, and to a lower level in colon cancer and lung cancer. The PSCA v1 / v.2 / v.4 PCR product has been detected in prostate cancer, bladder cancer, renal cancer, colon cancer, lung cancer, ovarian cancer, breast cancer, cancer metastases, and cancer of the prostate. pancreas. Among normal tissues, the PSCA v.l / v.2 / v.4 PCR product was detected only in the prostate, stomach and to a lesser extent in the kidney and lung, while PSCA v.5 was not detected in any normal tissue. The PCR product of PSCA v.3 was not detected in any of the samples tested.

Initiators were designed to differentiate between PSCA v.4 and PSCA v.5 (Figure 1J (a). PSCA v.4 led to a PCR product of 460 bp while PSCA v.5 led to a PCR product of 945 bp of first-strand cDNA was prepared from the bladder, brain, heart, kidney, liver, lung, prostate, spleen, skeletal muscle, testis, pancreas, colon, normal stomach, prostate cancer clusters, bladder cancer , and multi-xenograft cluster (prostate cancer xenografts, renal cancer, and bladder cancer) (Figure 1J (b).) Normalization was performed by PCR using actin primers, semi-quantitative PCR using the specific primers of variants was performed in 30 cycles of amplification.

The results show expression of PSCA v.4 in prostate cancer, bladder cancer, and multi-xenograft, kidney and normal prostate clustering. PSCA v.5 was detected only in normal prostate and bladder cancer. The restricted expression of PSCA variants in normal tissues and the expression detected in specimens of cancer patients indicates that PSCA variants are therapeutic, prognostic, laboratory, prophylactic, and diagnostic targets for human cancers.

Example 2

PSCA Splicing Variants

As used herein, the term variant includes transcript variants and single nucleotide polymorphisms (SNPs). Transcript variants are variants of mature mRNA from the same gene which arise by alternative transcription or alternative splicing. Alternative transcripts are transcribed from the same gene but initiate transcription at different points. Splicing variants are variants of mRNA spliced differently from the same transcript. In eukaryotes, when a multi-exon gene is transcribed from genomic DNA, the initial RNA is subjected to splicing to produce functional mRNA, which has only exons and is used for translation into an amino acid sequence. Accordingly, a given gene may have zero to several alternative transcripts and each transcript may have zero to several splice variants. Each transcript variant has a unique exon constitution, and may have different coding and / or noncoding portions (5 'or 3' ends) from the original transcript. Transcript variants may encode the same proteins, or similar or different proteins, such proteins having the same or similar function or a different function. The variant proteins may be expressed in the same tissue at the same time, in a different tissue at the same time, or in the same tissue at different times, or in a different tissue at a different time. Proteins encoded by a transcript variant may have similar or different subcellular or extracellular (e.g., secreted versus intracellular) locations.

Transcript variants are identified by a variety of methods accepted in the art. For example, alternative transcripts and splicing variants are identified through full-length cloning, or through the use of full length transcript and EST sequences. First, all human ESTs were grouped into clusters that show a direct or indirect identity between them. Second, ESTs from the same cluster were further pooled into sub-clusters and assembled into a consensus sequence. The original gene sequence is compared to the consensus sequence (s) or other full length sequences. Each consensus sequence is a potential splicing variant for that gene. Various confirmatory methods are known in the art, such as identification of the variant through Northern analysis, full length cloning or through the use of probe libraries, etc. Even when a variant is identified that is not yet a clone of length In addition, that portion of the variant is very useful as a research tool, for example, for the generation of antigens or for further cloning of the full length splicing variant, using techniques known in the art.

In addition, computer programs that identify variants of transcripts or genomic sequences are available in the art. Programs for the identification of variants of genomic-based transcripts include FgenesH (A. Salamov and V. Solovyev, &quot; Ab initio gene finding in Drosophila genomic DNA, &quot; Genome Research, April 2000; 10 (4): 516-22 ); Grail (URL comp-bio.ornl.gov/Grail-bin/EmptyGrailForm) and GenScan (URL genes.mit.edu/GENSCAN.html). For a general discussion of protocols for identification of splicing variants see, for example, Southan, C., A genomic perspective on human proteases, FEBS Lett. 8 Jun 2001; 498 (2-3): 214-8; de Souza, S.J., et al. , Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags, Proc. Natl Acad Sci USA. 7 nov 2000; 97 (23): 12690-3.

To further confirm the parameters of a transcript variant, a variety of techniques are available in the art, such as full-length cloning, proteomic validation, PCR-based validation, and 5 'RACE validation, etc. (see, for example, Proteomic validation: Brennan, SO, et al., Albumin banks peninsula: a new termination variant characterized by electrospray mass spectrometry, Biochem Biophys Acta 17 Aug 1999; 1433 (1-2): 321- 6, Ferranti P, et al., Differential splicing of pre-messenger RNA produces multiple forms of mature caprine alpha (si) -casein, Eur J Biochem 1 Oct 1997; 249 (1): 1-7. in PCR: Wellmann S, et al., Specific reverse transcription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCliner technology, Clin Chem, Apr. 2001; 47 (4): 654-60; For validation based on PCR and validation of 5'RACE: Brigle, KE, et al., "Discovery of new human beta-defensins using a genomics-based approach, Gene 24 Jan 2001; 263 (1-2): 211- et al., Organization of the Murine reduced folate carrier gene and identification of variant splice forms, Biochem Biophys Acta, 7 Aug 1997; 1353 (2): 191-8). It is known in the art that genomic regions are modulated in cancer. When the genomic region to which a gene is mapped is modulated in a particular cancer, alternative transcripts or splicing variants of the gene are also modulated. It is disclosed herein that PSCA has a particular expression profile related to cancer (see, for example, Table I). Alternative splicing transcripts and variants of PSCA are also involved in the cancers, for example, in one or more of these tissues and also in additional tissues. The variants thus serve as tumor associated markers / antigens.

Using the full-length PSCA gene along with EST sequences, four additional transcript variants were identified, designated as PSCA v.2, v.3, v.4 and v.5. The boundaries of the exons in the original transcript, PSCA v. 1 were set forth in Table VI. Sequences for PSCA and variants of PSCA are set forth in Figure 1.

Example 3

PSCA Single Nucleotide Polymorphisms

A Single Nucleotide Polymorphism (SNP) is a variation of a single base pair on a nucleotide sequence at a specific location. At any given point in the genome, there are four possible nucleotide base pairs: A / T, C / G, G / C, and T / A. As used herein, an allele is one of a number of alternate forms of a given gene, differing in the DNA sequence, and affecting a product (RNA and / or protein).

A SNP occurring in a cDNA is called a cSNP. This cSNP can alter amino acids of the protein encoded by the gene and thereby alter the function of the protein. Some SNPs cause hereditary diseases; others contribute to quantitative variations in the phenotype and reactions to environmental factors including diet and drugs among individuals. Therefore, the existence of a SNP and / or combinations of alleles (called haplotypes) has many useful applications, such as diagnosis of hereditary diseases, determination of reactions and dosages of drugs, identification of genes responsible for diseases, and analysis of the genetic relationship between individuals (P. Nowotny, JM Kwon and AM Goate, &quot; SNP analysis to dissect human traits, &quot; Curr. Opin. Neurobiol., 2001, 11 (5): 637-641; M. Pirmohamed and BK

Park, &quot; Genetic susceptibility to adverse drug reactions, &quot; Trends Pharmacol. Sci. Jun 2001; 22 (6): 298-305; J. H.

Riley, C.J. Allan, E. Lai and A. Roses, &quot; The use of single nucleotide polymorphisms in the isolation of common disease genes, &quot; Pharmacogenomics. Feb 2000; 1 (1): 39-47; R. Judson, J.C. Stephens and A. Windemuth, &quot; The predictive power of haplotypes in clinical response, &quot; Pharmacogenomics. Feb 2000; 1 (1): 15-26).

SNPs are identified by a variety of methods accepted in the art (P. Bean, &quot; The promising voyage of SNP target discovery, &quot; Am. Clin. Lab. Oct-Nov 2001; 20 (9): 18-20; KM Weiss, &quot; In search of human variation, &quot; Genome Res. Jul 1998; 8 (7): 691-697; Μ M. She, &quot; Enabling large-scale pharmacogenetic studies by high throughput mutation detection and genotyping technologies , Clin Chem Chem., 2001, 47 (2): 164-172). For example, SNPs are identified by the sequencing of DNA fragments that exhibit polymorphism through gel-based methods such as restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). They are also discovered by direct sequencing of clustered DNA samples from different individuals or by comparing sequences from different DNA samples. With the rapid accumulation of sequence data in public and private databases, SNPs are also discovered by comparing sequences using computer programs (Z. Gu, L. Hillier and PY Kwok, &quot; Single nucleotide polymorphism hunting in cyberspace, &quot; Hum. Mutat, 1998; 12 (4): 221-225). SNPs can be verified and the genotype or haplotype of an individual can be determined by a variety of methods including direct sequencing and high throughput microarrays (PY Kwok, &quot; Methods for genotyping single nucleotide polymorphisms, &quot; Annu Rev. Genomics J. Kokoris, K. Dix, K. Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines and A. Duesterhoeft, &quot; High throughput SNP genotyping with the Masscode system, &quot; Mol Diagn. Dec 2000, 5 (4): 329-340).

Using the methods described above, thirteen SNPs were identified in the transcript for PSCA v.2. Variant 2 was used, instead of for example variant 1, since it had less ambiguous bases than variant 1. Consequently, SNPs were identified for PSCA v.2 at positions 57 (t / c), 367 (c / t), 424 (w / w), 495 (w / w), 499 (w / w), 563 (w / w), 567 t / g), 835 (g / a), 847 (g / a), 878 (g / a), and 978 (c / g). Transcripts or proteins with alternative alleles were designated as variant of PSCA v.6 to v.18, as shown in Figure 1B and Figure 1G. The nucleotide change in v.6 altered the v.l initiation codon and therefore, the translation would not start until the next ATG (AUG in mRNA), resulting in a shorter 9A protein than the v.l protein. The nucleotide changes for v.7 and v.8 were protein-quiet.

Twelve of these 13 SNPs were also present in variant 4. The 12 variants of SNPs related to PSCA v.4 are designated PSCA v. 19 to v.30. Variants 19 to 27 encode alternative amino acids as shown in Figure 1H.

Reference Example 4

Production of Recombinant PSCA in Prokaryotic Systems

To express PSCA and recombinant PSCA variants in prokaryotic cells, PSCA cDNA or full length or partial PSCA variant sequences are cloned into any of a variety of expression vectors known in the art. One or more of the following regions of the PSCA variants are expressed: the full length sequence shown in Figure 1, or any of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from PSCA, variants, or analogs thereof. A. In vitro transcription and translation constructs: pCRII: To generate PSCA sense and antisense RNA probes for in situ RNA investigations, pCRII constructs (Invitrogen, Carlsbad CA) are generated by encoding all or all of the PSCA cDNA . The pCRII vector has the Sp6 and T7 promoters flanking the insert to conduct the transcription of PSCA RNA for use as probes in in situ RNA hybridization experiments. These probes are used to analyze PSCA expression of cells and tissues at the RNA level. Transcribed PSCA RNA representing the cDNA amino acid coding region of the PSCA gene is used in in vitro translation systems such as the TnT ™ Coupled Reticulolysate System (Promega, Corp., Madison, WI) to synthesize PSCA protein. B. Bacterial Constructions:

PGEX constructs: To generate recombinant PSCA proteins in bacteria that are fused to the glutathione S-transferase (GST) protein, all or parts of the PSCA cDNA protein coding sequence are cloned into the pGEX family of fusion vectors with GST (Amersham Pharmacia Biotech, Piscataway, NJ). These constructs allow controlled expression of the amino terminal fused GST recombinant PSCA protein sequences and an epitope of six histidines (6X His) at the carboxyl terminus. The GST and 6X His tags allow purification of the recombinant fusion protein from bacteria induced with the appropriate affinity matrix and allow recognition of the fusion protein with anti-GST and anti-His antibodies. The 6X His tag is generated by adding 6 histidine codons to the cloning primer at the 3 'end, for example, from the open reading frame (ORF). A proteolytic cleavage site, such as the PreScission ™ recognition site in pGEX-6P-1, may be employed so as to permit cleavage of the GST tag from the PSCA-related protein. The ampicillin resistance gene and pBR322 origin allow the selection and maintenance of pGEX plasmids in E. coli.

Constructions of pMAL; To generate, in bacteria, recombinant PSCA proteins that are fused to maltose binding protein (MBP), all or portions of the coding sequence of the PSCA cDNA protein are fused to the MBP gene by cloning into the vectors pMAL- c2X and pMAL-p2X (New England Biolabs, Beverly, MA). These constructs allow controlled expression of recombinant PSCA protein sequences with MBP fused at the amino terminus and a 6X His epitope at the carboxyl terminus. The MBP and 6X His tags allow purification of the recombinant protein from bacteria induced with the appropriate affinity matrix and allow recognition of the fusion protein with anti-MBP and anti-His antibodies. The 6X His epitope is generated by the addition of 6 histidine codons to the 3 'cloning primer. A Factor Xa recognition site allows cleavage of the label pMAL from PSCA. The pMAL-c2X and pMAL-p2X vectors are optimized to express the recombinant protein in the cytoplasm or periplasm respectively. The expression in the periplasm potentiates the folding of the proteins with disulfide bridges.

PET Constructs: To express PSCA in bacterial cells, all or parts of the PSCA cDNA protein coding sequence are cloned into the vector pET family (Novagen, Madison, WI). These vectors allow tightly controlled expression of PSCA protein in bacteria with and without solubility-enhancing fusion proteins, such as NusA and thioredoxin (Trx), and epitope tags, such as 6X His and S-Tag ™, which aid in purification and detection of the recombinant protein. For example, constructs are made using the 43.1 pET NusA fusion system so that regions of the PSCA protein are expressed as amino terminal fusions with NusA. C. Yeast Constructions:

PESC constructs: To express PSCA in the Saccharomyces cerevisiae yeast species for recombinant protein generation and functional studies, all or parts of the PSCA cDNA protein coding sequence are cloned into the pESC family of vectors each of which contains 1 to 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 cloned genes or sequences containing the Flag ™ or Myc epitope tags in the same yeast cell. This system is useful for confirming protein-protein interactions of PSCA. Additionally, expression in the yeast produces similar post-translational modifications, such as glycosylations and phosphorylations, which are found when expressed in eukaryotic cells.

Constructs of pESP: To express PSCA in the yeast species Saccharomyces pombe, all or parts of the PSCA cDNA protein coding sequence are cloned into the pESP family of vectors. These vectors allow a high level of controlled expression of a PSCA protein sequence that is fused at the amino or carboxy terminus to GST which aids in the purification of the recombinant protein. A Flag ™ epitope tag allows detection of the recombinant protein with an anti-Flag ™ antibody.

Reference Example 5

Production of Recombinant PSCA in Superior Eukaryotic Systems A. Mammalian Constructions:

To express PSCA in eukaryotic cells, full length or partial PSCA cDNA sequences, or variants thereof, may be cloned into any of a variety of expression vectors known in the art. One or more of the following regions of PSCA are expressed in these constructs, amino acids 1 to 123, or any of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids from PSCA v 1, PSCA variants, or analogues thereof.

The constructs may be transfected into any of a variety of mammalian cells such as 293T cells. Transfected 293T cell lysates can be probed with the polyclonal anti-PSCA serum described herein.

PcDNA4 / HisMax constructs: To express PSCA in mammalian cells, a PSCA ORF, or portions thereof, of PSCA are cloned into pcDNA4 / HisMax Version A (Invitrogen, Carlsbad, CA). Protein expression is conducted from the cytomegalovirus (CMV) promoter and the SP16 translation enhancer. The recombinant protein has the epitopes of Xpress ™ and six histidines (6X His) fused to the amino terminus. The pcDNA4 / HisMax vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance the stability of the mRNA together with the SV40 origin for episomal replication and single vector rescue in cell lines expressing the T large antigen. The Zeocin resistance gene allows the selection of mammalian cells expressing the protein and the ampicillin resistance gene and the ColEl origin allow the selection and maintenance of the plasmid in E. coli.

PcDNA3.1 / MycHis constructs: To express PSCA in mammalian cells, a PSCA ORF, or portions thereof, of PSCA with a consensus Kozak translation initiation site was cloned into pcDNA3.1 / MycHis Version A ( Invitrogen, Carlsbad, CA). Protein expression is conducted from the cytomegalovirus (CMV) promoter. Recombinant proteins have the epitope myc and 6X His epitope fused to the carboxyl terminus. The pcDNA3.1 / MycHis vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability, along with SV40 origin for episomal replication and single vector rescue in cell lines which express the large T antigen. The neomycin resistance gene can be used since it allows the selection of mammalian cells expressing the protein and the ampicillin resistance gene and the ColEl origin allow the selection and maintenance of the plasmid in E. coli.

Constructs of pcDNA3.1 / CT-GFP-TOPO: To express PSCA in mammalian cells and to allow detection of recombinant proteins using fluorescence, a PSCA ORF, or portions thereof, with a Kozak translation initiation site of pcDNA3.1 / CT-GFP-TOPO (Invitrogen, CA) are cloned into consensus. Protein expression is conducted from the cytomegalovirus (CMV) promoter. Recombinant proteins have Green Fluorescent Protein (GFP) fused at the carboxyl terminus facilitating noninvasive detection in vivo and cell biology studies. The vector pcDNA3.1CT-GFP-T0P0 also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with SV40 origin for episomal replication and single vector rescue in lines cells expressing the large T antigen. The neomycin resistance gene allows the selection of mammalian cells expressing the protein, and the ampicillin resistance gene and ColEl origin allow the selection and maintenance of the plasmid in E. coli. Additional constructs with an amino terminal GFP fusion are made of pcDNA3.1 / NT-GFP-T0P0 spanning the full length of a PSCA protein. PAPtag: A PSCA ORF, or portions thereof, is cloned into pAPtag-5 (GenHunter Corp. Nashville, TN). This construct generates an alkaline phosphatase fusion at the carboxyl terminus of a PSCA protein while fusing the IgGx signal sequence with the amino terminus. Constructs are also generated in which alkaline phosphatase with an amino terminal IgGx signal sequence is fused to the amino terminus of a PSCA protein. The resulting recombinant PSCA proteins are optimized for secretion into the medium of transfected mammalian cells and can be used to identify proteins such as ligands or receptors that interact with PSCA proteins. Protein expression is conducted from the CMV promoter and the recombinant proteins also contain myc and 6X His epitopes fused at the carboxyl terminus which facilitate detection and purification. The Zeocin resistance gene present in the vector allows the selection of mammalian cells expressing the recombinant protein and the ampicillin resistance gene allows selection of the plasmid in E. coli. ptagon; A PSCA ORF, or portions thereof, were cloned into pTag-5. This vector is similar to pAPtag but without alkaline phosphatase fusion. This construct generates a PSCA protein with an amino terminal IgGx signal sequence and epitope tags myc and 6X His at the carboxyl terminus that facilitate the detection and affinity purification. The resulting recombinant PSCA protein is optimized for secretion into the medium of transfected mammalian cells, and is used as an immunogen or ligand to identify proteins such as ligands and receptors that interact with the PSCA proteins. Protein expression is conducted from the CMV promoter. The Zeocin resistance gene present in the vector allows the selection of mammalian cells expressing the protein, and the ampicillin resistance gene allows selection of the plasmid in E. coli.

PsecFc: A PSCA ORF, or portions thereof, were cloned into psecFc. The psecFc vector was assembled by cloning the human immunoglobulin G1 (IgG) Fc (hinge regions, CH2, CH3) into pSecTag2 (Invitrogen, California). This construct generates an Fc fusion of IgG1 at the carboxyl terminus of the PSCA proteins, while fusing the IgGK signal sequence as the N-terminus. PSCA fusions using the murine IgG Fc region are also used. The resulting recombinant PSCA proteins are optimized for secretion into the medium of transfected mammalian cells, and can be used as immunogens or to identify proteins such as ligands and receptors that interact with the PSCA protein. Protein expression is conducted from the CMV promoter. The hygromycin resistance gene present in the vector allows the selection of mammalian cells expressing the recombinant protein, and the ampicillin resistance gene allows selection of the plasmid in E. coli. Figure 8 shows the expression and purification of PSCA.psecFc protein from 293T cells.

Constructs of pSRa: To generate mammalian cell lines expressing PSCA constitutively, a PSCA ORF, or portions thereof, of PSCA were cloned into pSRa constructs. Amphotropic and ecotropic retroviruses were generated by transfection of pSRa constructs into the 293T-10A1 packaging cell line or co-transfection of pSRα and an auxiliary plasmid (containing cleavage sequences deleted) into 293 cells, respectively. The retrovirus is used to infect a variety of mammalian cell lines, resulting in the integration of the cloned gene, PSCA, into host cell lines. Protein expression is conducted from a long terminal repeat (LTR). The neomycin resistance gene present in the vector allows the selection of mammalian cells expressing the protein, and the ampicillin resistance gene and ColEl origin allow selection and maintenance of the plasmid in E. coli. Retroviral vectors can subsequently be used for infection and generation of several cell lines using, for example, PC3, NIH 3T3, TsuPrl, 293 or rat-1 cells. Figure 6 shows the expression of PSCA in murine, rat and human recombinant cell lines using the PSCA.pSRa construct. The indicated murine, rat, and human cell lines were infected with retroviruses carrying the human PSCA cDNA and a neomycin resistance gene or with only the neomycin resistance gene. Stable recombinant cell lines were created by selection of G418 drugs. PSCA expression was determined by staining FACS with the anti-PSCA mAb 1G8 (5æg / ml). The FACS profile is shown in each cell line showing a fluorescent deviation in the PSCA-infected line indicating PSCA expression on the cell surface. These lines are useful in the development of MAb as immunogens, MAb screening reagents, and in functional assays. Additional pSRa constructs are made that fuse an epitope tag such as the FLAG ™ tag to the carboxyl terminus of PSCA sequences to allow detection using anti-Flag antibodies. For example, the FLAG ™ 5'gat tac aag gat gac gac aag 3 '(SEQ ID NO: 76) sequence is added to the cloning primer at the 3' end of the ORF. Additional pSRa constructs are made to produce GFP and myc / 6X His fusion proteins at the amino and carboxy terminus of the full length PSCA proteins.

Additional Viral Vectors: Additional constructs for virus-mediated PSCA delivery and expression are made. A high virus titer leading to a high level of PSCA expression is achieved in virus delivery systems such as adenoviral vectors and herpes amplicon vectors. A PSCA coding sequence or fragments thereof are amplified by PCR and subcloned into the AdEasy carrier vector (Stratagene). Recombination and viral packaging are performed according to the manufacturer's instructions for generating adenoviral vectors. Alternatively, the PSCA coding sequences or fragments thereof are cloned into the HSV-1 vector (Imgenex) to generate herpes viral vectors. Viral vectors are subsequently used for infection of various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

Regulated Expression Systems: To control the

expression of PSCA in mammalian cells, PSCA coding sequences, or portions thereof, are cloned into regulated mammalian expression systems such as the T-Rex system (Invitrogen), the GeneSwitch system (Invitrogen) and the strictly regulated Ecdysone system (Sratagene). These systems allow the study of time-dependent and concentration-dependent effects of recombinant PSCA. These vectors are subsequently used to control PSCA expression in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells. B. Baculovirus Expression Systems

To generate recombinant PSCA proteins in a baculovirus expression system, a PSCA ORF, or portions thereof, are cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a N-terminal His-tag. Specifically, pBlueBac -PSCA is co-transfected with the helper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda) insect cells to generate the recombinant baculovirus (see the Invitrogen instruction manual for details). The baculovirus is then collected from the cell supernatant and purified by plaque assay. Recombinant PSCA protein is then generated by infection of HighFive insect cells (Invitrogen) with purified baculovirus. Recombinant PSCA protein can be detected using anti-PSCA or anti-tag-His antibodies. The PSCA protein can be purified and used in various cell-based assays or as an immunogen to generate PSCA-specific monoclonal and polyclonal antibodies.

C. Expression Vectors for Orthologs of PSCA

Mouse and monkey PSA orthologs were cloned into pcDNA3.1 / MycHis Version A (Invitrogen, Carlsbad, CA). Protein expression is conducted from the cytomegalovirus (CMV) promoter. Recombinant proteins have the epitope myc and 6X His epitope fused to the carboxyl terminus. These vectors allow the expression of PSCA orthologs to test the cross-reactivity of human anti-PSCA monoclonal antibodies.

Mouse and monkey PSA orthologs were also cloned into pSRa constructs. The pSRa constructs allow the generation of mammalian cell lines that constitutively express PSCA orthologs. Protein expression is conducted from the cytomegalovirus (CMV) promoter. Recombinant proteins have the epitope myc and 6X His epitope fused to the carboxyl terminus. These vectors allow the expression of PSCA orthologs to test the cross-reactivity of human anti-PSCA monoclonal antibodies and to study the functional activity of PSCA orthologs. Amphotropic and ecotropic retroviruses were generated by transfection of pSRa constructs into the 293T-10A1 packaging cell line or co-transfection of pSRα and an auxiliary plasmid (containing cleavage sequences deleted) into 293 cells, respectively. The retrovirus is used to infect a variety of mammalian cell lines, resulting in integration of the cloned gene, PSCA ortholog, into host cell lines. Figure 7 shows the expression of mouse and simian PSCA.pcDNA3.1 / MycHis after transfection into 293T cells. 293T cells were transfected with mouse PSCA.pcDNA3.1 / MycHis or PSCA.pcDNA3.1 / MycHis of simian or control vector pcDNA3.1 / MycHis. Forty hours later, the cells were harvested and analyzed by flow cytometry using anti-PSCA monoclonal antibodies.

Example 6

Profiles of Antigenicity and Secondary Structure

The amino acid profiles of the PSCA variants 1, 3, and 4 were found by accessing the World Wide Web website at (.expasy.ch / cgi-bin / protscale.pl) on the ExPasy molecular biology server.

These profiles, Hydrophilicity, (Hopp T.P., Woods K.R., 1981. Proc Natl Acad Sci USA 78: 3824-3828); Hydropatibility, (Kyte J., Doolittle R.F., 1982. J. Mol.

Biol. 157: 105-132); Percentage of Accessible Residues (Janin J., 1979 Nature 277: 491-492); Medium Flexibility, (Bhaskaran R., and Ponnuswamy P.K., 1988. Int. J. Pept.

Protein Res. 32: 242-255); Links between beta sheets (Deleage, G., Roux B. 1987 Protein Engineering 1: 289-294); and optionally others available in the art, such as on the ProtScale website, were used to identify antigenic regions of each of the PSCA variant proteins. Each of the amino acid profiles of the PSCA variants were generated using the following ProtScale parameters for analysis: 1) A window size of 9; 2) 100% by weight of the window margins compared to the center of the window; and, 3) normalized amino acid profile values to be between 0 and 1.

Hydrophilicity, Hydropacity, and Percentage of Accessible Residues profiles were used to determine hydrophilic amino acid extensions (ie, values greater than 0.5 in the profile of Hydrophilicity and Percentage of Accessible Residues, and values lower than 0.5 in the profile of Hydropathy). Such regions are likely to be exposed to the aqueous environment, to be present on the surface of the protein, and therefore available for immune recognition, such as antibodies. The mean Flexibility and Binding profiles between beta sheets determine amino acid extensions (ie values greater than 0.5 in the Binding profile between beta sheets and Medium Flexibility profile) that are not restricted in secondary structures such as beta sheets and propellers alpha. Such regions are also more likely to be exposed in the protein and therefore to those accessible to immune recognition, such as antibodies.

Antigenic sequences of the PSCA variant proteins indicated, for example, by the profiles described above are used to prepare immunogens, peptides or nucleic acids encoding them, to generate therapeutic and diagnostic anti-PSCA antibodies. The immunogen may be any of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 35, 40, 45, 50 or more than 50 contiguous amino acids; or the corresponding nucleic acids encoding them, from the PSCA protein variants listed in Figure 1 of which the amino acid profiles can be inferred because the variant contains a sequence which is the same as the variant shown. In particular, the peptidic immunogens of the invention may comprise a peptide region of at least 5 amino acids of Figure 1 in any integer increment that includes an amino acid position having a value greater than 0.5 in the Hydrophilicity profile; a peptide region of at least 5 amino acids of Figure 1 in any integer increment which includes an amino acid position having a value less than 0.5 in the Hydropatibility profile; a peptide region of at least 5 amino acids of Figure 1 in any increment of whole number that includes an amino acid position having a value greater than 0.5 in the Percentage of Accessible Residue profiles; a peptide region of at least 5 amino acids of Figures 1 in any integer increment which includes an amino acid position having a value greater than 0.5 in the Mean Flexibility profiles; and a peptide region of at least 5 amino acids of Figure 1 in any increment of whole number that includes an amino acid position having a value greater than 0.5 in the profile of Binding of beta sheets. The peptide immunogens of the invention may also comprise nucleic acids encoding any of the foregoing.

All of the immunogens of the invention, peptides or nucleic acids, may be made in a unit dosage form for humans, or comprised of a composition including a pharmaceutical excipient compatible with human physiology. The secondary structure of the PSCA 1, 3, 4, and 6 protein variants, namely the presence and predicted location of the alpha helices, extended chains, and random spirals, is predicted from the primary amino acid sequence using the HNN-Hierarchical method Neural Network (NPS: Network Protein Sequence Analysis TIBS March 2000 Vol. 25, No. 3 [291]: 147-150 Combet C., Blanchet C., Geourjon C. and Deléage G., http: // pbil. ibcp.fr/cgi-bin/npsa_automat.pi?page=npsa_nn.html), accessed from the ExPasy molecular biology server located on the World Wide Web at (.explasy.ch / tools /). The analysis indicates that variant 1 of PSCA is composed of 30.89% of alpha helices, 21.95% of extended chain, and 47.15% of random spirals. Variant 3 of the PSCA protein is composed of 14.89% alpha helices, 8.51% extended chain, and 76.60% random spirals. Variant 4 of the PSCA protein is composed of 9.52% alpha helices, 8.99% extended chain, and 81.48% random spirals. Variant 6 of the PSCA protein is composed of 24.56% alpha helices, 21.93% extended chain, and 53.51% random spirals. Analysis of the potential presence of the transmembrane domains in the PSCA variant proteins was carried out using a variety of transmembrane prediction algorithms accessed from the ExPasy molecular biology server located on the World Wide Web at (.expasy.ch / tools /).

Example 7

Generation of Polyclonal Antibodies for PSCA

Polyclonal antibodies may be produced in a mammal, for example, through one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and / or adjuvant will be injected into the mammal through multiple subcutaneous or intraperitoneal injections. In addition to immunization with a full-length PSCA protein variant, computational algorithms are employed in the design of immunogens which, based on amino acid sequence analysis, contain antigenic characteristics and are available for recognition by the immune system of the immunized host ( see the Example entitled &quot; Antigenicity Profiles and Secondary Structure &quot;). Such regions would be predicted to be hydrophilic, flexible, in conformations of beta-leaf bonds, and exposed on the surface of the protein.

For example, recombinant bacterial fusion proteins and peptides containing hydrophilic, flexible, beta-sheet bound regions of PSCA protein variants are used as antigens to generate polyclonal antibodies in New Zealand white rabbits or monoclonal antibodies as described in the Example entitled &quot;; Generation of Monoclonal Antibodies (MAbs) for PSCA &quot;. For example, in PSCA variant 1 such regions include, but are not limited to, amino acids 28-56 and amino acids 66-94. For variant 3, such regions include, but are not limited to, amino acids 7-39 and amino acids 70-94. For variant 4 such regions include, but are not limited to, amino acids 6-18, amino acids 27-39, amino acids 103-133, and 177-189. For variant 6, such regions include, but are not limited to, amino acids 19-35 and amino acids 57-85. It is useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal to be 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 103-133 of PSCA variant 4 is conjugated to KLH and used to immunize a rabbit. Alternatively, the immunizing agent may include all or portions of the variant PSCA proteins, analogs or fusion proteins thereof. For example, the amino acid sequences of the PSCA variants can be fused using recombinant DNA techniques for any of a variety of fusion protein partners which are well known in the art, such as glutathione-S-transferase (GST) and proteins merged with HIS. In one embodiment, PSCA variant 1 sequence, amino acids 18-98, was fused to GST using recombinant techniques in the expression vector pGEX, purified, and used to immunize both rabbits and mice to generate polyclonal and monoclonal antibodies respectively . Such fusion proteins are purified from bacteria induced using the appropriate affinity matrix.

Other recombinant bacterial fusion proteins that may be employed include maltose binding protein, LacZ, thioredoxin, NusA, or an immunoglobulin constant region (see the section entitled &quot; Production of PSCA in Prokaryotic Systems &quot; and Current Protocols in Molecular Biology, Volume 2, Unit 16, Frederick M.

Ausubel et al. eds., 1995; Linsley, P.S., Brady, W., Urnes, M., Grosmaire, L., Damle, N., and Ledbetter, L. (1991) J.Exp. Med. 174, 561-566).

In addition to fusion proteins derived from bacteria, mammalian expressed protein antigens are also used. These antigens are expressed from mammalian expression vectors such as Tag5 and Fc fusion vectors (see the section entitled &quot; Production of Recombinant CA PS in Eukaryotic Systems &quot;), and retain post-translational modifications such as glycosylations found in native protein. In one embodiment, the PSCA variant 1 cDNA, minus the N-terminal leader peptide and GPI anchor at the C-terminus was cloned into the mammalian secretion vector Tag5, and expressed in 293T cells. Recombinant protein was purified by metal chelate chromatography from tissue culture supernatants of 293T cells stably expressing the recombinant vector. The purified Tag5 PSCA protein was then used as an immunogen.

During the immunization protocol, it is useful to mix or emulsify the antigen in adjuvants that enhance the immune response of the host animal. Examples of adjuvants include, but are not limited to, Freund's complete adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A, trehalose dicorinomicolate).

In a typical protocol, rabbits are initially subcutaneously immunized with up to 200æg, typically 100-200æg of fusion protein or peptide conjugated to KLH mixed in complete Freund's adjuvant (CFA). The rabbits are then injected subcutaneously every two weeks with up to 200 pg, typically 100-200 pg, of the immunogen in incomplete Freund's adjuvant (IFA). Test bleeds are taken approximately 7-10 days after each immunization and are used to monitor the titer of the antiserum by ELISA.

To test the reactivity and specificity of immune serum, such as rabbit serum derived from immunization with a PSCA variant 3 or 4 protein, the full-length PSCA variant cDNA is cloned into the expression vector pCDNA 3.1 myc-his (Invitrogen, see the Example entitled &quot; Production of Recombinant PSCA in Eukaryotic Systems &quot;). After transfection of the constructs into 293T cells, the cell lysates are probed with the anti-variant and anti-His antibody serum (Santa Cruz Biotechnologies, Santa Cruz, CA) to determine the specific reactivity for the denatured variant protein using the Western blot. In addition, the immune serum is tested by fluorescence microscopy, flow cytometry and immunoprecipitation against 293T and other cells expressing recombinant PSCA variant to determine the specific recognition of native protein. Western blot techniques, immunoprecipitation, fluorescence microscopy and flow cytometry using endogenously PSCA-expressing cells are also carried out to test for reactivity and specificity.

Antiserum from rabbits immunized with PSCA variant fusion proteins, such as GST and MBP fusion proteins, are purified by depletion of antibodies reactive with the fusion partner sequence by passage over an affinity column containing the fusion partner alone or in the context of an irrelevant fusion protein. For example, antiserum derived from a PSCA-GST variant 1 fusion protein is first purified by passing over a GST protein column covalently coupled to an Affigel matrix (BioRad, Hercules, Calif.). The antiserum is then affinity purified by passage over a column composed of a MBP-PSCA fusion protein covalently coupled to an Affigel matrix. The serum is further purified by protein G affinity chromatography to isolate the IgG fraction. Serums from other rabbits immunized with His tagged peptides and antigens as well as immobilized sera from fusion partners are affinity purified by passage over a column matrix composed of the parent protein immunogen or free peptide.

Example 8

Generation of Monoclonal Antibodies (MAbs) for PSCA

In one embodiment, therapeutic Monoclonal Antibodies (&quot; MAbs &quot;) for PSCA and PSCA variants comprise those that react with specific epitopes for each protein or specific for sequences in common among the variants that would bind, internalize, disrupt or modulate the function PSCA or PSCA variants, for example, those that would disrupt interaction with ligands and binding partners. Immunogens for the generation of such MAbs include those designed to encode or contain the extracellular domain of the complete PSCA protein sequence, regions which are expected to contain functional motifs, and regions of the PSCA protein variants predicted to be antigenic from the analysis of the amino acid sequence. Immunogens include peptides, recombinant bacterial proteins such as GST-PSCA fusion proteins (Figure 8) and His tagged PSCA peptide vector protein (Figure 6) and mammalian expressed purified His tagged proteins (Figure 7) and fusion of human and murine IgG Fc. Additionally, cells engineered through retroviral transduction to express high levels of PSCA variant 1, such as RAT1-PSCA, 293T-PSCA, 3T3-PSCA or 300.19-PSCA are used to immunize mice (Figure 5).

To generate monoclonal antibodies to PSCA, mice were first immunized in the plantar cushion (FP) with typically 5-50æg of protein immunogen or between 106 and 107 cells expressing mixed PSCA in a suitable adjuvant. Examples of adjuvants suitable for PF immunizations are TiterMax (Sigma) for the initial injection into FP followed by alum gel with Immuneasy (Qiagen). After the initial injection the mice were subsequently immunized twice weekly until the time they were sacrificed and the B cells obtained from the lymph node used for fusion.

During the immunization protocol, test bleeds were taken to monitor the titre and specificity of the immune response. In most cases, once appropriate reactivity and specificity were obtained as determined by ELISA, Western blot, immunoprecipitation, fluorescence microscopy or flow cytometry analyzes, fusion and generation of hybridomas was then carried out using Electrocell fusion (BTX , ECM2000).

In one embodiment, the invention provides monoclonal antibodies designated Hal-1.16, Hal-5.99, Hal-4.117, Hal-4.120, Hal-4.121, and Hal-4.37. Antibodies have been identified and are shown to react and bind to cell surface or immobilized PSCA. MAbs for PSCA were generated using XenoMouse® technology in which the murine heavy and light chain loci were inactivated and a majority of human heavy chain and heavy chain immunoglobulin loci were inserted: Hal-1.16 was generated after Xenomice immunization human gamma 1 (13-fold with PSCA-GST); Hal-5.99 was generated after immunization of Xenomice human gamma 2 producers 6 times with Ratl-PSCA cells followed by two injections with PSCA-tag5; Hal-4.117, HA1-4.37, Hal-4.120, and Hal-4.121 were generated after Xenomice immunization of human gamma 1 6 times with Ratl-PSCA cells followed by 4 injections with PSCA-tag5. The anti-PSCA MAbs, Hal-1.16, Hal-5.99, Hal-4.117, Hal-4.120, and Hal-4.121 bind to endogenous cell surface PSCA expressed in prostate cancer xenograft cells.

Antibodies designated Hal-5.99, Hal-4.117, Hal-4.120, Hal-4.37, Hal-1.16 and Hal-4.121 were sent (via Federal Express) to the American Type Culture Collection (ATCC), PO Box 1549, Manassas, VA 20108 on May 4, 2004 and assigned with accession numbers PTA-6703 and PTA-6699 and PTA-6700 and PTA-6702 and PTA-6698 and PTA-6701 respectively.

The DNA coding sequences for the anti-PSCA MAbs Hal-1.16, Hal-5.99, Hal-4.117, Hal-4.120, Hal-4.121, and Hal-4.37, were determined after isolation of the mRNA from the respective hybridoma cells with Trizol reagent (Life Technologies, Gibco BRL). Total RNA was purified and quantified. First strand cDNA was generated from total oligo (dT) 12-18 start RNA using the Gibco BRL Superscript system

Preamplification. First-strand cDNA was amplified using human immunoglobulin variable heavy chain primers, and human immunoglobulin variable light chain primers. PCR products were cloned into the vector pCRScript vector (Stratagene, La Jolla). Several clones were sequenced and the determined variable light and heavy chain regions. The nucleic acid and amino acid sequences of the variable light and heavy chain regions are listed in Figure 2 and Figure 3. Alignment of antibodies to PSCA with germline V-D-J Sequences is shown in Figure 4A - Figure 4M.

Example 9

Antibody Identification and Screening for PSCA

Antibodies generated using the procedures set forth in the example entitled &quot; Generation of Monoclonal Antibodies (MAbs) for PSCA &quot; were screened and identified using a combination of assays including ELISA, FACS, epitope clustering, and affinity for PSCA expressed on the cell surface. A. Screening of MAb for human PSCA by FACS. Screening of primary hybridomas for MAbs for PSCA was performed by FACS. The protocol was as follows: 50 ul / well of hybridoma supernatant (neat) or purified antibodies (serial dilutions) were added to 96-well FACS plates and mixed with PSCA (endogenous and recombinant cells, 50,000 cells / well). The mixture was incubated at 4 ° C for two hours. At the end of the incubation, the cells were washed with FACS Buffer and incubated with 100 μl of detection antibody (anti-hlgG-PE) for 45 minutes at 4 ° C. At the end of the incubation, cells were washed with FACS Buffer, fixed with Formaldehyde and analyzed using FACscan. Data were analyzed using CellQuest Pro software. The filled histograms indicate data from negative control cells, and unfilled histograms represent data from PSCA positive cells (Figure 9).

Positive hybridomas identified from primary screenings were transferred to 24-well plates and the supernatants were collected for confirmatory screenings. Confirmatory screenings included FACS analysis on B300.19-PSCA / 300.19-neo, Rat1-PSCA / Ratl-neo, PC3-PSCA / neo, SW780 (bladder cancer cell line), LAPC9AI (cancer cell line prostate), HPAC (pancreatic cancer cell line), and ELISA assay using Tag5-PSCA, GST-PSCA, GST-PSCA N-term, Med.C-Term, and pET-PSCA. B. Relative Affinity Analysis of MAb for human PSCA

Hybridoma supernatants were tested for determination of their binding affinity relative to cell surface PSCA. Hybridoma supernatants were serially diluted in FACS Buffer (FB), from pg / ml to sub-ng / ml; and evaluated in a FACS binding assay using LAPC9AI cells. Antibodies with high affinity provided high MFI values. The MFI values for each point were obtained using the CellQuest Pro software and used for affinity calculation using the Graphpad Prism software (Table VII and Table VIII): Sigmoid Dose-Response (variable slope) equation. The results of the relative affinity analyzes are set forth in Figure 10. C. Epitope Grouping

Antibodies to PSCA were pooled according to the epitopes by assessing their binding pattern in LAPC9AI cells. Briefly, a small amount of each of the antibodies was biotinylated; then each of the biotinylated antibodies were incubated with LAPC9AI in the presence of excess amount (100x) of non-biotinylated antibodies at 4øC for 1 hour during incubation. Generally, an excess amount of antibodies will compete with biotinylated antibodies if they bind to the same epitope. At the end of the incubation, the cells were washed and incubated with Streptavidin-PE for 45 min. at 4 ° C. After washing off the unbound streptavidin-PE, the cells were analyzed using FACS. MFI determinations were used for data analysis (Table VII). As shown in Table XI, cells highlighted in yellow indicate self-competition (100% competition), MFI in these cells constitutes a background control for each biotinylated antibody. The colorless cells indicate that the two antibodies compete with one another (low MFI), an elevated MFI (highlighted in blue) indicates that the two antibodies bind to two distinct epitopes. Antibodies having the same binding pattern bind to the same epitope between the antibodies. There are 6 groups of epitopes within the antibodies tested. Table XI shows that PSCA 4.121 binds to its unique epitope.

Example 10

Characterization and Expression of Antibodies to PSCA A. Cross Reactivity with Monkey PSCA and Mouse PSCA

MAbs were screened and characterized for their ability to react with simian and mouse origin PSCA. This property is useful for understanding the consequences of coupling of MAb to PSCA in cells and tissues when using animal models of simian and mouse. The mouse and monkey Cynomologian monkey PSCA genes were cloned, expressed in a retrovirus, and transiently infected in 293-T cells. 293-T cells were incubated with the respective antibodies using the following protocol. Test antibodies were incubated with 293-T cells expressing murine or monkey PSCA Cinomologo or 293T cells expressing the neo gene only as a negative control. Specific recognition was determined using an anti-glgG-PE secondary detection antibody. A representative histogram illustrating the cross-reactivity of species is shown in Figure 11. The summary shown in Table X shows that all but one of the human anti-PSCA antibodies reacted crosswise with monkey PSCA and only one human anti-PSCA antibody reacted cross-linked with mouse PSCA.

B. Determination of Affinity by FACS

A panel of seven (7) human anti-PSCA antibodies was tested for their PSCA binding affinity in SW780 cells, a bladder cancer cell line expressing high levels of PSCA. 23 serial 1: 2 serial dilutions of purified antibodies were incubated with SW780 cells (50,000 cells per well) overnight at 4 ° C with a final concentration of 167 nM to 0.01 pM. At the end of the incubation, the cells were washed and incubated with anti-hlgE-PE detection antibody for 45 min. at 4 ° C.

After washing the unbound detection antibodies, the cells were analyzed by FACS; the MFI values for each point were obtained using the CellQuest Pro software and were used for affinity calculation using the Graphpad Prism software: Dose-Response equation

Sigmoid (variable slope) (Table VII and Table VIII). The affinity values of the seven (7) antibodies are set forth in Table IX.

Example 11

Internalization of Antibodies to PSCA The internalization of Hal-4.121 was studied using PC3-PSCA cells. Briefly, Hal-4.121 was incubated with cells at 4øC for 90 min to allow binding of the antibodies to the cell surface. The cells were then divided into two groups and incubation continued at 37 ° C to allow for the internalization of the antibodies or at 4 ° C as controls (without internalization). An acidic wash after incubation at 37øC / 4øC was employed to remove PSCA 4.121 bound to the cell surface. Subsequent permeabilization enabled the detection of internalized PSCA-bound antibodies. After incubation with secondary detection antibodies, the cells were analyzed using FACS or observed under a fluorescence microscope.

Approximately 30% of Hal-4121 was internalized after incubation at 37 ° C for two hours (Figure 12).

Example 12

Secondary antibody-mediated death

Antibodies to PSCA mediate saporin-dependent death in cells expressing PSCA. Cells B300.19 expressing PSCA (750 cells / well) were seeded in a 96-well plate on day 1. The next day an equal volume of medium containing a 2x concentration of the indicated primary antibody together with an excess of 2x polyclonal antibody anti-human (Hum-Zap) or anti-goat (Goat-Zap) conjugated to saporin toxin (Advanced Targeting Systems, San Diego, CA) was added to each well. Cells were allowed to incubate for 5 days at 37 degrees C. At the end of the incubation period, MTS (Promega) was added to each well and incubation was continued for an additional 4 hours. The OD at 450 nm was determined. The results in Figure 13 (A) show that the antibodies to PSCA HA1-4.121 and HA1-4.117 mediate saporin-dependent cytotoxicity in B300.19-PSCA cells while a non-specific human IgG1 control antibody had no effect. The results in Figure 13 (B) show that the addition of a secondary saporin-conjugated antibody that did not recognize human Fc was not able to mediate cytotoxicity (Figure 13 (A) and Figure 13 (B)). These results indicate that cytotoxic drugs or proteins may be selectively delivered to cells expressing PSCA using an appropriate anti-PSCA MAb. Example 13

Antibody Immunomedocyte Cytotoxicity

Antibodies to PSCA were evaluated to determine their ability to mediate immunity-dependent cytotoxicity. Antibodies to PSCA (0-50 pg / ml) were diluted with RHB buffer (RPMI 1640, Gibco Life Technologies, 20 mM HEPES). Cells expressing B300.19-PSCA were washed with RHB buffer and resuspended at a density of 106 cells / ml. In a typical assay, 50 μΐ of PSCA antibody, 50 μΐ rabbit complement serum (Cedarlane, Ontario, Canada), and 50 μΐ of a cell suspension were added together to a bottom 96 well tissue culture plate plan. The mixture was incubated for 2 h at 37øC in a 5% CO2 incubator to facilitate complement mediated cell lysis. Subsequently, 50 μl of Alamar Blue (Biosource Inti. Camarillo, CA) were added to each well and incubation was continued for an additional 4-5 h at 37 ° C. Fluorescence in each well was read using a 96-well fluorometer with excitation at 530 nm and emission at 590 nm. The results show that PSCA antibodies having an IgG1 (HA1-4,121) or IgG2 (HA1-5.99.1) isotype but not an IgG4 isotype (HA1-6.46) were able to mediate complement-dependent lysis (Figure 14). ADCC (Cell-Dependent Cell Cytotoxicity

Antibodies) is an immunometric mediated attack on cells bound with antibody directed to a specific cell surface antigen. In this case it is PSCA. The immune cells recognize the Fc portion of the antibody by binding to Fcy receptors on the surface of leukocytes, monocytes and NK cells triggering a lytic attack that results in cell death. The ability of the antibodies to PSCA to mediate this reaction can be assessed by labeling tumor cells in vitro with chromium, europium or a fluorescent molecule and incubating them in the presence of MAbs for human PSCA together with peripheral blood mononuclear cells. Specific lysis of the tumor cells can be determined by measuring the percentage lysis of the target tumor cells. Common goals that are determined include the release of radioactivity, europium or fluorescent dye from the dead cells using an appropriate detection method. Alternatively, the release of an intracellular enzyme such as lactate dehydrogenase (LDH) can be measured.

Example 14

The generation of F (Ab ') 2 fragments of MAbs is useful for studying the effects of MAb molecules that retain their bivalent antigen binding site but lack the Fc effector immune domain in therapeutic models in vitro and in vivo. 20 mg of MAb Hal-4.121 in 20 mM sodium acetate buffer pH 4.5 were incubated with and without immobilized pepsin (Pierce, Rockford IL) for the indicated periods of time. Intact MAb and digested Fc fragments were removed by protein A chromatography. Shown in Figure 15 is a non-reduced, undigested and intact MAb SDS-PAGE stained gel, unaltered aliquots of digested material taken from indicated times, and a reduced sample of the final digested F (ab ') 2 product. This reagent can be used to treat animals bearing tumors that express PSCA. The antitumor activity observed with this antibody fragment can distinguish the intrinsic biological activity of the activity mediated by immunodependent mechanisms. Example 15

Expression of Human Antibodies using Recombinant DNA Methods

To express anti-PSCA mAbs recombinantly in transfected cells, anti-PSCA variable heavy and light chain sequences were cloned upstream of the constant heavy chain IgG1 and human light chain Igx constant regions respectively. Complete anti-PSCA human heavy chain and light chain cassettes were cloned downstream of the CMV promoter / enhancer into a cloning vector. A polyadenylation site was included downstream of the Mab coding sequence. The recombinant anti-PSCA Mabs expressing constructs were transfected into 293T, Cos and CHO cells. The HA1-4.121 antibody secreted from 293-T cells was evaluated for cell surface PSCA binding in Figure Pia-3A and compared to the same antibody produced from the original hybridoma (Figure 16).

Reference Example 16

Class I and Class II HLA Binding Assays HLA class I and class II binding assays are performed using HLA molecules purified according to the disclosed protocols (e.g.

PCT Publication 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, MHC molecules (5 to 500 nM) are incubated in various unlabeled peptidic inhibitors and 1251-labeled radiolabeled probe peptides at 1-10 nm as described. After incubation, the MHC-peptide complexes are separated from the free peptides by gel filtration and the bound peptide fraction is determined. Typically, in preliminary experiments, each MHC preparation is titrated in the presence of fixed amounts of radiolabeled peptides to determine the concentration of HLA molecules required 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] &lt; [HLA] and IC50- [HLA], the measured IC50 values are reasonably approximations of the true values of KD. Peptidic inhibitors are typically tested at concentrations ranging from 120 mg / 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 number for each peptide is calculated by dividing the IC50 of a positive control for inhibition by the IC50 for each peptide tested (typically unlabeled versions of the radiolabeled probe peptide) . For purposes of databases, and comparisons between experiments, relative binding values are compiled. These values can subsequently be transformed to nm values of IC 50 by dividing the IC 50 nm of the positive controls for inhibition by the relative binding of the peptide of interest. This method of data collection is accurate and consistent for comparing peptides that have been tested on different days, or with different batches of purified MHC.

Binding assays as outlined above may be used to analyze peptides bearing HLA supermotives and / or HLA motifs.

Reference Example 17

Construction of Multi-Epitope DNA Plasmids &quot; Minigene &quot;

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

A minigene expression plasmid typically includes various CTL and HTL peptide epitopes. In the present example, peptides epitopes carrying HLA-A2, -A3, -B7 supermotifs and epitopes of peptides bearing HLA-A1 and -A24 motifs are used in conjunction with epitopes bearing DR supermotives and / or DR3 epitopes. Peptide epitopes bearing HLA class I motifs or supermotifs derived from PSCA are selected so that various motifs / supermotifs are represented to ensure wide population coverage. Similarly, HLA class II epitopes from PSCA are selected to provide broad population coverage, i.e. both epitopes carrying HLA DR-1-4-7 supermotifs and DR-3 motif bearing epitopes are selected for inclusion in the minigene building. 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 directing the HTL epitopes to the endoplasmic reticulum. For example, the 1β protein may be fused to one or more HTL epitopes as described in the art, wherein the CLIP sequence of the 1β protein is removed and replaced with an HLA class II epitope so that the HLA class II epitope is directed to the endoplasmic reticulum, where the epitope binds to an HLA class II molecule.

This example illustrates the methods to be used for the construction of a minigene-bearing expression plasmid. Other expression vectors that may be used for minigene compositions are available and are known to those skilled in the art. The minigene DNA plasmid of the present example contains a consensus Kozak sequence and a mouse Ig kappa light chain signal sequence followed by CTL and / or HTL epitopes selected according to the principles disclosed herein. The sequence encodes an open reading frame fused to the Myx and His antibody epitope tag encoded by part of the Myc-His pcDNA 3.1 vector. Overlapping oligonucleotides are synthesized which may, for example, be on average about 70 nucleotides in length with 15 nucleotide overlaps, and are purified by HPLC. The oligonucleotides encode the selected peptide epitopes as well as suitable linker nucleotides, Kozak sequence, and signal sequence. The final multi-epitope minigene is assembled by extending the overlapping oligonucleotides into 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: 95øC for 15 sec, hybridization temperature (5øC below the calculated lowest 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 mg of each of two oligonucleotides are hybridized and extended: In one example using eight oligonucleotides, i.e., four pairs of primers, 1 + 2 oligonucleotides , 3 + 4, 5 + 6, and 7 + 8 in 100 ml reactions containing Pfu polymerase buffer (1x = 10 mM KCL, 10 mM NBS +, 20 mM Trischloride, pH 8.75 , 2 mM MgSO4, 0.1% Triton-X100, 100 pg / ml BSA), 0.25 mM dNTPs each, and 2.5 U Pfu polymerase. Full-length dimer products are purified by gel, and two reactions containing the product of 1 + 2 and 3 + 4, and the product of 5 + 6 and 7 + 8, are hybridized, and extended for 10 cycles. 5 cycles of hybridization and extension prior to the addition of flanking primers to amplify the full-length product. and are cloned into pCR-blunt (Invitrogen) and individual clones are screened by sequencing. Example 18 The Construction of Plasmid and the Degree Which Induces Immunogenicity. The degree to which a plasmid construct, for example a plasmid constructed according to the preceding Example, is capable of inducing immunogenicity is confirmed in vitro by determining the epitope presentation through APC followed by the transduction or transfection of the APC with a construct nucleic acid expressing epitope. Such a study determines &quot; antigenicity &quot; 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. The quantification may be carried out by directly measuring the amount of peptide eluted from the APC (see, for example, 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 subsequent determination of the peptide concentration required to obtain equivalent levels of lysis or release of lymphokines (see, for example, Kageyama et al., J.

Immunol. 154: 567-576, 1995).

Alternatively, immunogenicity is confirmed by in vivo injections into mice and subsequent in vitro evaluation of CTL and HTL activity, which are analysed using cytotoxicity and proliferation assays, respectively, as detailed for example in Alexander et al., Immunity 1: 751-761, 1994.

For example, to confirm the ability of a minigene construct of DNA containing at least one HLA-A2 supermotif peptide to induce CTLs in vivo, HLA-A2.1 / Kb transgenic mice, for example, are immunized intramuscularly with 100æg of CDNA nude. As a means of comparing the level of CTLs induced by cDNA immunization, an animal control group with an actual peptidic composition is also immunized which comprises several epitopes synthesized as a single polypeptide as they would be encoded by the minigene. Splenocytes are stimulated from animals immunized twice with each of the respective compositions (peptide epitopes encoded in the minigene or the polyepitopic peptide), and then evaluated for peptide-specific cytotoxic activity in a 51 Cr release assay. The results indicate the magnitude of the CTL response directed against the A2 restricted epitope as such indicating the in vivo immunogenicity of the minigene vaccine and the polyepitopic vaccine. As such, it has been found that minigene elicits immune responses directed to HLA-A2 supermotif peptide epitopes in the same way as the polyepitopic peptide vaccine. A similar analysis is also performed using other HLA-A3 and HLA-B7 transgenic mouse models to evaluate the induction of CTL by HLA-A3 and HLA-B7 motif epitopes, so it is also found that the minigene elicits responses immune responses directed to the provided epitopes.

To confirm the ability of a minigene encoding class II epitope to induce HTLs in vivo, DR transgenic mice, or for those epitopes which cross-react with the appropriate mouse MHC molecule, mice with restricted I-Ab, for example, are immunized intramuscularly with 100 Âμg of plasmid DNA. As a means of comparing the level of HTLs induced by DNA immunization, a group of control animals with an actual peptide composition emulsified in complete Freund's adjuvant is also immunized. CD4 + T cells, i.e., HTLs, are purified from splenocytes from animals immunized and stimulated with each of the respective compositions (peptides encoded in the minigene). The HTL response is measured using a 3 H-thymidine incorporation proliferation assay (see, for example, Alexander et al., Immunity 1: 751-761, 1994). The results indicate the magnitude of the HTL response, as such demonstrating the in vivo immunogenicity of the minigene.

DNA minigenes, constructed as described in the previous Example, may also be confirmed as a vaccine in combination with a boosting agent through a priming protocol. The boosting agent may consist of recombinant protein (for example, 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 protein (see, for example, Hanke et al., Vaccine 16: 439-445, 1998, Sedegah et al., Proc Natl Acad Sci USA 95: 7648-53, 1998, Hanke and McMichael, Immunol Letters 66: 177-181, 1999; and Robinson et al., Nature Med 5: 526-34, 1999).

For example, the efficacy of the DNA minigene used in a priming enhancement protocol is initially evaluated in transgenic mice. In this example, A2.1 / Kb transgenic mice are immunized with 100æg of a DNA minigene encoding the immunogenic peptides including at least one HLA-A2 supermotif carrier peptide. After an incubation period (ranging from 3-9 weeks), mice are boosted with 107 pfu / mouse IP of a recombinant vaccinia virus expressing the same sequence encoded by the DNA minigene. Control mice are immunized with 100 pg of recombinant DNA or vaccinia without the minigene sequence, or with DNA encoding the minigene, but without the vaccinia boost. After an additional two-week incubation period, splenocytes are immediately assayed from the mice for peptide-specific activity in an ELISPOT assay. In addition, in vitro splenocytes are stimulated with A2-restricted peptide epitopes encoded in minigene and recombinant vaccinia, and subsequently assayed for peptide-specific activity in an IFN alpha, beta and / or gamma ELISA. It is found that the minigene used in a priming enhancement protocol triggers greater immune responses to HLA-A2 supermotif peptides than to DNA alone. A similar analysis can also be performed using other HLA-A11 or HLA-B7 transgenic mouse models to evaluate CTL induction by HLA-A3 and HLA-B7 motif or supermotif epitopes. The use of priming protocols in humans is described below in the Example titled &quot; Induction of CTL Responses Using a Priming Reinforcement Protocol &quot;.

Reference Example 19

Polyepitopic Vaccine Compositions from Various Antigens

The PSCA peptide epitopes of the present invention are used in conjunction with epitopes from other antigens associated with a target tumor to create a vaccine composition that is useful for the prevention or treatment of cancer expressing PSCA and such other antigens. For example, a vaccine composition may be provided as a single polypeptide incorporating various epitopes from PSCA as well as tumor associated antigens which are often expressed with a target cancer associated with PSCA expression, or may be administered as a composition comprising a cocktail of one or more discrete epitopes. Alternatively, the vaccine may be administered as a minigene construct or as dendritic cells which were loaded with the peptide epitopes in vitro. Reference Example 20

Use of peptides to evaluate an immune response

Peptides of the invention can be used to analyze an immune response for the presence of specific antibodies, CTL or HTL targeted to PSCA. Such an assay may be carried out in a manner described by Ogg et al., Science 279: 2103-2106, 1998. In this Example, peptides according to the invention are used as a reagent for diagnostic or prognostic purposes, not as an immunogen.

In this example, human antigen-leukocyte tetrameric complexes (&quot; tetramers &quot;) are used for cross-sectional analysis of, for example, HLA-A * 0201 specific CTLA frequencies of PSCA from subjects with HLA A * 0201 positive at different stages of disease or after immunization comprising a PSCA peptide containing an A * 0201 motif. Tetrameric complexes are synthesized as described (Musey et al., N. Engl. J. Med. 337: 1267, 1997).

Briefly, purified HLA heavy chain (A * 0201 in this example) and p2-microglobulin are synthesized by means of a prokaryotic expression system. The heavy chain is modified by elimination of the transmembrane cytosolic tail and addition at the COOH terminus of a sequence containing an enzymatic biotinylation site. The heavy chain, β2-microglobulin, and peptide are refolded by dilution. The refolded 45 kD product is isolated by flash protein liquid chromatography and subsequently biotinylated by BirA in the presence of biotin (Sigma, St. Louis, Missouri), adenosine 5 'triphenylte and magnesium. Streptavidin-phycoerythrin conjugate in a 1: 4 molar ratio is added, and the tetrahydro product is concentrated to 1 mg / ml. The resulting product is referred to as tetrameromer-phycoerythrin.

For analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300g for 5 minutes and resuspended in 50 ml of cold phosphate buffered saline. Tricolor analysis with tetramer-phycoerythrin, along with Tricolor anti-CD8, and anti-CD38 are performed. PBMCs are incubated with tetramer and antibodies on ice for 30 to 60 min and then washed twice prior to fixation with formaldehyde. Ratings are applied to contain &gt; 99.98% of the control samples. Controls for the tetramers include both subjects with A * 0201 negative and non-A * 0201 positive donors. The percentage and cells stained with the tetramor are then determined by flow cytometry. The results indicate the number of cells in the sample of PBMCs containing epitope-restricted CTLs, thus readily indicating the degree of immune response to the PSCA epitope, and therefore the state of exposure to PSCA, or exposure to a vaccine that triggers a protective or therapeutic response.

Example 21

Induction of Immune Responses Using a Priming Reinforcement Protocol

A similar priming protocol in its principle underlying that used to confirm the efficacy of a DNA vaccine in transgenic mice, as described above in the Example entitled &quot; The Construction of Plasmid and the Degree Which Induces Immunogenicity &quot; may also be used for administration of the vaccine to humans. Such a vaccine regimen may 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 mixture of peptides administered in an adjuvant.

For example, the initial immunization can be performed using an expression vector, such as that constructed in the Example titled &quot; Construction of Multi-Epitope DNA Plasmids &quot; Minigene &quot; in the form of naked nucleic acid administered IM (or SC or ID) in amounts of 0.5-5 mg at various sites. Nucleic acid (0.1 to 1000 mg) can also be administered using biobalistics. After an incubation period of 3-4 weeks, a booster dose is then administered. The boost may be recombinant avian pox virus administered in a dose of 5-107 to 5x109 pfu. An alternative recombinant virus, such as an MVA, canary fowlpox, adenovirus, or adeno virus associated with the booster may be used, or the polyepitopic protein or a mixture of the peptides may be administered. For the evaluation of vaccine efficacy, blood samples are obtained from the patients prior to immunization as well as at intervals after administration of the initial vaccine and booster doses of the vaccine. Peripheral blood mononuclear cells are isolated from freshly heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted into freezing mediums and stored frozen. Samples are evaluated for CTL and HTL activity. Analysis of the results indicates that a sufficient magnitude of response is generated to achieve therapeutic or protective immunity against PSCA.

Example 22

Complementary polynucleotides Sequences complementary to the PSCA coding sequences (Figure 1 or Figure 3), or any parts thereof, are used to detect, decrease, or inhibit naturally occurring PSCA expression. Although the use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or larger sequence fragments. Suitable oligonucleotides are designed using, for example, OLIGO 4.06 (National Biosciences) software and the PSCA coding sequence. To inhibit transcription, a complementary oligonucleotide is drawn from the most unique 5 'sequence and used to prevent binding of the promoter to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to a PSCA-encoding transcript.

Example 23

Purification of Natural or Recombinant Occurrence PSCA Using Natural or Recombinant PSCA PSCA Specific Antibodies is substantially purified by immunoaffinity chromatography using antibodies specific for PSCA. An immunoaffinity column is constructed by covalently coupling an anti-PSCA antibody with an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After coupling, the resin is blocked and washed according to the manufacturer's instructions.

PSCA-containing media are passed over the immunoaffinity column, and the column is washed under conditions permitting preferential absorbance of PSCA (e.g., high ionic strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibody / PSCA binding (for example, a buffer of pH 2 at pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and GCR.P.

Example 24

Identification of Molecules that Interact with PSCA PSCA, or biologically active fragments thereof, are labeled with Bolton-Hunter 121.1 reagent. (See, for example, Bolton et al (1973) Biochem J. 133: 529) Candidate molecules previously arranged in the wells of a multiwell plate are incubated with labeled PSCA, washed, and any complex wells PSCA labeled. Data obtained using different concentrations of PSCA are used to calculate values for the number, affinity, and association of PSCA with the candidate molecules.

Reference Example 25

In Vivo Assay for Promoting Growth of PSCA Tumors The effect of PSCA protein on tumor cell growth is evaluated in vivo by assessing tumor development and growth of cells expressing or lacking PSCA. For example, SCID mice are injected subcutaneously into each 1 x 106 or 3T3 flank, or prostate cancer cell lines (e.g. PC3 cells) containing empty vector tkNeo or PSCA. At least two strategies may be used: (1)

Constitutive expression of PSCA under regulation of a promoter such as a constitutive promoter obtained from virus genomes such as polyoma virus, avian pox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis B virus and more preferably Syrian Virus 40 (SV40), or from heterologous mammalian promoters, for example, the actin promoter or a promoter (2) Regulated expression under the control of an inducible vector system, such as ecdysone, tetracycline, etc., provided such promoters are compatible with the systems of the immunoglobulin, such that such promoters are compatible with the host cell systems. host cell. The tumor volume is then monitored by gauge measurement on the appearance of palpable tumors and followed over time to determine whether PSCA expressing cells grow at a faster rate and whether tumors produced through PSCA expressing cells exhibit altered aggression characteristics ( for example enhanced metastasis, vasculature, reduced responsiveness to chemotherapeutic drugs).

In addition, 1 x 105 mice from the same cells can be implanted orthotropically to determine whether PSCA has an effect on local prostate growth, and whether PSCA affects the ability of metastatic cells, specifically lymph nodes, and bone (Miki T et al., Oncol Res 2001; 12: 209; Fu X et al, Int J Cancer, 1991, 49: 938). The effect of PSCA on the formation and growth of bone tumors can be assessed by intratibial injection of prostate tumor cells. The assay is also useful for determining the PSCA inhibitory effect of candidate therapeutic compositions, such as, for example, PSCA intrabodies, PSCA antisense molecules and ribozymes.

Example 26

Inhibition of PSCA Monoclonal Antibody-Mediated Tumors In Vivo Significant expression of PSCA on the cell surface of tumor tissues, together with their restrictive expression in normal tissues converts PSCA into a good target for antibody therapy. Similarly, PSCA is a target for T cell-based immunotherapy. Thus, the therapeutic efficacy of anti-PSCA MAbs in human prostate cancer xenograft mouse models and human pancreatic cancer xenograft mouse models is evaluated using recombinant cell lines such as PC3-PSCA, and 3T3-PSCA (see, for example, Kaighn, ME, et al., Invest Urol, 1979, 17 (1): 16-23), as well as xenograft human prostate such as LAPC 9AD (Saffran et al PNAS 1999, 10: 1073-1078). The effectiveness of antibodies in tumor growth and formation of metastases, for example in mouse orthotropic prostate or pancreatic cancer xenograft models, is studied. The antibodies may be unconjugated as discussed in the present Example, or may be conjugated to a therapeutic modality as appreciated in the art. The anti-PSCA MAbs inhibit the formation of both pancreatic and prostate xenografts. The anti-PSCA MAbs also delay the growth of established orthrope tumors and prolong the survival of tumor bearing mice. These results indicate the utility of anti-PSCA MAbs in the treatment of local and advanced stages of prostate cancer, pancreatic cancer and those cancers set forth in Table 1 (see, for example, Saffran, D., et al., PNAS 10 : 1073-1078 or URL world wide web pnas.org/cgi/doi/10.1073/pnas.051624698). Administration of anti-PSCA mAbs led to delayed growth of established orthotropic tumors and to inhibition of metastases at distant sites, resulting in a significant prolongation of the survival of tumor bearing mice. These studies indicate that PSCA is an attractive target for immunotherapeutics and demonstrate the therapeutic potential of anti-PSCA MAbs in the treatment of local and metastatic prostate and pancreatic cancer. This example demonstrates that unconjugated PSCA monoclonal antibodies are effective to inhibit the growth of human prostate tumor xenografts grown in SCID mice; therefore a combination of such effective monoclonal antibodies is also effective.

Tumor inhibition using various MAbs for PSCA

Materials and methods

Monoclonal Antibodies for PSCA:

Monoclonal antibodies against PSCA were produced as described in the Example entitled &quot; Generation of PSCA Monoclonal Antibodies (MAbs) &quot;. the antibodies are characterized by ELISA, Western blot, FACS, and immunoprecipitation for their ability to bind PSCA. Epitope mapping data for anti-PSCA MAbs, as determined by ELISA and Western analysis, recognize epitopes on the PSCA protein. Immunohistochemical analysis of prostate cancer tissues and cells with these antibodies is carried out.

Monoclonal antibodies are purified from hybridoma tissue culture ascites or supernatants by Protein G- or Protein A-Sepharose chromatography, dialyzed against PBS, sterilized by filtration, and stored at -20 ° C. Protein determinations are performed by a Bradford assay (Bio-Rad, Hercules, CA). A therapeutic monoclonal antibody or a cocktail comprising a mixture of individual monoclonal antibodies is used and is used for the treatment of mice receiving subcutaneous or orthotropic injections of LAPC9 AD and HPAC tumor xenografts. Cell Lines and Xenografts

The prostate cancer cell lines, PC3 and LNCaP cell line as well as the NIH 3T3 fibroblast line (American Type Culture Collection) are maintained in RPMI and DMEM respectively, supplemented with L-glutamine and 10% FBS.

The PC3-PSCA and 3T3-PSCA cell populations are generated by retroviral gene transfer as described in Hubert, RS, et al., Proc Natl Acad Sci USA, 1999. 96 (25): 14523. The LAPC-9 xenograft, which expressing a wild-type androgen receptor and producing prostate-specific antigen (PSA), is passed into 6-to 8-week-old male Combined Immunodeficient (SCID) mice (Taconic Farms) via a subcutaneous trocar implant (Craft , N., et al., Nat Med 1999, 5: 280). Single cell suspensions of LAPC-9 tumor cells are prepared as described in Craft, et al. Xenograft Mouse Models. Subcutaneous (s.c.) tumors are generated by injection of 1 x 10 6 cancer cells mixed at a 1: 1 dilution with Matrigel (Collaborative Research) on the right flank of male SCID mice. To test the effectiveness of antibodies in tumor formation, i.e., injections of antibodies are initiated on the same day as injections of tumor cells. As a control, they are injected into mice or purified mouse IgG (ICN) or PBS; or a purified monoclonal antibody that recognizes an irrelevant antigen not expressed in human cells. In preliminary studies, no difference was found between mouse IgG or PBS in tumor growth. Tumor sizes are determined by gauge measurements, and the tumor volume is calculated as length x width x height. Mice with subcutaneous tumors larger than 1.5 cm in diameter are sacrificed. Orthotropic injections are made under anesthesia using ketamine / xylazine. For orthotropic prostate studies, an incision is made through the abdomen to expose the prostate and LAPC or PC3 (2 x 106) tumor cells mixed with Matrigel are injected into the prostate capsule in a volume of 10 ml. To monitor tumor growth, mice are palpated and blood is collected weekly to measure PSA levels. Mice are separated into groups for appropriate treatments, with anti-PSCA MAbs or control being injected i.p.

Anti-PSCA MAbs Inhibit Growth of Xenograft Carcinogenic Tumors Expressing PSCA The effect of anti-PSCA MAbs on tumor formation is tested using HPAC and LAPC9 orthotropic models. As compared to the sc tumor model, the orthotropic model, which requires injection of tumor cells directly into the pancreas or mouse prostate, respectively, results in local tumor growth, development of distal site metastasis, deterioration of the mouse's health, and subsequent death (Saffran, D., et al., PNAS supra). These characteristics make the orthotropic model more representative of the progression of human disease and allowed to follow the therapeutic effect of MAbs at clinically relevant endpoints.

Accordingly, tumor cells are injected into the prostate gland of the mouse, and 2 days later, the mice are separated into two groups and treated with either: a) 250-1000 mg of anti-PSCA Ab, or b) control antibody three times per mouse. week for two to five weeks.

A major advantage of orthotropic cancer models is the ability to study the development of metastases. The formation of metastases in mice bearing established orthrope tumors is studied by IHC analysis in lung sections using an antibody against a tumor-specific cell surface protein such as anti-CK20 for prostate cancer (Lin et al., Cancer Detect Prev (2001) 25: 202).

Another advantage of xenograft cancer models is the ability to study neovascularization and angiogenesis. Tumor growth depends partly on the development of new blood vessels. Although the capillary system and developing blood network is of host origin, the initiation and architecture of the neovasculature is regulated by the xenograft tumor (Davidoff et al., Clin Cancer Res. (2001) 7: 2870; Solesvik et al. Eur J Cancer Clin Oncol (1984) 20: 1295). The effect of antibodies and small molecules on neovascularization is studied according to procedures known in the art, such as through IHC analysis of tumor tissues and their surrounding microenvironment.

Mice bearing established orthotopic tumors are administered with injections of anti-PSCA MAb or control antibody over a 4 week period. Mice in both groups are allowed to establish a high tumor burden to ensure a high frequency of metastasis in the lungs of the mice. The mice are then sacrificed and their bladders, livers, bones and lungs are analyzed for the presence of tumor cells by IHC analysis. These studies demonstrate broad antitumor efficacy of anti-PSCA antibodies at the onset and progression of prostate cancer in xenograft mouse models. Anti-PSCA antibodies inhibit tumor formation of tumors while delaying the growth of established tumors and prolong the survival of treated mice. In addition, anti-PSCA MAbs have demonstrated a dramatic inhibitory effect on the spread of tumor from the local prostate to distal sites, even in the presence of a large tumor burden. Thus, anti-PSCA MAbs are effective at major clinically relevant goals (tumor growth), survival prolongation, and health.

Effect of MAbs for PSCA on the Growth of Human Prostate Cancer in Mice

Using the above methodology, LAPC-9AI tumor cells (2.0 x 106 cells) were injected subcutaneously into male SCID mice. Mice were randomized into groups (n = 10 in each group) and treatment was started intraperitoneally (i.p.) on Day 0 with HA1-4,120 or isotype control Mab as indicated. The animals were treated twice weekly for a total of 7 doses by day 28 of the study. Tumor growth was monitored using one gauge measurements every 3 to 4 days as indicated. The results show that the human anti-PSCA monoclonal antibody Hal-4.120 significantly inhibited the growth of human prostate cancer xenografts implanted subcutaneously in SCID mice (p <0.05) (Figure 18).

In another experiment, LAPC-9AI tumor cells (2.0 x 106 cells) were injected subcutaneously into male SCID mice. When the tumor volume reached 50 mm 3, the mice were randomized into groups (n = 10 in each group) and the treatment was started intraperitoneally (i.p.) with HA1-5.99.1 or isotype control Mab as indicated. The animals were treated twice weekly for a total of 5 doses by day 14 of the study. Tumor growth was monitored using one gauge measurements every 3 to 4 days as indicated. The results show that the fully human anti-PSCA monoclonal antibody Hal-5.99 significantly inhibited the growth of established androgen independent human prostate cancer xenografts implanted subcutaneously in SCID mice (p <0.05). (Figure 19).

In another experiment, LAPC-9AD tumor cells (2.5 x 106 cells) were injected subcutaneously into male SCID mice. When the tumor volume reached 40 mm 3, the mice were randomized into groups (n = 10 in each group) and treatment was started intraperitoneally (i.p.) with increasing concentrations of HA1-4.121 or isotype control Mab as indicated. The animals were treated twice weekly for a total of 7 doses by day 21 of the study. Tumor growth was monitored using one gauge measurements every 3 to 4 days as indicated. The results of this study demonstrate that HA1-4,121 inhibited the growth of subcutaneous human androgen dependent prostate cancer xenografts established in SCID mice. The results were statistically significant for the 300æg dose group at days 14, 17 and 21 (p <0.05, Kruskal-Wallis test, bilateral at a = 0.05) and at the dose group of 700æg on days 10, 14, 17 and 21 (p <0.05, Kruskal-Wallis test, bilateral with a = 0.05) (Figure 20).

In another experiment, androgen dependent patient derived LAPC-9AD tumor cells (2.0 x 106 cells) were injected into the dorsal lobes of the prostates of male SCID mice. The tumors were allowed to grow for approximately 10 days, at which time the mice were randomized into clusters. Treatment with 500 mg of HA1-4.117, HA1-4.121 human or isotype control MAb was started 10 days after tumor implantation. Antibodies were administered intraperitoneally twice weekly in a total of 7 doses. Four days after the last dose, the animals were sacrificed and the primary tumors were excised and weighed. The results show that the human anti-PSCA monoclonal antibodies Hal-4.121 (p <0.01) and Hal-4.117 (p &lt; 0.05) significantly inhibited the growth of orthotopically implanted LAPC-9AD prostate cancer xenografts in SCID mice (Figure 21).

In another experiment, androgen dependent patient derived LAPC-9AD tumor cells (2.0 x 106 cells) were injected into the dorsal lobes of the prostates of male SCID mice. The tumors were allowed to grow for approximately 9 days, at which time the mice were randomized into clusters. Randomized animals in the survival groups include 11 mice in the isotype control MAb and 12 mice in the HA1-4,121 treatment group. The animals were treated i.p. with 1000æg of Hal-4.121 or 1000æg of isotype control MAb twice weekly for a total of 9 doses. The results demonstrated that HA1-4.121 significantly (log-rank test: p <0.01) significantly prolonged survival of SCID mice with human androgen dependent prostate tumors. Two mice in the HA1-4,121 treated group remained free of palpable tumors at day 110, the last day of the experiment (Figure 22).

Effects of MAbs for PSCA in combination with Taxotere in mice

In another experiment, LAPC-9AI tumor cells (2 x 106 cells per animal) were injected subcutaneously into male SCID mice. When the tumor volume reached 65 mm3, the animals were randomized and assigned to four different groups (n = 10 in each group) as indicated. Starting at day 0, Hal-4.121 or isotype control MAb were administered i.p. twice a week at a dose of 500æg for a total of 6 doses. The last dose was given on day 17. Taxotere was given intravenously at a dose of 5 mg / kg on days 0, 3 and 7. Tumor growth was monitored every 3-4 days using one-gauge measurements. The results of this study demonstrate that HA1-4,121 as the sole agent inhibited the growth of androgen-independent prostate xenografts in SCID mice by 45% compared to the control antibody treatment alone at day 28 (ANOVA / Tukey's test: p < 0.05). Administration of the taxotere isotype control MAb inhibited tumor growth by 28% compared to the control antibody treatment alone, which was not statistically significant. Administration of HA1-4,121 in combination with taxotere potentiated the effect and resulted in inhibition of 69% tumor growth compared to the control antibody alone (ANOVA / Tukey's test: p <0.01). A statistically different difference was also demonstrated when the combination group of HA1-4,121 plus taxotere was compared with the HA-4,121 or isotype control MAb plus taxotere groups (ANOVA / Tukey's test: p <0.05) (Figure 23).

Effect of MAbs for PSCA on the Growth of Human Pancreatic Cancer in Mice

In another experiment, human HPAC human pancreatic cancer cells (2x106 / mouse) were injected subcutaneously into immunodeficient SCID ICID mice (Taconic Farm, Germantown, NI). Mice were randomized into groups (n = 10 animals / group) and treatment with indicated human PSCA monoclonal antibody initiated the same day. Antibodies (500 mg / mouse) were administered intraperitoneally twice weekly for a total of 8 doses. The results demonstrated that the anti-PSCA monoclonal antibodies Hal-4.121, Hal-4.117 and Hal-1.16 significantly inhibited the growth of human pancreatic cancer xenografts implanted subcutaneously in SCID mice. Statistical analyzes were performed using a t-test (bilateral, a = 0.05) (Figure 24).

In another experiment, HPAC cells (3.0 x 106 cells) were orthopedically implanted into the pancreas of SCID mice. Mice were randomly assigned to three groups (n = 9 in each group) as indicated. Treatment with HA1-4,121 (250æg or 1000æg) isotype control Mab (1000æg) was started on the day of implantation. Antibodies were given i.p twice a week for a total of 10 doses. Thirty days after the last dose, the animals were sacrificed and the primary tumors were excised and weighed. The results of this study demonstrate that HA1-4,121 significantly inhibited the orthotopic growth of human pancreatic cancer xenografts in SCID mice at both dose levels examined. Treatment with 250æg and 1000æg of AGS-PSCA inhibited tumor growth by 66% and 70%, respectively (Kruskal-Wallis / Tukey's test: p <0.01 and p <0.01, respectively) (Figure 25 ).

At necropsy, visible metastases in the lymph nodes and distal organs were observed in the control antibody treated group. No visible metastases were observed in both HA1-4.121 treated groups. Lymph nodes, lungs and livers were removed from all animals and examined histologically for the presence of metastatic tumor. Cuts of the lungs and lymph nodes removed from each animal were stained for human cytokeratin and the number of metastases was determined microscopically. Histological analysis results demonstrated a significant reduction in lymph node metastases (NB) in animals treated with HA1-4,121 (p = 0.0152 as detected by Fisher's exact test). The incidence of metastases and invasion was also significantly decreased in animals treated with both concentrations of HA1-4,121 (p = 0.0152 as detected by Fisher's exact test). The number of lung metastases decreased significantly in mice treated with the 1.0 mg dose of HA1-4.121 alone (p = 0.0498 as detected by Fisher's exact test) (Figure 26).

Effect of MAbs for PSCA on the Growth of Human Bladder Cancer in Mice

In another experiment, human bladder cancer cells (2x106 / mouse) were injected subcutaneously into immunodeficient SCID ICID mice (Taconic Farm, Germantown, NI). Mice were randomized into groups (n = 10 animals / group) and treatment with indicated human PSCA MAb initiated on the same day. Antibodies (250 mg / mouse) were administered intraperitoneally twice weekly for a total of 7 doses. The results showed that HA1-4.117 (p = 0.014), HA1-4.37 (p = 0.0056), HA1-1.78 (p = 0.001), Hal-5.99 (p = 0.0002) and HA1-4.5 (p = 0.0008) significantly inhibited the growth of SW780 bladder tumors implanted subcutaneously in SCID mice. Statistical analyzes were performed using a t-test (bilateral, a = 0.05) (Figure 27).

The results of these experiments show that MAbs for PSCA can be used for therapeutic and diagnostic purposes for treatment and management of cancers set forth in Table I.

Example 27

Therapeutic Use and Diagnosis of Anti-PSCA Antibodies in Humans.

Anti-PSCA monoclonal antibodies are safely and effectively used for diagnostic, prophylactic, prognostic and / or therapeutic purposes in humans. Western blot analysis and immunohistochemistry of cancerous tissues and cancer xenografts with anti-PSCA MAbs show extensive extensive staining in carcinoma but significantly lower or undetectable levels in normal tissues. The detection of PSCA in carcinoma and in metastatic disease demonstrates the usefulness of MAb as a diagnostic and / or prognostic indicator. Anti-PSCA antibodies are therefore used in diagnostic applications such as immunohistochemistry of renal biopsy specimens for detection of cancer from suspected patients.

As determined by flow cytometry, the anti-PSCA MAb binds specifically to carcinoma cells. Thus, anti-PSCA antibodies are used in diagnostic whole-body imaging applications such as radioimmuno-mytiocytosis and radioimmunotherapy, (see, for example, Potamianos S., et al., Anti-cancer Res 20 (2A): 925-948 (2000)) for the detection of localized and metastatic cancers exhibiting PSCA expression. The dissemination or release of an extracellular domain of PSCA to the extracellular medium, such as that observed for alkaline phosphodiesterase 10 (Meerson, NR, Hepatology 27: 563-568 (1998)), permits the diagnostic detection of PSCA by anti- -PSCA in serum and / or urine samples from suspected patients.

Anti-PSCA antibodies that bind specifically to PSCA are used in therapeutic applications for the treatment of cancers that express PSCA. Anti-PSCA antibodies are used as a non-conjugated and conjugated form in which the antibodies are linked to one of a variety of imaging or therapeutic modes well known in the art, such as prodrugs, enzymes or radioisotopes. In preclinical studies, conjugated and unconjugated anti-PSCA antibodies are tested for the efficacy of tumor prevention and inhibition of tumor growth in the cancer xenograft models of SCID mice, for example, AGS-K3 and AGS renal cancer models -K6, (see, for example, the Example entitled &quot; Inhibition of Tumors Monoclonal Antibody Mediated by PSCA In Vivo &quot;). Conjugated and unconjugated anti-PSCA antibodies are used as a therapeutic modality in clinical trials of humans either alone or in combination with other treatments as described in the following Examples.

Example 28

Human Clinical Assays for the Treatment and Diagnosis of Human Carcinomas by Using Anti-PSCA In Vivo Antibodies Antibodies of the present invention are used that recognize an epitope on PSCA, and are used in the treatment of certain tumors such as those listed in Table I. Based on a number of factors, including levels of PSCA expression, tumors such as those listed in Table I are currently preferred indications. In connection with each of these indications, three clinical approaches are pursued successfully. I). Adjuvant Therapy: In adjuvant therapy, patients are treated with anti-PSCA antibodies in combination with a chemotherapeutic or antineoplastic agent and / or radiation therapy. Primary cancer targets, such as those listed in Table I, are treated under standard protocols by the addition of anti-PSCA antibodies to standard first and second line therapy. The protocol designs address efficacy as assessed by reducing tumor mass as well as the ability to reduce standard chemotherapy standard doses. These dose reductions allow for additional and / or prolonged therapy by reducing the dose-related toxicity of the chemotherapeutic agent. Anti-PSCA antibodies are used in several adjuvant assays in combination with the chemotherapeutic or antineoplastic agents adriamycin (advanced prostate carcinoma), cisplatin (advanced head and neck and lung carcinomas), taxol (breast cancer), and doxorubicin -clinical). II. ) Monotherapy: In connection with the use of anti-PSCA antibodies in tumoral monotherapy, the antibodies are administered to patients without a chemotherapeutic or antineoplastic agent. In one embodiment, the monotherapy is conducted clinically in terminal cancer patients with extensive metastatic disease. The patients present some destabilization of the disease. The assays demonstrate an effect in refractory patients with cancerous tumors. III. ) Imaging agent: By binding a radionuclide (iodine or yttrium (I131, Y90) to the anti-PSCA antibodies, radiolabeled antibodies are used as a diagnostic and / or imaging agent. As such, labeled antibodies localize both solid tumors as well as metastatic lesions of PSCA-expressing cells. In connection with the use of anti-PSCA antibodies as imaging agents, the antibodies are used as an adjuvant to the surgical treatment of solid tumors as well as a pre-clinical tracer and as a post-operative follow-up to determine which tumor remains and / or returns. In one embodiment, an antibody (211In) -PSCA is used as an imaging agent in the assay (see, for example, Divgi et al., J. Natl Cancer Inst 83: 97-104 (1991)). followed with a standard anterior and posterior chamber range. The results indicate that primary lesions and metastatic lesions are identified.

Dose and Route of Administration

As appreciated by those skilled in the art, dosage considerations can be determined by comparison with the analogous products found in the clinic. Thus, anti-PSCA antibodies can be administered at doses in the range of 5 to 400 mg / m 2, with the lower doses used, for example in connection with safety studies. The affinity of the anti-PSCA antibodies to the affinity of a known antibody for its target is a parameter used by those skilled in the art to determine analogous dosage regimens. In addition, anti-PSCA antibodies that are fully human antibodies, compared to the chimeric antibody, have a slower elimination; consequently, the dosage in patients with such fully human anti-PSCA antibodies may be lower, perhaps in the range of 50 to 300 mg / m 2, and still remain effective. The dosage in mg / m 2, as opposed to the conventional dose measurement in mg / kg, is a surface-based measurement and is a convenient dosage measurement which is designed to include patients of all sizes from infants to adults.

Three distinct administration approaches are useful for administration of anti-PSCA antibodies. Conventional intravenous administration is a standard delivery technique for many tumors. However, in connection with tumors in the peritoneal cavity, such as ovarian tumors, bile duct, other duets, and the like, intraperitoneal administration may prove to be favorable for obtaining a high dose of antibody in the tumor and also for minimizing antibody clearance. Similarly, certain solid tumors have a vasculature that is appropriate for regional perfusion. The regional perfusion allows a high dose of antibodies at the site of a tumor and minimizes the short term elimination of the antibody.

Clinical Development Plan (CDP)

Overview: CDP follows and develops anti-PSCA antibody treatments in conjunction with adjuvant therapy, monotherapy, and as an imaging agent. The assays initially demonstrate safety and subsequently confirm the efficacy at repeated doses. Assays are opened comparing standard chemotherapy with standard therapy plus anti-PSCA antibodies. As will be appreciated, one criterion that can be used in connection with patient enrollment is the levels of PSCA expression in their tumors as determined by biopsy.

As with any therapy based on infusion of proteins or antibodies, safety issues are primarily related to (i) cytokine release syndrome, i.e., hypotension, fever, tremors, chills; (ii) the development of an immunogenic response against the material (i.e., development of human antibodies by the patient against antibody therapy, or HAHA response); and, (iii) toxicity to normal cells expressing PSCA. Standard tests and follow-ups are used to monitor each of these safety issues. Anti-PSCA antibodies are considered to be safe following administration to humans.

Example 29

Clinical Trial in Humans: Monotherapeutic with

Anti-PSCA Human Antibody

Anti-PSCA antibodies are safe in connection with the adjuvant assay discussed above, a human Phase II clinical trial confirms the efficacy and optimum dosage for monotherapy. Such an assay is achieved, and involves the same safety and outcome analyzes, which the adjuvant assay described above with the exception that patients do not receive chemotherapy concurrently with receipt of doses of anti-PSCA antibodies.

Example 30

Clinical Trial in Human Beings; Production of Diagnostic Imaging with Anti-PSCA Antibodies

Once again, as the adjuvant therapy discussed above is safe within the safety criteria discussed above, a human clinical trial is carried out in connection with the use of anti-PSCA antibodies as a diagnostic imaging agent. The protocol is designed in a manner substantially similar to those described in the art, such as in Divgi et al. J. Natl. Cancer Inst. 83: 97-104 (1991). Antibodies are considered to be both safe and efficacious when used as a diagnostic modality.

Example 31

Human Clinical Assay Adjuvant Therapy with Human Anti-PSCA Antibody and Chemotherapy, Radiation, and / or Hormone Ablation Therapy

A human phase I clinical trial is started to evaluate the safety of six intravenous doses of a human anti-PSCA antibody in connection with the treatment of a solid tumor, for example a cancer of a tissue listed in Table I. In the study, the safety of single doses of anti-PSCA antibodies when used as adjuvant therapy to an antineoplastic or chemotherapeutic or hormonal ablation agent as defined herein, such as, without limitation: cisplatin, topotecan, doxorubicin, adriamycin, taxol, Lupron, Zoladex, Eulexin, Casodex, Anandron or the like is evaluated. The assay design includes administration of approximately six single doses of an anti-PSCA antibody with dosage of the antibody increasing from about 25 mg / m 2 to about 275 mg / m 2 over the course of the treatment according to the following regimen, or a similar one:

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

MAb dose 25 75 125 175 225 275 mg / m2 mg / m2 mg / m2 mg / m2 mg / m2 mg / m2

Chemotherapy + + + + + + (standard dose)

Patients are followed closely for one week after each antibody and chemotherapy administration. In particular, patients assessed for safety issues mentioned above: (i) cytokine release syndrome, ie, hypotension, fever, tremors, chills; (ii) the development of an immunogenic response against the material (i.e., development of human antibodies by the patient against human antibody therapy, or HAHA response); and, (iii) toxicity to normal cells expressing PSCA. Standard tests and follow-ups are used to monitor each of these safety issues. Patients are also assessed for clinical outcome, and particularly for reduction in tumor mass as evidenced by MRI or other imaging technique.

Anti-PSCA antibodies are shown to be safe and effective. Phase II assays confirm efficacy and refine optimal dosing.

Reference Example 32 RNA Interference (RNAI) RNA interference technology (RNAI) is implemented in a variety of cell assays relevant to oncology. IRNA is a double-stranded RNA activated post-transcriptional gene silencing mechanism (dsRNA). IRNA induces the specific degradation of mRNA leading to changes in protein expression and subsequently in gene function. In mammalian cells, these dsRNAs called short interfering RNAs (siRNAs) have the correct composition to activate the degradation-directed iRNA pathway, specifically some mRNAs. See, Elbashir S.M., et al., Duplexes of 21-nucleotide RNAs Mediate RNA interference in Cultured Mammalian Cells, Nature 411 (6836): 494-8 (2001). Thus, iRNA technology is successfully used in mammalian cells to silence target genes.

Loss of control of cell proliferation is a characteristic of cancer cells; thus, the evaluation of the role of PSCA in cell survival / proliferation assays is relevant. Accordingly, iRNA was used to investigate the function of the PSCA antigen. To generate siRNA for PSCA, algorithms were used to predict oligonucleotides that exhibit critical molecular parameters (G: C content, melt temperature, etc.) and have the ability to significantly reduce expression levels of PSCA protein when introduced into cells. According to this Example, PSCA siRNA compositions comprising siRNA (short-chain double-stranded interfering RNA) corresponding to the PSCA protein nucleic acid ORF sequence or subsequences thereof are used. Thus, the subsequences of siRNAs that are used in this way generally have 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 , 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35 or more than 35 contiguous RNA nucleotides in length. These siRNA sequences are complementary and not complementary to at least a portion of the mRNA coding sequence. In a preferred embodiment, the subsequences are 19-25 nucleotides in length, more preferably 21-23 nucleotides in length. In preferred embodiments, these siRNAs reach the PSCA antigen knockdown in cells expressing the protein and have functional effects as described below. The selected siRNA (oligo PSCA.b) was tested in numerous cell lines in the survival / proliferation MTS assay (measures cell metabolic activity). Tetrazolium-based colorimetric assays (i.e., MTS) detect only viable cells, since living cells are metabolically active and can therefore reduce the tetrazolium salts in colored formazan compounds; dead cells, however, can not. In addition, this oligo PSCA.b achieved knockdown of PSCA antigen on cells expressing the protein and had functional effects as described below using the following protocols.

Mammalian siRNA transfections: On the day prior to siRNA transfection, the different cell lines were plated on medium (RPMI 1640 with 10% FBS without antibiotics) at 2x103 cells / well in 80 μΐ (96-well plate format ) for the survival / MTS assay. In parallel with the PSCA-specific siRNA oligo, the following sequences were included in all experiments as controls: a) cells transfected by Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and hybridization buffer (no siRNA); b) luciferase-4 specific siRNA (target sequence: 5'-AAGGGACGAAGACGAACACUUCT-3 ') (SEQ ID NO: 77); and c) Eg5 specific siRNA (target sequence: 5'-AACT-GAAGACCTGAAGACAATAA-3 ') (SEQ ID NO: 78). SiRNAs were used at a final concentration of lipofectamine 2000 at 10 nM and 1 pg / ml. The procedure was as follows: The siRNAs were first diluted in OPTIMEM (0.1 × 10% concentrated serum-free transfection medium, Invitrogen) and incubated at RT for 5-10 min. Lif of ectamine 2000 was diluted to 10æg / ml (10-fold concentrated) for the total number of transfections and incubated for 5-10 minutes at room temperature (RT). Appropriate amounts of concentrated 10 times diluted Lipofectamine 2000 were mixed 1: 1 with concentrated 10-fold diluted siRNA and incubated at RT for 20-30 &quot; (5 fold concentrated solution). 20 μl of the 5 fold concentrated transfection solutions were added to respective samples and incubated at 37 ° C for 96 hours prior to analysis.

MTS assay: The MTS assay is a colorimetric method for determining the number of viable cells in proliferation, cytotoxicity or chemosensitivity assays based on a tetrazolium compound [3- (4,5-dimethylthiazol-2-yl) -5- ( 3-carboxymethoxyanil) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt; MTS (b)] and an electron coupling reagent (phenazine ethosulfate; PES). Assays were performed by adding a small amount of the Solution Reagent directly to culture wells, incubating for 1-4 hours and then recording the absorbance at 490 nm with a 96 well plate reader. The amount of colored formazan product as measured by the amount of absorbance at 490 nm is directly proportional to the mitochondrial activity and / or number of living cells in culture.

In order to address PSCA function in cells, the PSCA is quenched by the transfection of endogenously PSCA-expressing cell lines.

Another embodiment of the invention is a method for analyzing cell proliferation related to PSCA is the measurement of DNA synthesis as a marker for proliferation. The labeled DNA precursors (i.e., 3 H-Thymidine) are used and their incorporation into the DNA is quantified. Incorporation of the labeled precursor into the DNA is directly proportional to the amount of cellular cell division that occurs in the culture. Another method used to measure cell proliferation is the performance of clonogenic assays. In these assays, a defined number of cells are plated in the appropriate matrix and the number of colonies formed after a growth period after the siRNA treatment is counted.

In validation of PSCA cancer targets, complementation of cell survival / proliferation analysis with apoptosis studies and production of cell cycle profiles is considered. The biochemical characteristic of the apoptotic process is the genomic fragmentation of DNA, an irreversible event that commits the cell to die. One method of observing fragmented DNA in cells consists of the immunological detection of histone-complexed DNA fragments by an immunoassay (i.e., cell death detection ELISA) which measures the enrichment of histone complexed DNA fragments (mono- and oligonucleosomes) in the cytoplasm of apoptotic cells. This assay does not require pre-labeling of the cells and can detect DNA degradation in non-proliferating cells in vitro (i.e., newly isolated tumor cells).

The most important effector molecules to trigger the death of apoptotic cells are caspases. Caspases are proteases which when cleaved cleave numerous substrates at the carboxy-terminal site of an aspartate residue mediating very early stages of apoptosis upon activation. All caspases are synthesized as proenzymes and activation involves the cleavage of aspartate residues. In particular, caspase 3 appears to play a central role in the initiation of cellular apoptosis events. Assays for the determination of capsase 3 activation detect early events of apoptosis. Following iRNA treatments, Western blot detection of the presence of active caspase 3 or proteolytic cleavage of products (i.e., PARP) found in apoptotic cells further supports an active induction of apoptosis. Since the mechanisms that result in apoptosis are complex, each has its advantages and limitations. Consideration of other criteria / targets such as cell morphology, chromatin condensation, membrane vesicle formation, apoptotic bodies helps to further support cell death as apoptotic. Since not all gene targets regulating cell growth are anti-apoptotic, the DNA content of permeabilized cells is measured to obtain the DNA profile profile or cell cycle profile. Nuclei of apoptotic cells contain less DNA due to extravasation out of the cytoplasm (sub-Gl population). In addition, the use of DNA strands (i.e., propidium iodide) also differentiates between different cell cycle phases in the cell population due to the presence of different amounts of DNA in G0 / G1, S and G2 / M. In these studies subpopulations can be quantified.

For the PSCA gene, iRNA studies facilitate understanding of the contribution of the gene product to the cancer pathways. Such active iRNA molecules have utility in screening assays to screen for MAbs that are active antitumor therapies. In addition, siRNAs are administered as therapeutics to cancer patients to reduce the malignant growth of various types of cancer, including those listed in Table 1. When the PS CA plays a role in cell survival, cell proliferation, tumorigenesis or apoptosis, it is used as a target for diagnostic, prognostic, preventive and / or therapeutic purposes.

Reference Example 33

PSCA protein detection in specimens of patients with IHC cancer

Expression of the PSCA protein in tumor specimens from cancer patient was detected using the HA1-4,117 antibody. Paraffin-embedded formalin-fixed tissues were cut into 4 micron sections and mounted on glass slides. The sections were dewaxed, rehydrated and treated with antibody recovery solution (Antigen Retrieval Citra Solution; BioGenex, 4600 Norris Canyon Road, San Ramon, CA, 94583) at elevated temperature. The sections were then incubated in fluorescein-conjugated anti-PSCA monoclonal antibody, Hal-4.117, for 16 hours at 4øC. The slides were washed three times in buffer and further incubated with rabbit anti-fluorescein antibody for 1 hour and, after washing in buffer, immersed in DAKO EnVision + ™ peroxidase-conjugated rabbit anti-immunoglobulin goat secondary antibody (DAKO Corporation , Carpenteria, CA) for 30 minutes. The sections were then washed in buffer, developed using the DAB kit (SIGMA Chemicals), counterstained using hematoxylin, and analyzed by light field microscopy. The results show the expression of PSCA in prostate adenocarcinoma tumor cells (A, B), transitional bladder carcinoma (C) and ductal adenocarcinoma of the pancreas (D). These results indicate that PSCA is expressed in human cancers and that antibodies directed against this antigen are useful as diagnostic reagents (Figure 17).

These results indicate that PSCA is a target for diagnostic, prognostic and / or therapeutic applications in cancer.

Throughout the present application, various data contents of websites, publications, patent applications and patents are referenced. (The websites are referenced through their Uniform Resource Locator addresses, or URL, on the World Wide Web).

Frames

Table I: Tissues expressing PSCA when malignant.

Prostate Pancreas

Bladder

Kidney colon

Lung

Ovary

Mama

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id ΰ f'P &lt; ': iTT &gt; ... Χί ίΧ · &quot;! Ρ7: Ω i Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ο Ο Ο Ο Ο Ο Ο Ο Ο Ο Ο (1), (2), (2) and (4) and (4) and (4) and (4) '' '' '' '' '' '' '' '' '' '' '' '' '' '' '' '' '' '' (1), (2) and (2) and (2) are as follows: 7, 7, 7, 7, 7, 7, 7, 7, 7, 7, 7, 7, 7, 7, s vs I &amp; 7 ^ 1 W 7 O: 7 and to 7 to 7 7: 7 to 7 m ** O iS φ 7 &quot; Cs Pd Φ '7 &quot; S: § A (S): ## STR1 ## wherein X is as defined in claim 1, Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ Φ.

.

Tj v * H?

(S) - (-) - - - - - - - - - - - - - - -

(1), (2) and (4). -: -S, · &quot; (f-s,. &Quot; ν '· ---, &quot;

(S). I l · &quot; C I I W

Gt; gt; &lt; / RTI &gt; g &lt; / RTI &gt; ? Y ® T? · - h -§ and a 7

h g 1 ^ 77 7 ^ 77 ^ g 7 ^ &quot; I S 3

Ή Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Ώ Î ±: j 1 vt ·, 7¾ g 7 7 1 -1 w! 17 s *

Pi-5-β-β-O-β- [Î ±] -7- [Î ±] -7- [ (1), (2), (3) and (4) and (4)

&lt; &quot;

TABLE IV (G):

Coverage of the calculated population achieved through

different combinations of HLA supertypes

HLA π "supertypes Phenotypic frequency

CaucasianaNegraJaponesa ChinesePhysical HispanicNa 83, 86, 1 87.5 88.4 86.3 86.2 A2, A3 and B7 99.5 98.1 100.0 99.5 99.4 99.3 A2, A3, 99 , 9 99.6 100.0 99.8 99.9 99.8 B7, A24, 844 and AI2, A3, B7, A24, B44, Al, 827, B62, and B58

Table VIII: Affinity calculated using Graphpad Prism software: Sigmoid Dose-Response Equation (variable slope).

Table IX: Affinity based on

FACS on Mabs for fully human PSCA

Table X: Antibodies that cross-react with Monkey PSCA and / or Mouse PSCA. Cross Reaction ID with Cross Reaction

PSCA Monkey Hybridoma with PSCA from

Hl-1.10 Mouse

Hal-1.16 +

Hal-1.78 +

Hal-1.41 +

Hal-4.5 +

Hal-4.37 +

Hal-4.117 + +

Hal-4.120 +

Hal-4.121 +

Hal-5.99 +

Coverage of the calculated population achieved through

different combinations of HLA supertypes

Supertypes ",. . rTT, Phenotype frequency

of HLA

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

Table XI: PSCA: clustering of epitopes through FACS analysis

Table XI: PSCA: clustering of epitopes via FACS analysis

LIST OF SEQUENCES &lt; 110 &gt; Agensys, Inc. Gudas, Jean Jakobovits, Aya Xiao-Chi, Jia Morrison, Robert Kendall Morrison, Karen Jane Meyrick Shao, Hui Challita-Eid, Pia M. Raitano, Arthur B.

&lt; 120 &gt; ANTIBODIES AND RELATED MOLECULES CONNECTING PSCA PROTEINS &lt; 130 &gt; 51158-20088.41 &lt; 140 &gt; Not yet signed &lt; 141 &gt; Presented together with the present &lt; 150 &gt; 60/616 381 &lt; 151 &gt; 05-10-2004 &lt; 150 &gt; 60 / 617,881 &lt; 151 &gt; 10-12-2004 &lt; 150 &gt; 60 / 621,310 &lt; 151 &gt; 10/21/2004 &lt; 150 &gt; 60 / 633,077 &lt; 151 &gt; 02-12-2004 &lt; 150 &gt; 10 / 857,484 &lt; 151 &gt; 28-05-2004 &lt; 150 &gt; 60 / 475,064 &lt; 151 &gt; 30-05-2003 &lt; 160 &gt; 78 &lt; 170 &gt; FastSEQ for Windows Version 4.0

&lt; 210 &gt; 1 &lt; 211 &gt; 990 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 220 &gt; &lt; 221 &gt; CDS &lt; 222 &gt; (18) .. (389) &lt; 221 &gt; misc_feature &lt; 222 &gt; 543 &lt; 223 &gt; n = a, t, c or g <4 0 0> 1 agggagaggc agtgacc atg aag get gtg ctg ett gcc ctg ttg atg gca 50

Met Lys Ala Val Leu Leu Ala Leu Leu Met Ala 1 5 10 ggc ttg gcc ctg cag cca ggc act gcc ctg ctg tgc tac tcc tgc aaa 98

Gly Leu Ala Leu Gin Pro Gly Thr Ala Leu Leu Cys Tyr Ser Cys Lys 15 20 25 gcc cg gtg age aac gag gac tgc ctg cag gtg gag aac tgc acc cag 14 6

Ala Gin Val Ser Asn Glu Asp Cys Leu Gin Val Glu Asn Cys Thr Gin 30 35 40 ctg ggg gag cag tgc tgg acc geg ege to ege gca gtt ggc etc. ctg 194

Leu Gly Glu Gin Cys Trp Thr Ala Arg He Arg Ala Val Gly Leu Leu 45 50 55 acc gtc until age aaa ggc tgc age ttg aac tgc gtg gat gac tea cag 242

Thr Val lie Ser Lys Gly Cys Ser Leu Asn Cys Val Asp Asp Ser Gin '60 65 70 75 gac tac t gt g ggc aag aag aac tg tg t gt gac acc gac ttg 290

Asp Tyr Tyr Val Gly Lys Lys Asn lie Thr Cys Cys Asp Thr Asp Leu 80 85 90 tgc aac gcc age ggg gcc cat gcc ctg cag ccg gcc gcc ate ett 338

Cys Asn Ala Ser Gly Ala His Ala Leu Gin Pro Ala Ala Ala lie Leu • 95 100 105 cg gt etc. cct gca etc ggc ctg ctg etc tgg gga ccc ggc cag eta 386

Ala Leu Leu Pro Ala Leu Gly Leu Leu Leu Trp Gly Pro Gly Gin Leu 110 115 120 tag gctctggggg gceccgctgc agcccacact gggtgtggtg ccccaggcet 439 * ttgtgccact cctcacagaa cctggcccag tgggagcctg tcctggttcc tgaggcacat 499 cctaacgcaa gtttgaccat gtatgtttgc accccttttc cccnaaccct gaccttccca 559 tgggcctttt ccaggattcc cacccggcag atcagtttta gtgacacaga tccgcctgca 619 gatggcccct ccaacccttt ctgttgctgt ttccatggcc cagcattttc cacccttaac 679 cctgtgttca ggeaettett cccccaggaa gccttccctg cccaccccat ttatgaattg 739 agccaggttt ggtccgtggt gtcccccgca cccagcaggg gacaggcaat caggagggcc 799 cagtaaaggc tgagatgaag tggactgagt agaactggag gacaagagtt gacgtgagtt 859 cctgggagtt tccagagatg gggcctggag gcctggagga aggggccagg cctcacattt 919 gtggggctcc egaatggcag cctgagcaca gcgtaggccc ttaataaaca cctgttggat 979 to 990 aagccaaaaa

&lt; 210 &gt; 2 &lt; 211 &gt; 123 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 2

Met Lys Ala Val Leu Leu Ala Leu Leu Met Ala Gly Leu Ala Leu Gin IS 10 15

Pro Gly Thr Ala Leu Leu Cys Tyr Ser Cys Lys Ala Gin Val Ser Asn 20 25 30

Glu Asp Cys Leu Gin Val Glu Asn Cys Thr Gin Leu Gly Glu Gin Cys 35 40 45

Trp Thr Ala Arg Ile Arg Ala Val Gly Leu Leu Thr Val Ile Ser Lys 50 55 60

Gly Cys Ser Leu Asn Cys Val Asp Asp Ser Gin Asp Tyr Tyr Val Gly 65 70 75 80

Lys Lys Asn lie Thr Cys Cys Asp Thr Asp Leu Cys Asn Ala Ser Gly 85 90 95

Wing His Wing Leu Gin Pro Wing Wing Wing wing Leu Wing Leu Pro Wing 100 105 110

Leu Gly Leu Leu Leu Trp Gly Pro Gly Gin Leu 115 &lt; 210 &gt; 3 &lt; 211 &gt; 1020

&lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 220 &gt;

&lt; 221 &gt; CDS &lt; 222 &gt; (56) .. (427) &lt; 400 &gt; 3 tttgaggcca tataaagtca cctgaggccc tctccaccac agcccaccag tgacc atg 58

Met 1 aag get gtg ctg ett gee ctg ttg atg gea ggc ttg gee ctg cca cca 106

Lys Ala Vai Leu Leu Ala Leu Leu Met Ala Gly Leu Ala Leu Gin Pro 5 10 15 ggc act gee ctg ctg tgc tac tee tgc aaa gee cag gtg age aac gag 154

Gly Thr Ala Leu Leu Cys Tyr Ser Cys Lys Ala Gin Will Be Asn Glu 20 25 30 gac tgc ctg cag gtg gag aac tgc acc cag ctg ggg gag cag tgc tgg 202

Asp Cys Leu Gin Vai Glu Asn Cys Thr Gin Leu GLy Glu Gin Cys Trp 35 40 45 acc gcg ege to gge gt gt cctg acc gtc to age aaa ggc 250

Thr Ala Arg Ile Arg Ala Gly Leu Leu Thr Ile Ile Ser Lys Gly 50 55 60 65 tgc age ttg aac tgc gtg gat gac tea cag gac tac tac gtg ggc aag 298

Cys Ser Leu Asn Cys Go Asp Asp Ser Gin Asp Tyr Tyr Go Gly Lyâ 70 75 80 aag aac tgt gac acc gac tg g a g g g g g g g g g g g e g 6

Lys Asn Ile Thr Cys Cys Asp Thr Asp Leu Cys Asn Ala Ser Gly Ala 85 90 95 cat gee ctg cag ccg get gee gee et ett gcg ctg etc cct gea etc. 394

His Ala Leu Gin Pro Ala Ala Ala lie Leu Ala Leu Leu Pro Ala Leu 100 105 110 ggc ctg ctg etc tgg gga ccc ggc cag eta tag gctctggggg gccccgctgc 44 7

Gly Leu Leu Leu Trp Gly Pro Gly Gin Leu * 115 120 agcccacact gggtgtggtg ccccaggcct ctgtgccact cctcacacac ccggcccagt 507 gggagcctgt cctggttcct gaggcacatc ctaacgcaag tctgaccatg tatgtctgcg 567 cccctgtccc ccaccctgac cctcccatgg ccctctccag gactcccacc cggcagatcg 627 gctctattga cacagatccg cctgcagatg gcccctccaa ccctctctgc tgctgtttcc 687 atggcccagc attctccacc cttaaccctg tgctcaggca cctcttcccc caggaagcct 747 tccctgccca ccccatctat gacttgagcc aggtctggtc cgtggtgtcc cccgcaccca 807 gcaggggaca ggcactcagg agggcccggt aaaggctgag atgaagtgga ctgagtagaa 867 ctggaggaca ggagtcgacg tgagttcctg ggagtctcca gagatggggc ctggaggcct 927 ggaggaaggg gccaggcctc acattcgtgg ggctccctga atggcagcct cagcacagcg 987 taggccctta ataaacacct gttggataag cca 1020

&lt; 210 &gt; 4 &lt; 211 &gt; 123 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 4

Met Lys Wing Val Leu Leu Wing Leu Leu Met Allah Gly Leu Wing Leu Gin 15 10 15

Pro Gly Thr Ala Leu Leu Cys Tyr Ser Cys Lys Ala Gin Val Ser Asn 20 25 30

Glu Asp Cys Leu Gin Val Glu Asn Cys Thr Gin Leu Gly Glu Gin Cys 35 40 45

Trp Thr Ala Arg Ile Arg Ala Val Gly Leu Leu Thr Val Ile Ser Lys 50 55 60

Gly Cys Ser Leu Asn Cys Val Asp Asp Ser Gin Asp Tyr Tyr Val Gly 65 70 75 80

Lys Lys Asn lie Thr Cys Cys Asp Thr Asp Leu Cys Asn Ala Ser Gly 85 90 95

Wing His Wing Leu Gin Pro Wing Wing Wing wing Leu Wing Leu Pro Wing 100 105 110

Leu Gly Leu Leu Leu Trp Gly Pro Gly Gin Leu 115 120

&lt; 210 &gt; 5 &lt; 211 &gt; 888 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 220 &gt;

&lt; 221 &gt; CDS &lt; 222 &gt; (423) .. (707) &lt; 400 &gt; 5 tttgaggcca tataaagtca cctgaggccc tctccaccac agcccaccag tgaccatgaa 60 ggctgtgctg cttgccctgt tgatggcagg cttggccctg cagccaggca ctgccctgct 120 gtgctactcc tgcaaagccc aggcgcagtt ggcctcctga ccgtcatcag caaaggctgc 180 agcttgaact gcgtggatga ctcacaggac tactacgtgg gcaagaagaa catcacgtgc 240 tgtgacaccg acttgtgcac tcggcctgct gctctgggga cccggccagc tataggctct 300 ggggg &lt; 3cccc gctgcagccc acactgggtg tggtgcccca ggcetctgtg ccactcctca 360 cacacccggc ccagtgggag cctgtcctgg ttcctgaggc acatcctaac gcaagtctga 420 cc atg tat gtc tgc gcc cct gtc ccc cac cct gac cct ccc atg gcc 4 67

Met Tyr Val Cys Ala Pro Val Pro His Pro Asp Pro Pro Met Ala 15 10 15 Etc agg act ccc acc egg cag ate ggc tet att gac aca gat ccg 515

Leu Ser Arg Thr Pro Thr Arg Gin Ile Gly Ser Ile Asp Thr Asp Pro 20 25 30 cct gca gat gcc ccc tcc aac cct etc tgc tgc tgt ttc cat ggc cca 563

Pro Ala Asp Gly Pro Ser Asn Pro Leu Cys Cys Cys Phe His Gly Pro 35 40 45 gca ttc tcc acc ett aac cct gtg etc agg cac etc. ttc ccc cag gaa 611

Ala Phe Ser Thr Leu Asn Pro Val Leu Arg His Leu Phe Pro Gin Glu 50 55 60 gcc ttc cct gcc cac ccc tat tat gac ttg age cag gtc tgg tcc gtg 659

Ala Phe Pro Ala His Pro He Tyr Asp Leu Ser Gin Val Trp Ser Val 65 70 75 gtg tcc ccc gca ccc age agg gga cag gca etc agg agg gcc egg taa 707

Val Ser Pro Ala Pro Ser Arg Gly Gin Ala Leu Arg Arg Ala Arg * 80 85 90 aggetgagat gaagtggact gagtagaact ggaggacagg agtcgacgtg agttcctggg 767 agtctccaga gatggggcct ggaggcctgg aggaaggggc caggcctcac attcgtgggg 827 ctccctgaat ggcagcctca gcacagcgta ggcccttaat aaacacctgt tggataagee 887 a 888

&lt; 210 &gt; 6 &lt; 211 &gt; 94 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 6

Met Tyr Val Cys Ala Pro Val Pro His Pro Asp Pro Pro Met Ala Leu 15 10 15

Ser Arg Thr Pro Thr Arg Gin Ile Gly Ser Ile Asp Thr Asp Pro Pro 20 25 30

Ala Asp Gly Pro Ser Asn Pro Leu Cys Cys Cys Phe His Gly Pro Ala 35 40 45

Phe Ser Thr Leu Asn Pro Val Leu Arg His Leu Phe Pro Gin Glu Ala 50 55 60

Phe Pro Ala His Proline Tyr Asp Leu Ser Gin Val Trp Ser Val Val 65 70 75 SO

Pro-Ala-Pro-Ser-Arg-Gly-Gin-Ala-Leu-Arg-Arg-

&lt; 210 &gt; 7 &lt; 211 &gt; 1174 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 220 &gt;

&lt; 221 &gt; CDS &lt; 222 &gt; (424) .. (993) &lt; 400 &gt; 7 gacagtgaac cctgcgctga aggcgttggg gctcctgcag ttctggggca gccacaggcg 60 cccagggttt cgtgccgatc agcccaggac ggtcttcccg gtgcagtttc tgatgcgggg 120 agggcagtgc tgccttccgg tcaccaggac cagtgct CAG cccgcctgct tgaccccctt 180 acttagctgg ggtccaatcc atacccaatt tagatgattc agacgatggg atttgaaact 240 tttgaactgg gtgcgactta agcactgccc tgctgtgcta ctcctgcaaa gcccaggtga 300 ctgcctgcag gtggagaact gcaacgagga gcacccagct gggggagcag tgctggaccg 360 cgcgcatccg cgcagttggc ctcctgaccg tcatcagcaa aggctgcagc ttgaactgcg 420 tgg atg act cac agg act act acg tgg gca aga aca tea cgt get 4 68

Met Thr His Arg Thr Thr Thr Trp Ala Arg Arg Thr Ser Arg Ala 15 10 15 gtg aca ccg act tgt gca acca cca geg ggg ccc atg ccc tgc age egg 516

Val Thr Pro Thr Cys Ala Thr Pro Ala Gly Pro Met Pro Cys Ser Arg 20 25 30 ctg cca cc tcc ctg cg cg teg gcc tgc tgc tet ggg 5 64

Leu Pro Pro Ser Leu Arg Cys Ser Leu His Ser Ala Cys Cys Ser Gly 35 40 45 gac ccg gcc age tat agg etc tgg ggg gcc ccg ctg cag ccc aca ctg 612

Asp Pro Ala Ser Tyr Arg Leu Trp Gly Ala Pro Leu Gin Pro Thr Leu 50 55 60 ggt gtg gcc ccc gcc gtg cca etc etc. aca cac ccg gee cag 660

Gly Val Val Pro Gin Ala Ser Val Pro Leu Leu Thr His Pro Ala Gin 65 70 75 tgg gag cct gtc ctg gtt cct gag gca cat cct aac gca agt ctg acc 708

Trp Glu Pro Val Leu Val Pro Glu Ala His Pro Asn Ala Ser Leu Thr 80 85 90 95 atg tat gtc tgc gcc cct gtc ccc cac cct gac cct ccc atg gcc etc 756

Met Tyr Val Cys Ala Pro Val Pro His Pro Asp Pro Pro Met Ala Leu 100 105 tcc agg act ccc acc egg cag ate ggc tct att gac aca gat ccg cct 804

Ser Arg Thr Pro Thr Arg Gin Ile Gly Ser Ile Asp Thr Asp Pro Pro 115 120 125 gca ggc ccc tcc aac cct etc tgc tgc tgt ttc cat ggc cca gca 852

Ala Asp Gly Pro Ser Asn Pro Leu Cys Cys Cys Phe His Gly Pro Ala 130 135 140 ttc tcc acc ett aac cct gtg etc. agg cac etc. ttc ccc cag gaa gcc 900

Phe Ser Thr Leu Asn Pro Val Leu Arg His Leu Phe Pro Gin Glu Ala 145 150 155 ttc ccc gcc cac ccc tat tat gac ttg age cag gtc tgg tcc gtg gtg 948

Phe Pro Ala His Proline Tyr Asp Leu Ser Gin Val Trp Ser Val Val 160 165 170 175 tcc ccc gca ccc age agg gga cag gca etc agg agg gcc egg taa 993

Ser Pro Ala Pro Ser Arg Arg Gly Gin Ala Leu Arg Arg Ala Arg * i 180 185 aggetgagat gaagtggact gagtagaact ggaggacagg agtcgacgtg agttcctggg 1053 agtctccaga gatggggcct ggaggcctgg aggaaggggc caggcctcac attcgtgggg 1113 ctccctgaat ggcagcctca gcacagcgta ggcccttaat aaacacctgt tggataagee 1173 a 1174

&lt; 210 &gt; 8 &lt; 211 &gt; 189 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 8

Met Thr His Arg Thr Thr Thr Trp Ala Arg Arg Thr Ser Arg Al Val 15 10 15

Thr Pro Thr Cys Ala Thr Pro Ala Gly Pro Met Pro Cys Ser Arg Leu 20 25 30

Pro-Ser-Ser-Arg-Ser-Ser-Ser-Ser-Ser-Ser-Arg

Pro Wing Wing Tyr Arg Leu Trp Gly Wing Pro Leu Gin Pro Thr Leu Gly 50 55 60

Val Val Pro Gin Ala Ser Val Pro Leu Leu Thr His Pro Ala Gin Trp 65 70 75 80

Glu Pro Val Leu Val Pro Glu Ala His Pro Asn Ala Ser Leu Thr Met 85 90 95

Tyr Val Cys Ala Pro Val Pro His Pro Asp Pro Pro Met Ala Leu Ser 100 105 110

Arg Thr Pro Thr Arg Gin Ile Gly Ser He Asp Thr Asp Pro Pro Ala 115 120 125

Asp Gly Pro Ser Asn Pro Leu Cys Cys Cys Phe His Gly Pro Ala Phe 130 135 140

Ser Thr Leu Asn Pro Val Leu Arg His Leu Phe Pro Gin Glu Ala Phe 145 150 155 160

Pro Ala His Proline Tyr Asp Leu Ser Gin Val Trp Ser Val Val Ser 165 170 175

Pro Ala Pro Ser Arg Gly Gin Ala Leu Arg Arg Ala Arg 180 185 &lt; 210 &gt; 9 &lt; 211 &gt; 1660

&lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 220 &gt;

&lt; 221 &gt; CDS &lt; 222 &gt; (910). . (1479) &lt; 400 &gt; 9 gacagtgaac cctgcgctga aggcgttggg gctcctgcag ttctggggca gccacaggcg 60 cccagggttt egtgccgatc agcccaggac ggtettcccg gtgcagttte tgatgcgggg 120 agggcagtgc tgccttccgg tcaccaggac cagtgctcag cccgcctgct tgaccccctt 180 acttagctgg ggtccaatcc atacccaatt tagatgattc agacgatggg atttgaaact 240 tttgaactgg gtgcgactta agcactgccc tgctgtgcta ctcctgcaaa gcccaggtga 300 ctgcctgeag gtggagaact gcaacgagga gcacccagct gggggagcag tgctggaccg 360 cgcgeatccg tgagtggggg gaegacagec gccaggccta ggtctctgcc aetgaaetat 420 taatctttct ggccatctgt ccgcatctgt gtgctgtttt ccttccacct gtccccgaec 480 cgtcccgcac ctgcaccccc aacaatcacc cagcatctgt ccctccagcc atcctcctcc 540 atctgccaet cctccactca tctgtccctc cccatcctcc atcttccact cctccaccca 600 tctgtccctc cccatccctg agctcactta ctcactcacc ccatttctga cgctcagcgg 660 gtggtccatc tgccteggac atctggatag ggctgagacc agggccgaga ccaggccctc 720 gcactgcttg caatcctgag gccagcccag ggggactcta gagcattagg cagggtggga 780 caggaggagg cctggggcag gtcaggcagg tgagcacaca gggcagcccc atccccggat 840 occgctgctc cccaggcg gtgcgcccgcgtcat cagcaaaggc tgcagcttga 900 actgcgtgg atg act cc agg act actg tgg gc aga aga cgt 951 Met Thr His Arg Thr Thr Thr Trp Ala Arg Arg Thr Ser Arg 15 10 get gtg aca ccg act tgt gca acg cca geg ggg ccc atg ccc tgc age 999

Ala Val Thr Pro Thr Cys Ala Thr Pro Ala Gly Pro Met Pro Cys Ser 15 20 25 30 egg ctg ccg cca tcc tgg tgc tee ctg cg teg gee tgc tgc tet 1047

Arg Leu Pro Pro Ser Leu Arg Cys Ser Leu His Ser Ala Cys Cys Ser 35 40 45 ggg gac ccg gee age tat agg etc. tgg, ggg gee ccg ctg cag ccc aca 1095

Gly Asp Pro Ala Ser Tyr Arg Leu Trp Gly Ala Pro Leu Gin Pro Thr 50 55 60 ctg ggt gtg gtg ccc cag gee tet gtg cca etc etc aca cac ccg gee 1143

Leu Gly Val Val Pro Gin Ala Ser Val Pro Leu Leu Thr His Pro Ala 65 70 75 cag tgg gag cct gtc ctg gtt cct gag gca cat cct aac gca agt ctg 1191

Gin Trp Glu Pro Val Leu Val Pro Glu Ala His Pro Asn Ala Ser Leu 80 85 90 acc atg tat gtc tgc gee cct gtc ccc cac cct gac cct ccc atg gee 1239

Thr Met Tyr Val Cys Ala Pro Val Pro His Pro Asp Pro Pro Met Ala 95 100 105 110 etc tcc agg act ccc acc egg cag ate ggc tet att gac aca gat ccg 12Θ7

Leu Ser Arg Thr Pro Thr Arg Gin Ile Gly Ser Ile Asp Thr Asp Pro 115 120 125 cct gca gat ccc tec aac cct et tgc tgc tgt ttc cat ggc cca 1335

Pro Ala Asp Gly Pro Ser Asn Pro Leu Cys Cys Cys Phe His Gly Pro 130 135 140 gca ttc tec acc ett aac cct gtg etc. agg cac etc. ttc ccc cag gaa 1383

Ala Phe Ser Thr Leu Asn Pro Val Leu Arg His Leu Phe Pro Gin Glu 145 150 155 gee ttc cct gee cac ccc tat tat gac ttg age cag gtc tgg tec gtg 14 31

Ala Phe Pro Ala His Proline Tyr Asp Leu Ser Gin Val Trp Ser Val 160 165 170 gtg ccc gca ccc agg agg gag cca gca etc agg agg gee egg taa 14 79

Val Ser Pro Ala Pro Ser Arg Gly Gin Ala Leu Arg Arg Ala Arg * 175 180 185 aggetgagat gaagtggact gagtagaact ggaggacagg agtcgacgtg agttcctggg 1539 agtctccaga gatggggcct ggaggcctgg aggaaggggc caggcctcac attcgtgggg 1599 ctccctgaat ggcagcctca gcacagcgta ggcccttaat aaacacctgt tggataagee 1659 a 1660

&lt; 210 &gt; 10 &lt; 211 &gt; 189 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 4 0 0 &gt; 10

Met Thr His Arg Tht Thr Thr Trp Ala Arg Arg Thr Set Arg Ala Val

IS 10 IS

Thr Pro Thr Cys Ala Thr Pro Ala Gly Pro Met Pro Cys Ser Arg Leu 20 25 30

Pro-Ser-Ser-Arg-Ser-Ser-Ser-Ser-Ser-Ser-Arg

Pro Wing Wing Tyr Arg Leu Trp Gly Wing Pro Leu Gin Pro Thr Leu Gly SO 55 60

Val Val Pro Gin Ala Ser Val Pro Leu Leu Thr His Pro Ala Gin Trp 65 70 75 80

Glu Pro Val Leu Val Pro Glu Ala His Pro Asn Ala Ser Leu Thr Met 85 90 95

Tyr Val Cys Ala Pro Val Pro His Pro Asp Pro Pro Met Ala Leu Ser 100 105 110

Arg Thr Pro Thr Arg Gin Ile Gly Ser Ile Asp Thr Asp Pro Pro Ala 115 120 125

Asp Gly Pro Ser Asn Pro Leu Cys Cys Cys Phe His Gly Pro Ala Phe 130 135 140

Ser Thr Leu Asn Pro Val Leu Arg His Leu Phe Pro Gin Glu Ala Phe 145 150 155 160

Pro Wing His Pro He Tyr Asp Leu Ser Gin Val Trp Ser Val Val Ser 165 170 175

Pro Ala Pro Ser Arg Arg Gly Gin Ala Leu Arg Arg Ala Arg 180 185

&lt; 210 &gt; 11 &lt; 211 &gt; 1020 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 220 &gt; &lt; 221 &gt; CDS &lt; 222 &gt; (83) .. (427) &lt; 4 0 0 &gt; 11 tttgaggcca tataaagtca cctgaggccc tctccaccac agcccaccag tgaccatgaa 60 ggctgtgctg cttgccctgt tg atg gca ggc ttg gcc ctg cag cca ggc act 112

Met Ala Gly Leu Ala Leu Gin Pro Gly Thr 1 5 10 gcc ctg ctg tgC tac tcc tgc aaa gcc cag gtg age aac gag gac tgc 160

Ala Leu Leu Cys Tyr Ser Cys Lys Ala Gin Val Ser Asn Glu Asp Cys 15 20 25 ctg cg gtg gag aac tgc acc cag ctg ggg gag cag tgc tgg acc geg 208

Leu Gin Val Glu Asn Cys Thr Gin Leu Gly Glu Gin Cys Trp Thr Ala 30 35 40 ege gca gtt gg cg gt cg gtc acc gtc till age aaa ggc tgc age 256

Arg Ile Arg Ala Val Gly Leu Leu Thr Val Ile Ser Lys Gly Cys Ser 45 50 55 ttg aac tgc gtg gat gac gag gac tac gtg ggc aag aag aac 304

Leu Asn Cys Val Asp Asp Ser Gin Asp Tyr Tyr Val Gly Lys Lys Asn 60 65 70 tgt tgt tgt tgt tgt tgt tgt tg tt tt tt tt tt tt tt tg tt tt tt tt tt tt tt tt tt tt tt tt tt tt tt tt tt tt tt tt tt tt tt His Wing 75 80 85 90 ctg cag ccg get gcc gcc ate ett geg ctg etc cct gca etc ggc ctg 400

Leu Gin Pro Ala Ala Ala lie Leu Ala Leu Leu Pro Ala Leu Gly Leu 95 100 105 ctg etc tgg gga ccc ggc cag eta tag gctctggggg gccccgctgc 447

Leu Leu Trp Gly Pro Gly Gin Leu * 110 agcccacact gggtgtggtg ccccaggcct ctgtgccact cctcacacac ccggcccagt 507 gggagcctgt cctggttcct gaggcacatc ctaacgcaag tctgaccatg tatgtctgcg 567 cccctgtccc ccaccctgac cctcccatgg ccctctccag gactcccacc eggeagateg 627 getetattga cacagatccg cctgcagatg gcccctccaa ccctctctgc tgctgtttcc 687 atggcccagc attctccacc cttaaccctg tgctcaggca cctcttcccc caggaagcct 747 tccctgccca ccccatctat gaettgagee aggtctggte cgtggtgtcc cccgeaccca 807 gcaggggaca ggcactcagg agggcccggt aaaggctgag atgaagtgga ctgagtagaa 867 ctggaggaca ggagtcgacg tgagttcctg ggagtctcca gagatggggc ctggaggcct 927 ggaggaaggg gccaggcctc acattcgtgg ggctccctga atggcagcct cagcacagcg 987 taggccctta ataaacacct gttggataag cca 1020

&lt; 210 &gt; 12 &lt; 211 &gt; 114 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 4 0 0 &gt; 12

Met Ala Gly Leu Ala Leu Gin Pro Gly Thr Ala Leu Leu Cys Tyr Ser 1 5 10 15

Cys Lys Ala Gin Val Ser Asn Glu Asp Cys Leu Gin Val Glu Asn Cys 20 25 30

Thr Gin Leu Gly Glu Gin Cys Trp Thr Ala Arg Ile Arg Ala Val Gly 35 40 45

Leu Leu Thr Val lie Ser Lys Gly Cys Ser Leu Asn Cys Val Asp Asp 50 55 60

Ser Gin Asp Tyr Tyr val Gly Lys Lys Asn lie Thr Cys Cys Asp Thr 65 70 75 80

Asp Leu Cys Asn Ala Ser Gly Ala His Ala Leu Gin Pro Ala Ala Ala 85 90 95

He Leu Wing Leu Leu Pro Wing Leu Gly Leu Leu Leu Trp Gly Pro Gly 100 105 110

Gin Leu

&lt; 210 &gt; 13 &lt; 211 &gt; 148 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 4 0 0 &gt; 13

Val Ala Ala Pro Arg Trp Val Leu Ser Gin Val Gin Leu Gin Glu Ser 15 10 15

Gly Pro Gly Leu Val Lys Pro Ser Gin Thr Leu Ser Leu Thr Cys Thr 20 25 30

Val Ser Gly Gly Ser is Ser Gly Gly Tyr Tyr Trp lie Trp lie 35 40 45

Arg Gin His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr 50 55 60

Asn Gly Asn Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Met 65 70 75 80

Ser Val Asp Thr Ser Lys Asn Gin Phe Ser Leu Lys Leu Ser Ser Val 85 90 95

Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Glyl Thr 100 105 110

Met lys Arg Arg Gly Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gin Gly Thr 115 120 125

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Lys Gly 130 135 140

Pro Ser Val Phe &lt; 210 &gt; 14 &lt; 211 &gt; 155

&lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 4 0 0 &gt; 14

Gin Leu Thr Gin Ser Pro Ser Ser Ser Val Ser Ala Ser Val Gly Asp Arg 15 10 15

Val Thr He Thr Cys Arg Ala Ser Arg Gly Ile Ser Ser Trp Leu Ala 20 25 30

Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie Tyr Thr 35 40 45

Ala Ser Ser Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 50 55 60

Ser Gly Thr Asp Phe Thr Leu Thr He Ser Ser Leu Gin Pro Glu Asp 65 70 75 80

Phe Ala Thr Tyr Tyr Cys Gin Gin Ala Tyr Ser Phe Pro Arg Thr Phe 85 90 95

Gly Gin Gly Thr Lys Val Glu He Lys Arg Thr Val Ala Ala Pro Ser 10 10 105 110

Val Phe He Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr Ala 115 120 125

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 130 135 140

Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly 145 150 155

&lt; 210 &gt; 15 &lt; 211 &gt; 136 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 4 0 0 &gt; 15

Gin Cys Val Wing Ala Pro Arg Trp Val Leu Gin Gin Gin Leu Gin 15 10 15

Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin Thr Leu Ser Leu Thr 20 25. 30

Cys Thr Val Ser Gly Gly Ser He Ser Ser Gly Gly Tyr Tyr Trp Ser 35 40 45

Trp Ile Arg Gly His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile 50 55 60

Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val 65 70 75 80

Thr Ile Ser Val Asp Thr Ser Lys Asn Gin Phe Ser Leu Lys Leu Ser 85 90 95

Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg 100 105 110

He Thr Met Val Arg Gly Gly He Pro Ser Gly Met Asp Val Trp Gly 115 120 125

Gin Gly Thr Thr Val Thr Val Ser 130 135

&lt; 210 &gt; 16 &lt; 211 &gt; 139 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 4 0 0 &gt; 16

Ser Pro Phe Thr Cys Arg Ala Ser Gin Ser lie Thr Asn Tyr Leu Asn 15 10 15

Trp Tyr Gin Gin Lys Pro Gly Glu Ala Pro Lys Leu Leu He His Val 20 25 30

Ala Ser Ser Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 35 40 45

Ser Gly Arg Asp Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro Glu Asp 50 55 60

Phe Ala Thr Tyr Tyr Cys Gin Gin Ser His Ser is Pro Arg Thr Phe 65 70 75 80

Gly Gin Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser 85 90 95

Val Phe He Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr Ala 100 105 110

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 115 120 125

Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly 130 135 &lt; 210 &gt; 17 &lt; 211 &gt; 138

&lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 4 0 0 &gt; 17

Gin Val His Leu Gin Gin Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 15 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25. 30

Tyr Trp Ser Trp Ile Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp lie 35 40. 45

Gly Glu Ile Asn His Ser Gly Ser Thr Ser Tyr Lys Pro Ser Leu Lys 50 55 60

Ser Arg Val Thr Val Ser Val Asp Thr Ser Lys Asn Gin Phe Ser Leu 65 70 75 80

Lys Leu Ser Tyr Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95

Arg Asp Arg Gly Asp Tyr Gly Asp Phe Leu Phe Asp Tyr Trp Gly Gin 100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125

Phe Pro Leu Ala pro Ser Ser Lys Ser Thr 130 135

&lt; 210 &gt; 18 &lt; 211 &gt; 141 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 4 0 0 &gt; 18

Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Ile 15 10 15

Gly Ser Thr Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro 20 25 30

Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Glu 35 40 45

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 50 55 60

Gly Leu Glu Pro Glu Asp Phe Ala Go Phe Tyr Cys Gin Gin Cys Gly 65 70 75 80

Ser Ser Pro Pro Thr Phe Gly Gin Gly Thr Lys Go Glu Ile Lys Arg 85 90 95

Thr Will Ala Pro Will Be Phe Will Pro Phe Pro Ser Asp Glu Gin 100 105 110

Leu Lys Ser Gly Thr Ala Ser Vai Vai Cys Leu Leu Asn Asn Phe Tyr 115 120 125

Pro Arg Glu Ala Lys Go Gin Trp Lys Go Asp Asn Ala 130 135 140

&lt; 210 &gt; 19 &lt; 211 &gt; 139 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 4 0 0 &gt; 19

Gly Leu Gin Cys Goes Ala Wing Pro Arg Trp Go Leu Gin Go Gin 15 10 15

Leu Gin Gin Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr Leu Ser 20 25 30

Leu Thr Cys Ala Ty Tyr Gly Gly Ser Phe Ser Gly Asn Tyr Trp Ser 35 40 45

Trp Ile Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile 50 55 60

Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val 65 70 75 80

Thr Ile Ser Vai Asp Thr Ser Lys Asn Gin Phe Ser Leu Lys Leu Ser 85 90 95

Ser Go Thr Ala Ala Asp Thr Ala Go Tyr Tyr Cys Ala Arg Gly Gly 100. 105 110

Ser Tyr Asn Tyr Phe Asp Tyr Trp Gly Gin Gly Thr Leu Vai Thr Go 115 120 125

Being Ser Ala Ser Thr Lys Gly Pro Ser Vai Lys 130 135

&lt; 210 &gt; 20 &lt; 211 &gt; 236 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 4Ο0 &gt; 20

Met Asp Met Arg Val Pro Ala Gin Leu Leu Gly Leu Leu Leu Leu Trp 15 10 15

Leu Ser Gly Ala Arg Cys Asp lie Gin Met Thr Gin Ser Pro Ser Ser 20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gin Ala Ser 35 40 45

Gin Asp lie Ser Asn Tyr Leu Asn Trp Tyr Gin Gin Ser Pro Gly Lys 50 55 60

Ala Pro Lys Phe Leu lie Ser Asp Ala Ser Asn Leu Lys Thr Gly Val 65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Phe Thr 85 90 95 Ser Ser Leu Gin Pro Glu Asp lie Ala Thr Tyr Cys Cys Gin Gin 100 105 110

Tyr Asp Ser Leu Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp He 115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140

Glu Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu 165 170 175

Gin Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp 180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser 210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 23S

&lt; 210 &gt; 21 &lt; 211 &gt; 239 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 21

Met Arg Leu Pro Ala Gin Leu Leu Gly Leu Leu Met Leu Trp Val Ser 15 10 15

Gly Ser Ser Gly Asp lie Val Met Thr Gin Ser Pro Leu Ser Leu Pro 20 25 30

Val Thr Pro Gly Glu Pro Ala Serine Ser Cys Arg Ser Ser Gin Ser 35 40 45

Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Val Trp Tyr Leu Gin Lys 50 55 60

Pro Gly Gin Ser Pro Gin Leu Leu lie Tyr Leu Gly Ser lie Arg Ala 65 70 75 SO

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95

Thr Leu Lys He Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110

Cys Met Gin Pro Leu Gin Thr Prole Thr Phe Gly Gin Gly Thr Arg 115 120 125

Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe He Phe Pro 130 135 140

Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp 165 170 175

Asn Ala Leu Gin Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp 180 185 190

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gin 210 215 220

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235

&lt; 210 &gt; 22 &lt; 211 &gt; 141 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 22

Gin Val Gin Gin Gin Gly Pro Gly Leu Val Lys Pro Ser Gin 1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30

Be Ala Ala Trp Asn Trp lie Arg Gin Be Pro Be Arg Gly Leu Glu 35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Gly Tyr Ala 50 55 60

Val Ser Val Lys Ser Arg Met Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80

Gin Phe Ser Leu Gin Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95

Tyr Tyr Cys Ala Arg Glu Arg Leu Gly Glu Leu Tyr Gly Met Asp Val 100 105 110

Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115. 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 130 135 140

&lt; 210 &gt; 23 &lt; 211 &gt; 161 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 23

Gin Leu Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg 15 10 15

Ala Thr lie Asn Cys Lys Ser Ser Gin Asn lie Leu Tyr Arg Ser Ser 20 25 30

Lys Lys Asn His Leu Val Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro 35 40 45

Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Ala 50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser 65 70 75 80

Thr Leu Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Gin Tyr Tyr 85 90 95

Ser Thr Pro Thr Phe Gly Gin Gly Thr Arg Leu Glu Ile Lys Arg 100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin 115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140

Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser 145 150 155 160

Gly &lt; 210 &gt; 24

&lt; 211 &gt; 137 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 220 &gt; &lt; 221 &gt; VARIANT &lt; 222 &gt; (0). . (0) &lt; 223 &gt; Xaa = Any amino acid &lt; 400 &gt; 24

Gly Pro Gly Xaa Xaa Lys Pro Ser Gin Xaa Leu Ser Leu Thr Gly Thr 15 10 15

Val Ser Gly Gly Ser is Ser Gly Gly Tyr Tyr Trp Ser Trp lie 20 25 30

Arg Gin His Pro Gly Lys Gly Leu Glu Trp lie Gly Asn lie Tyr Tyr 35 40 45

Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile SO 55 60

Ser Val Asp Thr Ser Lys Asn Gin Phe Ser Leu Lys Leu Ser Ala Val 65 70 75 80

Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Asn lie Thr 85 90 95

Met Val Arg Gly Val Tyr Tyr Gly Met Asp Val Trp Gly Gin Gly Thr 100 105 110

Thr Val Thr Val Ser Ser Al Th Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125

Leu Wing Pro Ser Ser Lys Ser Thr Tyr 130 135

&lt; 210 &gt; 25 &lt; 211 &gt; 107 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 25

Tyr Ala Ala Ser Ser Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Giy 15 10 15

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ser Ser Leu Gin Pro 20 25 30

Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Ala His Ser Leu Pro Arg 35 .40 .45

Thr Phe Gly Gin Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 50 55 60

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 65 '70 75 80

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Lys Ala 85 90 95

Lys Val Gin Trp Lys Val Asp Asn Thr Leu Gin 100 105

&lt; 210 &gt; 26 &lt; 211 &gt; 144 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 26

Gin Val Gin Leu Val Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg 15 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg His 20 25 30

Gly Val His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ala Valine Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60

Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gly Gly Leu Ile Ala Val Arg Pro Gly Tyr Tyr Tyr Tyr Gly 100 105 HO

Met Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser Ala Ser 115 120 125

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 130 135 140

&lt; 210 &gt; 27 &lt; 211 &gt; 157 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 4Ο0 &gt; 27

Glu Met Gin Leu Thr Gin Ser Pro Ser Ser Ser Leu Ser Ala Set Val Gly 15 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gin Ser Ile Ser Ser Tyr 20 25 30

Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie 35 40 45

Tyr Ala Ala Ser Ser Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr He Ser Ser Leu Gin Pro 65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Ser Tyr Ser Thr Pro Leu 85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110

Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140

Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly 145 150 155

&lt; 210 &gt; 28 &lt; 211 &gt; 141 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 28

Cys Pro Gly Ala Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser 15 10 15

Gin Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser lie Ser Ser 20 25 30

Gly Gly Thr Tyr Tyr Trp lie Trp He Arg Arg His Pro Gly Lys Gly 35. 40 45

Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn 50 55 60

Pro Ser Leu Lys Ser Arg Val Thr He Ser Val Asp Thr Ser Lys Asn 55 70 75 80

Gin Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val 85 90 95

Tyr Tyr Cys Ala Arg Asp Gly Ile Thr Met Val Arg Arg Gly Ile Ser Gly 100 105 110

Gly Met Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser Ala 115 120 125

Ser Thr Lys Gly Pro Ser Val Lys Gly Pro Ser Val Phe 130 135 140

&lt; 210 &gt; 29 &lt; 211 &gt; 145 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 29

Ala Ser Gly Asp Arg Val Thr He Thr Cys Arg Ala Ser Gin Ser 15 10 15 Ile Ser His Hisu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro 20 25 30

Lys Leu Leu He Tyr Ala Ala Ser Ser Leu Gin Ser Gly Val Pro Ser 35 40 45

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser lie Ser 50 55 60

Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gin Gin Ser Tyr 65 70 75 80

Ser He Pro Arg Thr Phe Gly Gin Gly Thr Lys Val Glu He Thr Arg 85 90 95

Thr Val Wing Ala Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu Gin 100 105 HO

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 115 120 125

Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser 130 135 140

Gly 145

&lt; 210 &gt; 30 &lt; 211 &gt; 139 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 30

Gin Leu Gin Gin Ser Gly Pro Gly Leu Val Lys Pro Ser Gin Thr Leu 1 5 10 15

Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn Ser Ala 20 25 30

Ala Trp Asn Trp Ile Arg Gin Ser Pro Ser Arg Gly Leu Glu Trp Leu 35 40 45

Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Glu Tyr Ala Val Ser 50 55 60

Val Lys Ser Arg Met Thr Ile Asn Pro Asp Thr Ser Lys Asn Gin Phe 65 70 75 80

Ser Leu Gin Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr 85 90.95

Cys Ala Arg Glu Arg Leu Gly Glu Leu Tyr Gly Met Asp Val Trp Gly 100 105 110

Gin Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 130 135

&lt; 210 &gt; 31 &lt; 211 &gt; 128 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 31

Leu Gin Val Gin Pro Glu Cys Leu Tyr Thr Val Ser Asp Lys Asn Asn 1 5 10 15

Phe Leu Cys Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu 20 25 30

Met Tyr Trp Ala Ser Ile Arg Glu Ser Gly Val Pro Asp Arg Phe Ser 35 40 45

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin 50 55 60

Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Gin Tyr Tyr Ser Thr Pro

65 70 75 SO

Pro Thr Phe Gly Gin Gly Thr Arg Leu Glu He Lys Arg Thr Val Ala 85 90 95

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser 100 105 110

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 115 120 125

&lt; 210 &gt; 32 &lt; 211 &gt; 141 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 32

Gin Val Gin Gin Gin Gly Pro Gly Leu Val Lys Pro Ser Gin 1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30

Be Ala Ala Trp Asn Trp lie Arg Gin Be Pro Trp Arg Gly Leu Glu 35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Glu Tyr Ala 50 55 60

Val Ser Val Lys Ser Arg Met Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80

Gin Phe Ser Leu Gin Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95

Tyr Tyr Cys Ala Arg Glu Arg Leu Gly Glu Leu Tyr Gly Met Asp Val 100 105 110

Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 130 135 140

&lt; 210 &gt; 33 &lt; 211 &gt; 162 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 4Ο0 &gt; 33

Met Gin Leu Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu 15 10 15

Arg Ala Thr lie Asn Cys Lys Ser Ser Gin Asn Val Leu Tyr Arg Ser. 20 25 30

Asn Lys Lys Asn Phe Leu Val Trp Tyr Gin Gin Lys Pro Gly Gin Pro 35 40 45

Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg Glu Ser Gly Val Pro 50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie 65 70 75 80

Ser Ser Leu Gin Thr Glu Asp Val Ala Val Tyr Tyr Cys Gin Gin Tyr

85 90 9S

Tyr Ser Thr Pro Pro Thr Phe Gly Gin Gly Thr Arg Leu Glu Ile Lys, 100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu 115 120 125

Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140

Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin 145 150 155 160

Ser Gly

&lt; 210 &gt; &Lt; tb &gt; 445 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 34 gtggcagcac ccagatgggt cctgtcccag gtgcagctgc aggagtcggg cccaggactg 60 gtgaagcctt cacagaccct gtccctcacc tgcactgtct ctggtggctc catcagcagt 120 ggtggttact actggatctg gatccgccag cacccaggga agggcctgga gtggattggg 180 tacatctatt acaatgggaa cacctactac aacccgtccc tcaagagtcg agttaccatg 240 tcagtagaca cgtctaagaa ccagttctcc ctgaagctga gctctgtgac tgccgcggac 300 acggccgtgt attactgtgc gagagatggt attactatga tacgcggcta ctactacggt 360 atggacgtct ggggccaagg gaccacggtc accgtctcct cagcctccac caagggccca 420 tcggtcaagg gcccatcggt cttca 445

&lt; 210 &gt; &Lt; tb &gt; 148 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 4Ο0 &gt; 35

Go Ala Ala Pro Arg Trp Val Leu Ser Gin Val Gin Leu Gin Glu Ser 15 10 15

Gly Pro Gly Leu Val Lys Pro Ser Gin Thr Leu Ser Leu Thr Cys Thr 20 25 30

Val Ser Gly Gly Ser is Ser Gly Gly Tyr Tyr Trp lie Trp lie 35 40 45

Arg Gin His Pro Gly Lys Gly Leu Glu Trp He Gly Tyr lie Tyr Tyr 50 55 60

Asn Gly Asn Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Met 65 70 75 80

Ser Val Asp Thr Ser Lys Asn Gin Phe Ser Leu Lys Leu Ser Ser Val 85 90 95

Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Glyl Thr 100 105 110

Met lys Arg Arg Gly Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gin Gly Thr 115 120 125

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Lys Gly 130 13S 140

Pro Ser Val Phe 145

&lt; 210 &gt; 36 &lt; 211 &gt; 466 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 220 &gt; &lt; 221 &gt; misc_feature &lt; 222 &gt; (0) .. (0) &lt; 223 &gt; n = a, t, c or g &lt; 400 &gt; 36 cagctgacnc agtctccatc ttccgtgtct gcatctgtag gagacagagt caccatcact 60 tgtcgggcga gtcggggtat tagcagctgg ttagcctggt atcagcagaa accagggaaa 120 gcccctaagc tcctgatcta tactgcatcc agtttgcaaa gtggagtccc atcaaggttc 180 agcggcagtg gttctgggac agatttcact ctcaccatca gcagcctgca gcctgaagat 240 tttgcaactt actattgtca acaggcttac agtttccctc ggacgttcgg ccaagggacc 300 aaggtggaaa tcaaacgaac tgtggctgca ccatctgtct tcatcttccc gccatctgat 360 gagcagttga aatctggaac tgcctctgtt gtgtgcctgc tgaataactt ctatcccaga 420 gaggccaaag tacagtggaa ggtggataac gccctccaat cgggta 466

&lt; 210 &gt; 37 &lt; 211 &gt; 155 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 37

Gin Leu Thr Gin Ser Pro Ser Ser Ser Val Ser Ala Ser Val Gly Asp Arg 15 10 15

Val Thr Ile Thr Cys Arg Ala Ser Arg Gly He Ser Ser Trp Leu Ala 20 25 30

Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie Tyr Thr 35 40 45

Ala Ser Ser Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 50 55 60

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro Glu Asp 65 70 75 80

Phe Ala Thr Tyr Tyr Cys Gin Gin Ala Tyr Ser Phe Pro Arg Thr Phe 85 90 95

Gly Gin Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser 100 105 110

Val Phe He Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr Ala 115 120 125

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 130 135 140

Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly 145 150 155

&lt; 210 &gt; 38 &lt; 211 &gt; 409 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 38 cagtgtgtgg cagcacccag atgggtcctg tcccaggtgc agctgcagga gtcgggccca 60 ggactggtga agccttcaca gaccctgtcc ctcacctgca ctgtctctgg tggctccatc 120 agcagtggtg gttactactg gagctggatc cgccagcacc cagggaaggg cctggagtgg 180 attgggtaca tatattacag tgggagcacc tactacaacc cgtccctcaa gagtcgagtt 240 accatatcag tagacacgtc caagaaccag ttctccctga agctgagctc tgtgactgcc 300 gcggacacgg ccgtgtatta ctgtgcgaga gatcgaatta ctatggttcg gggaggtatt 360 cccagtggta tggacgtctg gggccaaggg accacggtca ccgtctcct 409 &lt; 210 &gt; 39 &lt; 211 &gt; 136

&lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 39

Gin Cys Val Wing Ala Pro Arg Trp Val Leu Gin Gin Gin Leu Gin 15 10 15

Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gin Thr Leu Ser Leu Thr 20 25 30

Cys Thr Val Ser Gly Gly Ser is Ser Gly Gly Tyr Tyr Trp Ser 35 40 45

Trp He Arg Gin His Pro Gly Lys Gly Leu Glu Trp lie Gly Tyr He 50 55 60

Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val 65 70 75 80

Thr He Ser Val Asp Thr Ser Lys Asn Gin Phe Ser Leu Lys Leu Ser 85 90 95

Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg 100 105 110

He Thr Met Val Arg Gly Gly Ile Pro Gly Met Asp Val Trp Gly 115 120 125

Gin Gly Thr Thr Val Thr Val Ser 130 135

&lt; 210 &gt; 40 &lt; 211 &gt; 417 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 40 tcaccattca cttgccggge aagtcagagc attaccaact atttaaattg gtatcagcag 60 aaaccagggg aagcccctaa gctcctgatc catgttgcat ccagtttgca aagtggggtc 120 ccatcaaggt tcagtggcag tggatctggg agagatttca ctctcaccat cagcagtctg 180 caacctgaag attttgcaac ttactactgt caacagagtc acagtatccc tcggacgttc 240 ggccaaggga ccaaggtgga aatcaaacga actgtggctg caccatctgt cttcatcttc 300 ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac 360 ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggt 417

&lt; 210 &gt; 41 &lt; 211 &gt; 139 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 4Ο0 &gt; 41

Ser Pro Phe Thr Cys Arg Ala Ser Gin Ser lie Thr Asn Tyr Leu Asn 15 10 15

Trp Tyr Gin Gin Lys Pro Gly Glu Ala Pro Lys Leu Leu Lie His Val 20 25 30

Ala Ser Ser Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 35 40 45

Ser Gly Arg Asp Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro Glu Asp SO 55 60

Phe Ala Thr Tyr Tyr Cys Gin Gin Ser His Ser is Pro Arg Thr Phe 65 * 70 75 80

Gly Gin Gly Thr Lys Val Glu He Lys Arg Thr Val Ala Ala Pro Ser 85 90 95

Val Phe Ile Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr Ala 100 105 110

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 115 120 125

Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly 130 135

&lt; 210 &gt; 42 &lt; 211 &gt; 416 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 42 caggtgcatc tacagcagtg gggcgcagga ctgttgaagc cttcggagac cctgtcccte 60 acctgcgctg tctatggtgg gteettcagt ggttactact ggagctggat ccgccagccc 120 ccggggaagg gactggagtg gattggggaa atcaatcata gtggaagcac cagctacaag 180 ccgtccctca agagtcgagt caccgtatca gtggacacgt ccaagaacca gttctccctg 240 aagctgagct atgtgaccgc cgcggacacg gctgtgtatt actgtgcgag agataggggt 300 gactacggtg acttcctctt tgactactgg ggccagggaa ccctggtcac cgtCtcctca 360 gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag caccta 416

&lt; 210 &gt; 43 &lt; 211 &gt; 138 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 43

Gin Val His Leu Gin Gin Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu 15 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25 30

Tyr Trp Ser Trp Ile Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp He 35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Ser Tyr Lys Pro Ser Leu Lys 50 55 60

Ser Arg Val Thr Val Ser Val Asp Thr Ser Lys Asn Gin Phe Ser Leu 65 70 75 80

Lys Leu Ser Tyr Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95

Arg Asp Arg Gly Asp Tyr Gly Asp Phe Leu Phe Asp Tyr Trp Gly Gin 100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 130 135

&lt; 210 &gt; 44 &lt; 211 &gt; 425 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 44 tctccagggg aaagagccac cctctcctgc agggccagtc agagtattgg cagcacctac 60 ttagcctggt accageagaa acctggccag gctcccaggc tcctçatcta tggtgcatcc 120 agcagggcca ctggcatccc agaaaggttc agtggcagtg ggtctgggac agacttcact 180 ctcaccatca gcggactgga gcctgaagat tttgcagtgt tttactgtca acagtgtggt 240 agctcacctc cgacgttcgg ccaagggacc aaggtggaaa tcaaacgaac tgtggctgca 300 ccatctgtct tcatcttccc gccatctgat gagcagttga aatctggaac tgcctctgtt 360 gtgtgectgc tgaataactt ctatcccaga gaggccaaag tacagtggaa ggtggataac 420 gccct 425

&lt; 210 &gt; 45 &lt; 211 &gt; 141 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 45

Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Ile 15 10 15

Gly Ser Thr Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro 20 25 30

Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Glu 35 40 45

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 50 55 60

Gly Leu Glu Pro Glu Asp Phe Ala Go Phe Tyr Cys Gin Gin Cys Gly 65 TO 75 80

Ser Ser Pro Pro Thr Phe Gly Gin Gly Thr Lys Go Glu Ile Lys Arg 85 90 95

Thr Will Ala Pro Will Be Phe Will Pro Phe Pro Ser Asp Glu Gin 100 105 110

Leu Lys Ser Gly Thr Ala Ser Vai Vai Cys Leu Leu Asn Asn Phe Tyr 115 120 125

Pro Arg Glu Ala Lys Go Gin Trp Lys Go Asp Asn Ala 130 135 140

&lt; 210 &gt; 46 &lt; 211 &gt; 417 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 46 ggtctgcagt gtgtggcagc acccagatgg gtcctgtccc aggtgcagct acagcagtgg 60 ggcgcaçgac tgttgaagcc ttcggagacc ctgtccctca cctgcgctgt ctatggtggg 120 tccttcagtg gtaactactg gagctggatc cgccagcccc cagggaaggg gctggagtgg 180 attggggaaa tcaatcatag tggaagcacc aactacaacc cgtccctcaa gagtcgagtc 240 accatatcag tagaeaeçte caagaaccag ttctccctga agctgagctc tgtgaccgcc 300 gcggacacgg ctgtgtatta ctgtgcgaga ggggggagct acaactactt tgactactgg 360 ggceagggaa ccctggtcac cgtctcctca gcctccacca agggcccatc ggtcaag 417

&lt; 210 &gt; 47 &lt; 211 &gt; 139 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 47

Gly Leu Gin Cys Val Wing Ala Pro Arg Trp Val Leu Ser Gin Val Gin 1 5 10 15

Leu Gin Gin Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr Leu Ser 20 25 30

Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Asn Tyr Trp Ser 35 40 45

Trp Ile Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile 50 55 60

Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val 65 70 75 00

Thr Ile Ser Val Asp Thr Ser Lys Asn Gin Phe Ser Leu Lys Leu Ser 85 90 95

Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Gly 100 '105 110

Ser Tyr Asn Tyr Phe Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val 115 120 125

Being Ser Ala Ser Thr Lys Gly Pro Ser Val Lys 130 135

&lt; 210 &gt; 48 &lt; 211 &gt; 933 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 48 aaaaaagtca gtcccaacca ggacacagca tggacatgag ggtccctgct cagctcctgg 60 ggctcctgct gctctggctc tcaggtgcca gatgtgacat ccagatgact cagtctceat 120 cetccctgtc tgeatctgta ggagacagag tcaccatcac ttgccaggeg agtcaggaea 180 ttagcaacta tttgaattgg tatcagcaga gtcccgggaa agcccctaag ttcctgatct 240 ccgatgcatc caatttaaaa acaggggtcc catcaaggtt cagtggaagt ggatctggga 300 cagatttttc tttcaccatc agcagcctac agcctgaaga tattgcgacc tattgctgtc 360 aacagtatga tagtctccca ttcactttcg gccctgggac caaagtggat atcaaacgaa 420 ctgtggctgc accatctgtc ttcatcttcc cgccatctga tgagcagCtg aaatctggaa 480 ctgcctctgt tgtgtgcctg ctgaataact tctatcccag agaggccaaa gtacagtgga 540 aggtggataa cgccctccaa tcgggtaact cccaggagag tgtcacagag caggacagca 600 aggacagcac ctacagcctc agcagcaccc tgacgctgag caaagcagac tacgagaaac 660 acaaagtcta cgcctgcgaa gtcacccatc agggcctgag ctcgcccgtc acaaagagct 720 tcaacagggg agagtgttag agggagaagt gcccccacct gctcctcagt tceagcctga 780 ccccctccca tcctttggcc tctgaccctt tttccacagg ggacctaccc etattgcggt 840 cctocagctc atctttc acc tcacccccct cctcctcctt ggctttaatt atgctaatgt 900 tggaggagaa tgaataaata aagtgaatct ttg 933

&lt; 210 &gt; 49 &lt; 211 &gt; 236 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 4Ο0 &gt; 49

Met Asp Met Arg Val Pro Ala Gin Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15

Leu Ser Gly Ala Arg Cys Asp lie Gin Met Thr Gin Ser Pro Ser Ser 20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gin Ala Ser 35 40 45

Gin Asp lie Ser Asn Tyr Leu Asn Trp Tyr Gin Gin Ser Pro Gly Lys 50 55 60

Ala Pro Lys Phe Leu lie Ser Asp Ala Ser Asn Leu Lys Thr Gly Val 65 70 75 90

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Phe Thr 85 90 95

He Be Ser Leu Gin Pro Glu Asp lie Ala Thr Tyr Cys Cys Gin Gin 100 105 110

Tyr Asp Ser Leu Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp lie 115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe lie Phe Pro Pro Ser Asp 130 135 140

Glu Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu 165 170 175

Gin Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp Ser Lys Asp 180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser 210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235

&lt; 210 &gt; 50 &lt; 211 &gt; 968 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 50 aaagatcagg actcctcagt tcaccttctc acaatgaggc tccctgctca gctcctgggg 60 ctgctaatgc tctgggtctc tggatccagt ggggatattg tgatgactca gtctccactc 120 tccctgcccg tcacccctgg agagccggcc tccatctcct gcaggtctag tcagagcctc 180 ctacatagta atggatacaa ctatttggtt tggtacctgc agaagccagg acagtctcca 240 cagctcctga tctatttggg ttctattcgg gcctccgggg tccctgacag gttcagtggc 300 agtggatcag gcacagattt tacactgaaa atcagcagag tggaggctga ggatgttggg 360 gtttattact gcatgcaacc tctacaaact ccgatcacct tcggccaagg gacacgactg 420 gagattaaac gaactgtggc tgcaccatct gtctteatct tcccgccatc tgatgagcag 480 ttgaaatctg gaactgcctc tgttgtgtgc ctgctgaata acttctatcc cagagaggcc 540 aaagtacagt ggaaggtgga taacgccctc caatcgggta actcccagga gagtgtcaca 600 gagcaggaca gcaaggacag cacctacagc ctcagcagca ccctgacgct gagcaaagca 660 gactacgaga aacacaaagt etacgcctgc gaagtcaccc atcagggcct gagctcgccc 720 gcttcaacag gggagagtgt tagagggaga gtcacaaaga agtgccccca cctgctcctc 780 agttccagcc tgaccccctc ccatcctttg gcctctgacc ctttttccac aggggaccta 840 cccctattgc ggtcctc cag ctcatctttc acctoacccc cctcctcctc cttggcttta 900 attatgctaa tgttggagga gaatgaataa ataaagtgaa tctttgeaaa aaaaaaaaaa 960 aaaaaaaa 968

&lt; 210 &gt; 51 &lt; 211 &gt; 239 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 51

Met Arg Leu Pro Ala Gin Leu Leu Gly Leu Leu Met Leu Trp Val Ser 15 10 15

Gly Ser Ser Gly Asp lie Val Met Thr Gin Ser Pro Leu Ser Leu Pro 20 25 30

Val Thr Pro Gly Glu Pro Ala Serine Ser Cys Arg Ser Ser Gin Ser 35 40 45

Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Val Trp Tyr Leu Gin Lys SO 55 60

Pro Gly Gin Ser Pro Gin Leu Leu lie Tyr Leu Gly Ser lie Arg Ala 65 70 75 80

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95

Thr Leu Lys He Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110

Cys Met Gin Pro Leu Gin Thr Prole Thr Phe Gly Gin Gly Thr Arg 115 120 125

Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140

Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp 165 170 175

Asn Ala Leu Gin Ser Gly Asn Ser Gin Glu Ser Val Thr Glu Gin Asp 180 185 190

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205

Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gin 210 215 220

Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235

&lt; 210 &gt; 52 &lt; 211 &gt; 425 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 52 caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60 acctgtgcca tctccgggga cagtgtctct agcaacagtg ctgcttggaa ctggatcagg 120 cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180 aatggttatg cagtatctgt gaaaagtcga atgaccatca acccagacac atccaagaac 240 cagttctccc tgcagctgaa ctctgtgact cccgaggaca cggctgtgta ttactgtgca 300 agagagaggt taggggagtt atacggtatg gacgtctggg gccaagggac cacggtcacc 360 gtctcctcag cctccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc 420 accta 425

&lt; 210 &gt; 53 &lt; 211 &gt; 141 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 53

Gin Val Gin Gin Gin Gin Gly Leu Gin Liu Pro Ser Gin 15 10 15

Thr Leu Ser Leu Thr Cys Ala He Ser Gly Asp Ser Val Ser Ser Asn 20 25 30

Be Ala Ala Trp Asn Trp lie Arg Gin Be Pro Be Arg Gly Leu Glu 35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Gly Tyr Ala 50 55 60

Val Ser Val Lys Ser Arg Met Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80

Gin Phe Ser Leu Gin Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95

Tyr Tyr Cys Ala Arg Glu Arg Leu Gly Glu Leu Tyr Gly Met Asp Val 100 105 110

Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 130 135 140

&lt; 210 &gt; 54 &lt; 211 &gt; 484 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 54 cagctgacgc agtctccaga ctccctggct gtgtctctgg gcgagagggc caccatcaac 60 tgcaagtcca gccagaatat tttatacagg tccagcaaga agaaccactt agtttggtac 120 cagcagaaac caggacagce tcctaagctg ctcatttact gggcatctac ccgggaatcc 180 ggggtccctg cccgâttcag tggcagcggg tctgggacag atttcactct caccatcagc 240 accctgcagg ctgaagatgt ggcagtttat tactgtcagc aatattatag tacteetccc 300 accttcggcc aagggacacg actggagatt aaacgaactg tggctgcacc atctgtcttc 360 atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgoctgctg 420 aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cotccaatcg 480 ggta 484

&lt; 210 &gt; 55 &lt; 211 &gt; 161 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 4Ο0 &gt; 55

Gin Leu Thr Gin Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg 15 10 15

Ala Thr He Asn Cys Lys Ser Ser Gin Asn lie Leu Tyr Arg Ser Ser 20 25 30

Lys Lys Asn His Leu Val Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro 35 40 45

Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Ala 50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser 65 70 75 80

Thr Leu Gin Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Gin Tyr Tyr 85 90 95

Ser Thr Pro Thr Phe Gly Gin Gly Thr Arg Leu Glu Ile Lys Arg 100 105

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin 115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140

Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser 145 150 155 160

Gly

&lt; 210 &gt; 56 &lt; 211 &gt; 409 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 220 &gt; &lt; 221 &gt; misc_feature &lt; 222 &gt; (0). . (0) &lt; 223 &gt; n = a, t, c or g &lt; 400 &gt; 56 ggcccaggac nggngaagcc ttcacagacc tgtcccteac cggcactgtc tetggtggcc 60 catcagcagt ggtggttatt actggagctg gatccgccag cacccaggga agggcctgga 120 gtggattggg aacatctatt acagtgggag cacctactac aacccgtccc tcaagagtcg 180 agttaccata tcagtagaca cgtctaagaa ccagttctcc ctgaagctga gcgctgtgac 240 tgccgcggac acggccgtgt attactgtgc gagagataat attactatgg ttcggggagt 300 ctactacggt atggacgtct ggggceaagg gaccacggtc accgtctcct cagcctccac 360 caagggccca tcggtcttcc ccctggcacc ctcctccaag 409 agcacctat

&lt; 210 &gt; 57 &lt; 211 &gt; 136 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 220 &gt; &lt; 221 &gt; VARIANT &lt; 222 &gt; (0). . (0) &lt; 223 &gt; Xaa = Any amino acid &lt; 400 &gt; 57

Ala Gin Asp Xaa Xaa Ser Leu His Arg Pro Val Pro His Arg His Cys 15 10 15

Leu Trp Trp Pro Ser Ser Gly Gly Tyr Tyr Trp Ser Trp Ile Arg 20 25 30

Gin His Pro Gly Lys Gly Leu Glu Trp lie Gly Asn lie Tyr Tyr Ser 35 40 45

Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr lie Ser 50 55 60

Val Asp Thr Ser Lys Asn Gin Phe Ser Leu Lys Leu Ser Ala Val Thr 65 70 75 80

Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Asn lie Thr Met 85 90 95

Val Arg Gly Val Tyr Tyr Gly Met Asp Val Trp Gly Gin Gly Thr Thr 100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125

Ala Pro Ser Ser Lys Ser Thr Tyr 130 135

&lt; 210 &gt; 58 &lt; 211 &gt; 321 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 58 tatgcagcat ccagtttgca aagtggggtc ccatcaaggt tcagcggcag tggatctgga 60 acagacttca ctctcaccat cagcagcctg cagcctgaag attttgcaac ttactattgt 120 caacaggctc acagtctccc tcggacgttc ggccaaggga ccaaggtgga aatcaaacga 180 aetgtggctg caccatctgt cttcatcttc ccgccatetg atgagcagtt gaaatctgga 240 actgcctctg ttgtgtgcct gctgaataac ttctatccca gaaaggccaa agtacagtgg 300 to 321 aaggtggata acaccctcca

&lt; 210 &gt; 59 &lt; 211 &gt; 107 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 59

Tyr Ala Ala Ser Ser Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly 15 10 15.

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin pro 20 25 30

Glu Asp Phe Ala Thr Tyr Tyr Cys Gin Gin Ala His Ser Leu Pro Arg 35 40 45

Thr Phe Gly Gin Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 50 55 60

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 65 70 75 80

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Lys Ala 85 90 95

Lys Val Gin Trp Lys Val Asp Asn Thr Leu Gin 100 105

&lt; 210 &gt; 60 &lt; 211 &gt; 433 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 6 0 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cgtccggatt caccttcagt cgccatggcg tgcactgggt ccgccaggct 120 ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaatactat 180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc gagaggaggc 300 cttatagcag ttcgtccggg gtactactac tacggtatgg acgtctgggg ccaagggacc 360 acggtcaccg tctcctcagc ctccaccaag ggcccatcgg tcttccccct ggcaccctec 420 tccaagagca cct 433

&lt; 210 &gt; 61 &lt; 211 &gt; 144 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 61

Gin Val Gin Leu Val Glu Ser Gly Gly Gly Val Val Gin Pro Gly Arg 1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg His 20 25 30

Gly Val His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ala Valine Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60

Lys Gly Arg Phe Thr Tie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gly Gly Leu He Ala Val Arg Pro Gl'y Tyr Tyr Tyr Tyr Gly 100 105 110

Met Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser Ala Ser 115 120 125

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 130 135 140

&lt; 210 &gt; 62 &lt; 211 &gt; 471 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 62 gaaatgcagc tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120 gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180 aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240 gaagattttg caacttacta ctgtcaacag agttacagta ccccgctcac tttcggcgga 300 gggaccaagg tggagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg t 471

&lt; 210 &gt; 63 &lt; 211 &gt; 157 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 63

Glu Met Gin Leu Thr Gin Ser Pro Ser Ser Ser Leu Ser Ala Ser Val Gly 15 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gin Ser He Ser Ser Tyr 20 25 30

Leu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro Lys Leu Leu lie 35 40 45

Tyr Ala Ala Ser Ser Leu Gin Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin Pro 65 70 75 Θ 0

Glu Asp phe Ala Thr Tyr Tyr Cys Gin Gin Ser Tyr Ser Thr Pro Leu 85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu He Lys Arg Thr Val Ala Ala 100 105 110

Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly 115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140

Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly 145 150 155

&lt; 210 &gt; 64 &lt; 211 &gt; 424 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 64 tgtccaggtg cactgcagga gtcgggccca ggactggtga ggccttcaca gaccctgtcc 60 ctcacctgca ctgtctctgg tggctccatc agcagtggtg gtacttacta ctggatctgg 120 atccgccagc acccagggaa gggcctggag tggattgggt acatctatta cagtgggagc 180 acctactaca acccgtccct caagagtcga gttaccatat cagtagacac gtctaagaac 240 cagttctccc tgaagctgag ctctgtgact gccgcggaca cggccgtgta ttactgtgcg 300 agagatggaa ttactatggt tcggggaatt agcgggggca tggacgtctg gggccaaggg 360 accacggtca ccgtctcctc agcctccacc aagggcccat cggtcaaggg cccatcggtc 420 ttca 424 &lt; 210 &gt; 65

&lt; 211 &gt; 141 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 65

Cys Pro Gly Ala Leu Gin Glu Ser Gly Pro Gly Leu Val Arg Pro Ser 1 5 10 15

Gin Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser lie Ser Ser 20 25 30

Gly Gly Thr Tyr Tyr Trp lie Trp He Arg Gin His Pro Gly Lys Gly 35 40 45

Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn 50 55 60

Pro Ser Leu Lys Ser Arg Val Thr He Ser Val Asp Thr Ser Lys Asn 65 70 75 80

Gin Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val 85 90 95

Tyr Tyr Cys Ala Arg Asp Glyl Thr Met Val Arg Arg Gly He Ser Gly 100 105 110

Gly Met Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser Ala 115 120 125

Ser Thr Lys Gly Pro Ser Val Lys Gly Pro Ser Val Phe 130 135 140

&lt; 210 &gt; 66 &lt; 211 &gt; 436 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 6 6 gcatctgtag gagacagagt caccatcact tgccgggcaa gtcagagcat tagtagtcat 60 ttaaattggt atcagcagaa accagggaaa gcccctaagc tcctgatcta tgctgcttcc 120 agtttgeaaa gtggggtccc atcaaggttc agtggcagtg gatctgggac agatttcact 180 ctctccatca gcagtctgca acetgaagat tttgcaactt acttetgtea acagagttac 240 agtatccctc ggacgttcgg ccaagggacc aaggtggaaa tcacacgaac tgtggctgca 300 ccatctgtct tcatcttccc gccatctgat gagcagttga aatctggaac tgcctctgtt 360 gtgtgcctgc tgaataactt ctatcccaga gaggccaaag tacagtggaa ggtggataac 420 gccctccaat cgggta 436

&lt; 210 &gt; 67 &lt; 211 &gt; 145 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 67

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gin Ser 15 10 15 Ile Ser His Hisu Asn Trp Tyr Gin Gin Lys Pro Gly Lys Ala Pro 20 25 30

Lys Leu Leu lie Tyr Ala Ala Ser Ser Leu Gin Ser Gly Val Pro Ser 35 40 45

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser lie Ser SO 55 60

Ser Leu Gin Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gin Gin Ser Tyr 65 70 75 80

Ser Ile Pro Arg Thr Phe Gly Gin Gly Thr Lys Val Glu He Thr Arg 85 90 95

Thr Val Ala Ala Pro Ser Val Phele phe Pro Pro Ser Asp Glu Gin 100 105 110

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 115 120 125

Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser 130 135 140

Gly 145

&lt; 210 &gt; 68 &lt; 211 &gt; 417 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 6 8 cagctgcagc agtcaggtcc aggactggtg aagccctcgc agaccctctc actcacctgt 60 gccatctccg gggacagtgt ctctagcaac agtgctgctt ggaactggat caggcagtcc 120 ccatcgagag gccttgagtg gctgggaagg acatactaca ggtocaagtg gtataatgaa 180 tatgcagtat ctgtgaaaag tcgaatgacc atcaacccag acacatccaa gaaccagttc 240 tccctgcagc tgaactctgt gactcccgag gacacggctg tgtattactg tgcaagagag 300 aggttagggg agttatacgg tatggacgtc tggggccaag ggaccatggt caccgtotcc 360 tcagcctcca ccaagggccc atcggtcttc cccctggcac cctcctccaa gagcacc 417

&lt; 210 &gt; 69 &lt; 211 &gt; 139 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 4Ο0 &gt; 6 9

Gin Leu Gin Gin Ser Gly Pro Gly Leu Val Lys Pro Ser Gin Thr Leu 15 10 15

Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn Ser Ala 20 25 30

Ala Trp Asn Trp Ile Arg Gin Ser Pro Ser Arg Gly Leu Glu Trp Leu 35 40 45

Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Glu Tyr Ala Val Ser 50 55 60

Val Lys Ser Arg Met Thr Ile Asn Pro Asp Thr Ser Lys Asn Gin Phe 65 '70 75 80

Ser Leu Gin Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr 85 90 95

Cys Ala Arg Glu Arg Leu Gly Glu Leu Tyr Gly Met Asp Val Trp Gly 100 105 110

Gin Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 130 135

&lt; 210 &gt; 70 &lt; 211 &gt; 384 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 70 ctgcaagtcc agccagagtg tctttataca gtgtccgaca agaacaactt cttatgttgg 60 taccagcaga aaccaggaca gcctcctaaa ctgctcatgt actgggcatc tatccgggaa 120 tccggggtcc ctgaccgatt cagtggcagc gggtctggga cagatttcac tctcaccatc 180 agcagcctgc aggctgaaga tgtggcagtt tattactgtc agcaatatta tagtactcct 240 cccaccttcg gccaagggac acgactggag actaaacgaa ctgtggctgc accatctgtc 300 ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 360 ctgaataact tctatcccag Agag 3B4

&lt; 210 &gt; 71 &lt; 211 &gt; 128 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 71

Leu Gin Val Gin Pro Glu Cys Leu Tyr Thr Val Ser Asp Lys Asn Asn 15 10 15

Phe Leu Cys Trp Tyr Gin Gin Lys Pro Gly Gin Pro Pro Lys Leu Leu 20 25 30

Met Tyr Trp Ale Ser is Arg Arg Glu Ser Gly Val Pro Asp Arg Phe Ser 35 40 45

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Gin 50 · 55 60

Ala Glu Asp Val Ala Val Tyr Tyr Cys Gin Gin Tyr Tyr Ser Thr Pro 65 70 75 80

Pro Thr Phe Gly Gin Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala 85 90 95

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser 100 105 110

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 115 120 125

&lt; 210 &gt; 72 &lt; 211 &gt; 425 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 72 caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60 acctgtgcca tctccgggga cagtgtctct agcaacagtg ctgcttggaa otggatcagg 120 cagtccccat ggagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180 aatgaatatg cagtatctgt gaaaagtcga atgaccatca acccagacac atccaagaac 240 cagttotccc tgcagctgaa ctctgtgact cccgaggaca cggctgtgta ttactgtgca 300 agagagaggt taggggagtt atacggtatg gacgtctggg gccaagggac cacggtcacc 360 gtctcctcag cctccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc 420 accta 425

&lt; 210 &gt; 73 &lt; 211 &gt; 141 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 73

Gin Val Gin Gin Gin Gly Pro Gly Leu Val Lys Pro Ser Gin 1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn 20 25 30

Be Ala Ala Trp Asn Trp He Arg Gin Be Pro Trp Arg Gly Leu Glu 35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Glu Tyr Ala 50 55 60

Val Ser Val Lys Ser Arg Met Thr Ile Asn Pro Asp Thr Ser Lys Asn 65 70 75 80

Gin Phe Ser Leu Gin Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val 85 90 95

Tyr Tyr Cys Ala Arg Glu Arg Leu Gly Glu Leu Tyr Gly Met Asp Val 100 105 110

Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 130 135 140

&lt; 210 &gt; 74 &lt; 211 &gt; 487 &lt; 212 &gt; DNA &lt; 213 &gt; Homo sapiens &lt; 220 &gt; &lt; 221 &gt; misc_feature &lt; 222 &gt; (0) .. (0) &lt; 223 &gt; n = a, t, c or g &lt; 400 &gt; 74 atgcagctga cncagtctcc agactccctg gctgtgtctc tgggcgagag ggccaccatc 60 aactgcaagt ccagccagaa tgttttatac aggtccaaca agaagaactt cttagtttgg 120 taccagcaga aaccaggaca gcctcctaag ctgctcattt actgggcatc tatccgggaa 190 tccggggtcc ctgaccgatt cagtggcagc gggtctggga cagatttcac tctcaccatc 240 agcagcctgc agactgaaga tgtggcagtt tattactgtc agcaatatta tagtactcct 300 cccaccttcg gccaagggac acgactggag attaaacgaa ctgtggctgc accatctgtc 360 ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 420 ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa 480 tcgggta 487

&lt; 210 &gt; 75 &lt; 211 &gt; 162 &lt; 212 &gt; PRT &lt; 213 &gt; Homo sapiens &lt; 400 &gt; 75

Met Gin Leu Thr Gin Ser Pro Asp $ le Leu Ala Val Ser Leu Gly Glu 15 10 15

Arg Ala Thr lie Asn Cys Lys Ser Ser Gin Asn Val Leu Tyr Arg Ser 20 25 30

Asn Lys Lys Asn Phe Leu Val Trp Tyr Gin Gin Lys Pro Gly Gin Pro 35 40 45

Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg Glu Ser Gly Val Pro 50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie 65 70 75 80

Ser Ser Leu Gin Thr Glu Asp Val Ala Val Tyr Tyr Cys Gin Gin Tyr 85 90 95

Tyr Ser Thr Pro Pro Thr Phe Gly Gin Gly Thr Arg Leu Glu Ile Lys 100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125

Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140

Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin 145 150 155 160

Ser Gly

&lt; 210 &gt; 76 &lt; 211 &gt; 24 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 22 0 &gt; &lt; 223 &gt; Flag Flag &lt; 400 &gt; 76 gattacaagg atgacgacga taag 24

&lt; 210 &gt; 77 &lt; 211 &gt; 24 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 22 0 &gt; &lt; 223 &gt; Control Sequence &lt; 400 &gt; 77 aagggacgaa gacgaacacu uctt 24

&lt; 210 &gt; 78 &lt; 211 &gt; 23 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 22 0 &gt; &lt; 223 &gt; Control Sequence &lt; 400 &gt; 78 aactgaagac ctgaagacaa taa 23

DOCUMENTS REFERRED TO IN THE DESCRIPTION

This list of documents referred to by the author of the present patent application has been prepared solely for the reader's information. It is not an integral part of the European patent document. Notwithstanding careful preparation, the IEP assumes no responsibility for any errors or omissions.

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Lisbon, October 6, 2015

Claims (30)

A monoclonal antibody or antigen-binding fragment thereof which comprises an antigen-binding site that specifically binds to a PSCA protein comprising the amino acid sequence of SEQ ID NO: 2, and wherein the monoclonal antibody comprises the amino acid sequence of the Vh region of SEQ ID NO: 47 and the VL region of SEQ ID NO: 51. An antibody or fragment according to claim 1, wherein the fragment is a Fab, F (ab ') 2, Fv or sFv fragment. An antibody or fragment according to claim 1 or 2, wherein the antibody or fragment is coupled to a detectable label, a toxin, or therapeutic agent, or a chemotherapeutic agent. A hybridoma producing a monoclonal antibody according to claim 1. A hybridoma according to claim 4, wherein the hybridoma is deposited under accession number PTA-6701 of the American Type Culture Collection (ATCC). A polynucleotide encoding a sequence comprising a light chain variable region and an heavy chain variable region of an antibody according to any one of claims 1 or 2. A polynucleotide according to claim 6, which encodes the light chain and the heavy chain of an antibody according to any one of claims 1 or 2. A vector comprising a polynucleotide according to claim 6 or 7. A cell transfected with a vector according to claim 8. A method for producing an antibody as defined in claim 1, or an antigen-binding fragment thereof, comprising: culturing a cell, wherein: (a) the cell has been transfected with a vector comprising a polynucleotide encoding a sequence comprising the light chain variable region of an antibody according to any one of claims 1 and 2 and a vector comprising a polynucleotide encoding a sequence comprising the heavy chain variable region of an antibody according to the invention. Claims 1 or 2; or (b) a cell transfected with a vector according to claim 8; wherein the antibody or an antigen-binding fragment thereof is produced. A pharmaceutical composition comprising an antibody, or Fab fragment, F (ab ') 2, Fv or sFv thereof according to any one of claims 1 to 3. A pharmaceutical composition according to claim 11, wherein the composition is for the treatment of cancers that express PSCA. A pharmaceutical composition according to claim 12, wherein the cancer is cancer of the prostate, pancreas, bladder, kidney, colon, lung, ovary, or breast. An anti-cancer agent for reducing tumor growth of PSCA-expressing tumor cells, wherein said anti-cancer agent comprises a combination of an antibody, or antigen-binding fragment thereof according to any one of claims 1 and 2, and a chemotherapeutic agent. An anti-cancer agent for use according to claim 14, wherein the tumor is a cancer tumor of the prostate, pancreas, bladder, kidney, colon, lung, ovary, or breast. An anticancer agent for use according to claim 14, wherein said antibody or fragment is coupled to a detectable label which is a radioisotope, a metal chelator, an enzyme, a fluorescent compound, a bioluminescent compound or a chemiluminescent compound. An anticancer agent for use according to claim 16, wherein the radioisotope is 212 Bi, 131 I, 90 Y, 186 Re, 211 At, 1251, 188 Re, 153 Sm, 213 B1, 32 P, or Lu. An anticancer agent for use according to claim 14, wherein the chemotherapeutic agent is ricin, ricin A chain, doxorubicin, daunorubicin, a maytansinoid, taxol, ethidium bromide, mitomycin, etoposide, tenophoside, vincristine, vinblastine, colchicine, dihydroxy anthracenedione, actinomycin, diphtheria toxin, Pseudomonas (PE) exotoxin A, PE40, abrinase, abrin A chain, modecin A chain, alpha sarcin, gelonin, mitogelin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Saponaria officinalis inhibitor, glucocorticoid, auristatin, auromycin, yttrium, bismuth, combrestatin, duocarmycins, dolostatin, ccl065, or a cisplatin. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 for the preparation of a medicament for use in a method of inhibiting the growth of a cancer cell expressing a PSCA protein which comprises the amino acid sequence of SEQ ID NO: 2 in a subject. Use according to claim 19, wherein the antibody or antigen-binding fragment thereof is coupled to a cytotoxic agent or a detectable label. Use according to claim 20, wherein said antibody or fragment is coupled to a detectable label which is a radioisotope, a metal chelator, an enzyme, a fluorescent compound, a bioluminescent compound or a chemiluminescent compound. Use according to claim 21, wherein said radioisotope is 212Bi, 131I, 90Y, 186Re, 211At, 1251, 188Re, 153Sm, 213Bi, 32P, or Lu. Use according to claim 20, wherein the cytotoxic agent is ricin, ricin A chain, doxorubicin, daunorubicin, a maytansinoid, taxol, ethidium bromide, mitomycin, etoposide, tenophoside, vincristine, vinblastine, colchicine, dihydroxy anthracenedione , actinomycin, diphtheria toxin, Pseudomonas exotoxin A (PE), PE40, abrin, abrin A chain, modecin A chain, alpha sarcin, gelonin, mitogelin, retstrictocin, phenomycin, enomycin, curicin, crotina, calicheamicin, Saponaria officinalis, glucocorticoid, auristatin, auromycin, yttrium, bismuth, combrestatin, duocarmycins, dolostatin, ccl065, or a cisplatin. Use according to any one of claims 19 to 23, wherein the cancer cell is a prostate, pancreatic, bladder, kidney, colon, lung, ovary, or breast cancer cell. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, for use in a method of treating cancer in a subject, wherein the cancer expresses the PSCA protein. An antibody or antigen-binding fragment thereof for use according to claim 25, wherein the cancer is prostate, pancreas, bladder, kidney, colon, lung, ovary, or breast cancer cancer. An antibody or antigen-binding fragment thereof for use according to claims 25 or 26, wherein said antibody or antigen-binding fragment thereof is administered together with a chemotherapeutic agent, radiation, or both. An antibody or antigen-binding fragment thereof for use according to claim 27, wherein the monoclonal antibody is administered prior to, during, or after initiation of the chemotherapeutic agent or radiation or both. An antibody or antigen-binding fragment thereof for use according to claim 28, wherein the monoclonal antibody is administered between 1 and 60 days prior to commencement of radiation therapy and / or chemotherapy. An antibody or antigen-binding fragment thereof for use according to claim 27, wherein the chemotherapeutic agent is selected from the group consisting of cisplatin, dacarbazine (DTIC), dactinomycin, meclorotamin (nitrogen mustard) , streptozocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel ), aldesleukine, asparaginase, bussulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, ifrofamide, alpha interferon, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide , testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil, taxol and combinations thereof. Lisbon, October 6, 2015