NO345322B1 - Antibodies and related molecules that bind to PSCA Proteins - Google Patents

Description

ANTIBODIES AND RELATED MOLECULES THAT BIND TO

PSCA PROTEINS

FIELD OF THE INVENTION

The present invention relates to antibodies, as well as binding fragments thereof and molecules constructed thereof, which bind proteins, designated PSCA.

The invention further relates to diagnostic, prognostic, prophylactic and therapeutic methods and compositions applicable in the treatment of cancer expressing PSCA.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of death in humans next to coronary heart disease. Millions of people worldwide die from cancer every year. In the United States alone, as indicated by the American Cancer Society, cancer causes the death of well over half a million people annually, with over 1.2 million new cases diagnosed per year. year. While deaths from heart disease have declined significantly, those from cancer are generally increasing. Early in the next century, cancer is predicted to become the leading cause of death.

Around the world, many forms of cancer stand out as the most important fatal diseases. In particular, carcinomas of the lung, prostate, breast, colon, pancreas, ovary and bladder represent the primary causes of cancer death. These and practically all other carcinomas have a common lethal factor. With very few exceptions, metastatic disease from a carcinoma is fatal. Furthermore, common experience has shown that even for those cancer patients who initially survive their primary cancer, their lives are dramatically changed. Many cancer patients experience strong anxiety driven by the knowledge of the risk of relapse or treatment failure. Many cancer patients experience physical impairment after treatment. Furthermore, many cancer patients experience a recurrence.

Worldwide, prostate cancer is the fourth most common form of cancer in men. In North America and Northern Europe, it is by far the most common form of cancer in men and is the second leading cause of cancer death in men. In the US alone, well over 30,000 men die annually from this disease - second only to lung cancer. Despite the magnitude of these numbers, there is still no effective treatment for metastatic prostate cancer. Surgical prostatectomy, radiation therapy, hormone ablation treatment, surgical castration and chemotherapy are still the most important forms of treatment. Unfortunately, these treatments are ineffective for many and are often associated with unwanted consequences.

In the diagnostic field, the lack of a prostate tumor marker that can accurately detect localized tumors at an early stage remains a significant limitation in the diagnosis and treatment of this disease. However, although the serum prostate specific antigen (PSA) assay has been a very useful tool, its specificity and overall utility are generally considered lacking in many important respects.

Progress in the identification of additional specific markers for prostate cancer has been enhanced by the production 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 have survived passage in immunodeficient ("severe combined immune deficient") (SCID) mice and have shown the ability to mimic the transition from androgen dependence to androgen independence (Klein et al., 1997 , Nat. Med. 3:402). Later identified prostate cancer markers include PCTA-1 (Su et al., 1996, Proc. Natl. Acad. Sci. USA 93: 7252), prostate specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res 1996 Sep 2 (9): 1445-51), STEAP (Hubert, et al., Proc Natl Acad Sci U S A.1999 Dec 7; 96(25): 14523-8) and prostate stem cell antigen (PSCA) (Reiter et al ., 1998, Proc. Natl. Acad. Sci. USA 95: 1735).

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

Renal cell carcinoma (RCC) accounts for approximately 3 percent of malignant diseases in adults. Once adenomas reach a diameter of 2 to 3 cm, the risk of malignancy exists. In adults, the two most important malignant kidney tumors are renal cell adenocarcinoma and transitional cell carcinoma of the renal pelvis or ureter.

The incidence of renal cell adenocarcinoma is estimated at more than 29,000 cases in the United States and more than 11,600 patients died of this disease in 1998.

Transitional cell carcinoma is less frequent, with an incidence of approximately 500 cases per year. year in the United States.

Surgery has been the primary treatment for renal cell adenocarcinoma for many decades. Until recently, metastatic disease has been resistant to any systemic treatment. With recent developments in systemic treatments, particularly immunotherapy, metastatic renal cell carcinoma can be approached aggressively in suitable patients with a possibility of durable responses. Even so, there is still a need for effective treatments for these patients.

Of all new cases of cancer in the United States, bladder cancer represents about 5 percent in men (fifth most common neoplasm) and 3 percent in women (eighth most common neoplasm). The incidence is increasing slowly, at the same time as an increasing elderly population. In 1998, there were an estimated 54,500 cases, comprising 39,500 in men and 15,000 in women. The age-adjusted incidence in the USA is 32 per 100,000 for men and eight per 100,000 for women. The historical male/female ratio of 3:1 may be decreasingly related to smoking patterns in women. There were an estimated 11,000 deaths from bladder cancer in 1998 (7,800 in men and 3,900 in women). Incidence and mortality of bladder cancer increase strongly with age and will be a growing problem as the population ages.

Most bladder cancers recur in the bladder. Bladder cancer is treated with a combination of transurethral resection of the bladder (TUR) and intravesical chemotherapy or immunotherapy. The multifocal and recurrent nature of bladder cancer points to the limitations of TUR. Most muscle-invasive cancers are not cured by TUR alone. Radical cystectomy and urinary diversion are the most effective methods to remove the cancer but have an undeniable impact on urine and sexual function. There is still a significant need for forms of treatment that are beneficial for bladder cancer patients.

There were an estimated 130,200 cases of colorectal cancer in 2000 in the United States, comprising 93,800 cases of colon cancer and 36,400 of rectal cancer. Colorectal cancer is the third most common form of cancer in men and women. Incidence rates decreased significantly during 1992-1996 (-2.1% per year). Research indicates that these declines are due to increased screening and removal of polyps, which prevents the development of polyps into invasive cancer. There were an estimated 56,300 deaths (47,700 from colon cancer, 8,600 from rectal cancer) in 2000, which accounts for approximately 11% of all cancer deaths in the United States.

Currently, surgery is the most common form of treatment for colorectal cancer, and for cancers that have not spread, it is often curative. Chemotherapy, or chemotherapy plus radiation, is given before or after surgery to most patients whose cancer has deeply perforated the bowel wall or has spread to the lymph nodes. A permanent colostomy (creation of an opening in the abdomen for the elimination of waste from the body) is occasionally necessary for colon cancer and is rarely necessary for rectal cancer. There is still a need for effective diagnostic and treatment methods 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 for cancer of the lungs and bronchi 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 incidence rate among women began to slowly decline. In 1996, the incidence rate in women was 42.3 per 100,000.

Lung and bronchial cancer caused an estimated 156,900 deaths in 2000, accounting for 28% of all cancer deaths. During 1992–1996, mortality from lung cancer declined significantly in men (-1.7% per year), while rates for women were still significantly increasing (0.9% per year). Since 1987, more women have died each year from lung cancer than from breast cancer, which, for over 40 years, was the leading cause of cancer death in women. The decreasing incidence and mortality rate of lung cancer was most likely due to reduced smoking rates over the previous 30 years; however, declining smoking patterns in women lag behind those of men. It is significant that, although the decline in tobacco use among adults has slowed, tobacco use among young people is increasing again.

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

Chemotherapy alone or combined with radiation is the treatment of choice for small cell lung cancer; in this regimen, a large percentage of patients experience remission, which in some cases is long-lasting. However, there is a continuing need for effective treatment and diagnostic methods for cancer of the lungs and bronchi.

According to calculations, 182,800 new invasive cases of breast cancer were expected to occur among women in the United States during the year 2000. In addition, approximately 1,400 new cases of breast cancer were expected to be diagnosed in men in 2000. After increasing approximately 4% per years in the 1980s, incidence rates for breast cancer in women leveled off in the 1990s to approximately 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 ranks second among cancer deaths in women. According to the most recent data, the death rate declined significantly during 1992–1996 with the largest reductions in younger women, both white and black. These reductions were likely the result of earlier detection and improved treatment.

Taking into account the medical circumstances and the patient's preferences, treatment of breast cancer may include lumpectomy (local removal of the tumor) and removal of the lymph nodes under the arm; mastectomy (surgical removal of the breast) and removal of the lymph nodes under the arm; radiation therapy; chemotherapy; or hormone therapy. Often two or more methods are used in combination. A number of studies have shown that, for early stage disease, the long-term survival rate after lumpectomy plus radiotherapy is similar to the survival rate after modified radical mastectomy. Significant advances in reconstruction techniques offer many options for breast reconstruction after mastectomy. Recently, such reconstruction has been performed at the same time as mastectomy.

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

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

Surgery, radiation therapy and chemotherapy are treatment options for ovarian cancer. Surgery usually involves the removal of one or both ovaries, the fallopian tube (salpingo-oophorectomy) and the uterus (hysterectomy). In some very early tumors, only the involved ovary will be removed, especially in young women who want to have children. In advanced disease, attempts are made to remove all intra-abdominal disease to improve the effect of chemotherapy. There is still 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 roughly constant but may have begun 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 reduction in the mortality rate among men (approximately -0.9% per year) while rates have increased slightly among women.

Surgery, radiation therapy and chemotherapy are treatment options for pancreatic cancer. These treatment options can prolong survival and/or alleviate symptoms in many patients, but probably do not prevent healing for most. There is a significant need for additional therapeutic and diagnostic options for cancer. These include the use of antibodies, vaccines and small molecules as forms of treatment. In addition, there is also a need to use these forms as research tools to diagnose, detect, monitor and promote the state of the 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 understood. Monoclonal antibodies have now been approved as treatment for transplantation, cancer, infectious disease, cardiovascular disease and inflammation. Different isotypes have different effector functions. Such differences in function are reflected in different 3-dimensional structures for the different immunoglobulin isotypes (P.M. Alzari et al., Annual Rev. Immunol., 6:555-580 (1988)).

Because mice are suitable for immunization and recognize most human antigens as foreign, mAbs against human targets with therapeutic potential have typically been of murine origin. However, murine mAbs have inherent disadvantages as human therapeutic agents. They require more frequent dosing as 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 foreign and producing a human anti-mouse antibody (HAMA) response. Such a HAMA response can result in an allergic reaction and rapid excretion of the murine antibody from the system, thereby rendering treatment with murine antibody useless. To avoid such impacts, attempts have been made to create human immune systems in mice.

In initial attempts, it was hoped to create transgenic mice capable of responding to antigens with antibodies having human sequences (See Bruggemann et al., Proc. Nat'l. Acad. Sci. USA 86:6709-6713 (1989) ), but was limited by the amount of DNA that could be stably maintained by available cloning vehicles. Application of "yeast artificial chromosome" (YAC) cloning vectors led to the introduction of large germ cell fragments of the human Ig locus into transgenic mammals. Essentially, most of the human v. D and J region genes were arranged with the same spacing as that found in the human genome and the human constant regions were introduced into mice using YACs. One such transgenic mouse strain is known as the XenoMouse® mouse and is commercially available from Abgenix, Inc. (Fremont CA).

EP 1414876 describes the prostate-specific cell surface antigen PSCA, which is widely overexpressed across all stages of prostate cancer.

SUMMARY OF THE INVENTION

The invention provides antibodies as well as binding fragments thereof and molecules constructed therefrom, which bind to PSCA proteins and polypeptide fragments of PSCA proteins. The invention encompasses polyclonal and monoclonal antibodies, murine and other mammalian antibodies, chimeric antibodies, humanized and fully human antibodies, and antibodies labeled with a detectable marker or therapeutic agent. In certain embodiments, it is a condition that the entire nucleic acid sequence according to Figure 3 is not encoded and/or the entire amino acid sequence of Figure 2 is not prepared. In certain embodiments, the entire nucleic acid sequence according to Figure 3 is encoded and/or the entire amino acid sequence according to Figure 2 is prepared, both of which are in respective human unit dosage forms.

The invention further provides 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. An embodiment according to the invention provides methods for monitoring PSCA gene products in a tissue or haematological sample which has or is suspected of having some form of misregulation of growth such as cancer.

The invention further provides various immunogens or therapeutic compositions and strategies for treating cancers that express PSCA such as cancers in tissues listed in Table I, including treatments directed at inhibiting the transcription, translation, processing or function of PSCA as well as cancer vaccines. Described herein are methods for treating 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 agents that inhibit the production or function of PSCA. In one aspect, the invention provides compositions 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 agents that inhibit the production or function of PSCA. Preferably, the carrier is a unique human carrier. In another aspect of the invention, the agent is a group that is immunoreactive with PSCA protein. Non-limiting examples of such moieties include, but are not limited to, antibodies (such as single chain, monoclonal, polyclonal, humanized, chimeric or human antibodies), functional equivalents thereof (whether naturally occurring or synthetic) and combinations thereof.

The antibodies may be conjugated to a diagnostic or therapeutic group. In another aspect, the agent is a small molecule as defined herein.

In one aspect, the invention provides an antibody or antigen-binding fragment thereof, which comprises a variable heavy chain region comprising an amino acid sequence according to SEQ ID NO:13 and a variable light chain region comprising an amino acid sequence according to SEQ ID NO:14, where the antibody or the antigen-binding fragment thereof binds specifically to PSCA protein (SEQ ID NO:2). In a further aspect, the invention provides a transgenic animal that produces a monoclonal antibody or antigen-binding fragment thereof, comprising a variable heavy chain region comprising an amino acid sequence according to SEQ ID NO:13 and a variable light chain region comprising an amino acid sequence according to SEQ ID NO :14, where the antibody or antigen-binding fragment thereof binds specifically to PSCA protein (SEQ ID NO:2). In a further aspect, the invention provides a hybridoma that produces a monoclonal antibody or antigen-binding fragment thereof, comprising a variable heavy chain region comprising an amino acid sequence according to SEQ ID NO:13 and a variable light chain region comprising an amino acid sequence according to SEQ ID NO:14, where the antibody or the antigen-binding fragment thereof binds specifically to PSCA protein (SEQ ID NO:2). In a further aspect, the invention provides a vector comprising a polynucleotide encoding a monoclonal antibody or antigen-binding fragment thereof, comprising a variable heavy chain region comprising an amino acid sequence according to SEQ ID NO:13 and a variable light chain region comprising an amino acid sequence according to SEQ ID NO:13 ID NO:14, where the antibody or the antigen-binding fragment thereof binds specifically to PSCA protein (SEQ ID NO:2). In a further aspect, the invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof, comprising a variable heavy chain region comprising an amino acid sequence according to SEQ ID NO:13 and a variable light chain region comprising an amino acid sequence according to SEQ ID NO: 14, wherein the antibody or antigen-binding fragment thereof binds specifically to PSCA protein (SEQ ID NO:2), in a human unit dosage form. In a further aspect, the invention provides an assay for detecting the presence of a PSCA protein in a biological sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof, comprising a variable heavy chain region comprising an amino acid sequence according to SEQ ID NO: NO:13 and a variable light chain region comprising an amino acid sequence according to SEQ ID NO:14, wherein the antibody or antigen-binding fragment thereof binds specifically to PSCA protein (SEQ ID NO:2), and detect the binding of PSCA protein (SEQ ID NO:2 ID NO:2) in the sample. In a further aspect, the invention provides an antibody or antigen-binding fragment thereof, which comprises a variable heavy chain region comprising an amino acid sequence according to SEQ ID NO:13 and a variable light chain region comprising an amino acid sequence according to SEQ ID NO:14, where the antibody or the antigen-binding fragment thereof specifically binds to PSCA protein (SEQ ID NO:2), for delivery of a cytotoxic agent or a diagnostic agent to a cell expressing a PSCA protein (SEQ ID NO:2), wherein the antibody or antigen-binding the fragment thereof is conjugated to the cytotoxic agent or diagnostic agent for delivery of a cytotoxic agent or diagnostic agent to a cell expressing a PSCA protein (SEQ ID NO:2), wherein the antibody or antigen-binding fragment thereof is conjugated to the the cytotoxic agent or the diagnostic agent. In a further aspect, the invention provides a method for detecting a PSCA protein (SEQ ID NO: 2) in a biological sample, comprising the steps of: providing the biological sample and a control sample; contacting the biological sample and the control sample an antibody or antigen-binding fragment thereof, which comprises a variable heavy chain region comprising an amino acid sequence according to SEQ ID NO:13 and a variable light chain region comprising an amino acid sequence according to SEQ ID NO:14, where the antibody or the antigen-binding fragment thereof specifically binds to PSCA protein (SEQ ID NO:2), which specifically binds to PSCA protein; and determining an amount of a complex of the substance with the PSCA protein and the antibody or antigen-binding fragment thereof present in the biological sample and the control sample. In a further aspect, the invention provides an antibody or antigen-binding fragment thereof, which comprises a variable heavy chain region comprising an amino acid sequence according to SEQ ID NO:13 and a variable light chain region comprising an amino acid sequence according to SEQ ID NO:14, where the antibody or the antigen-binding fragment thereof specifically binds to PSCA protein (SEQ ID NO:2), for use in inhibiting the growth of a cancer cell expressing PSCA (SEQ ID NO:2) in a subject.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. The cDNA and amino acid sequence of PSCA (also designated “PSCA v.1” or “PSCA variant 1”) is shown in Figure 1A. The starting 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 designated “PSCA v.2”) is shown in Figure 1B. The codon for start 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 designated “PSCA v.3”) is shown in Figure 1C. The codon for start methionine is underlined. The open reading frame extends from nucleic acid 423-707 including the stop codon. The cDNA and amino acid sequence of PSCA variant 4 (also designated “PSCA v.4”) is shown in Figure 1D. The codon for start methionine is underlined. The open reading frame extends from nucleic acid 424-993 including the stop codon.

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

The cDNA and amino acid sequence of PSCA variant 6 (also designated “PSCA v.6”) is shown in Figure 1F. The codon for start methionine is underlined. The open reading frame extends from nucleic acid 83-427 including the stop codon.

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

Figure 1H. SNP variants of PSCA v.4, PSCA v.19 through v.30. The PSCA v.19 through v.30 proteins have 189 amino acids. The variants PSCA v.19 to v.30 are variants with a single nucleotide difference from PSCA v.4. PSCA v.9, v.10, v.11, v.24 and v.25 proteins differ from PSCA v.1 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 transcript variants v.3 and v.4.

Figure 1I. Expression of PSCA variants. (1I(a)) Primers were designed to differentiate between the variants PSCA v.1/v.2/v.4, PSCA v.3 and PSCA v.5.

The primers A and B, indicated by small arrows above exons in the figure result in a PCR product of 425 bp for PSCA v.1/v.2/v4, a PCR product of 300 bp for PSCA v3 and a PCR product of 910 bp for PSCA v.5. (1I(b)) First strand cDNA was prepared from normal bladder, brain, heart, kidney, liver, lung, prostate, spleen, skeletal muscle, testis, pancreas, colon, stomach, collections of prostate cancer, bladder cancer, kidney cancer, colon cancer, lung cancer, ovarian cancer, breast cancer, cancer metastasis and pancreatic cancer. Normalization was performed by PCR using primers for actin. Semi-quantitative PCR, using the variant-specific primers was performed at 30 cycles of amplification. Results show expression of PSCA v.5 mainly in breast cancer, cancer metastasis and pancreatic cancer and at a lower level in colon cancer and lung cancer. PSCA v.1/v.2/v.4 PCR product was detected in prostate cancer, bladder cancer, kidney cancer, colon cancer, lung cancer, ovarian cancer, breast cancer, cancer metastasis and pancreatic cancer. In normal tissue, PSCA v.1/v.2/v.4 PCR product was detected only in prostate, stomach and at a lower level in kidney and lung, while PSCA v.5 was not detected in any normal tissue. PSCA v.3 PCR-detected product was not detected in any of the samples tested.

Figure 1J. Expression of PSCA v.4 and PSCA v.5.1J(a) Primers were designed to discriminate between PSCA v.4 and PSCA v.5 as indicated by the arrows labeled B and C in the figure. Primers specific for PSCA v.4 lead to a PCR product of 460 bp, while primers specific for PSCA v.5 lead to a PCR product with a size of 945 bp. 1J(b) First strand cDNA was prepared from normal bladder, brain, heart, kidney, liver, lung, prostate, spleen, skeletal muscle, testis, pancreas, colon, stomach, prostate cancer collections, bladder cancer and multi-xenograft collection (prostate cancer , kidney cancer and bladder cancer xenografts). Normalization was performed by PCR using primers for actin. Semi-quantitative PCR, using the variant-specific primers was performed at 30 cycles of amplification. Results show expression of PSCA v.4 in prostate cancer, bladder cancer and multi-xenograft collection, normal kidney and prostate. PSCA v.5 was detected only in normal prostate and bladder cancer.

Figure 2. Amino acid sequences of PSCA antibodies. Figure 2A.

The amino acid sequence of H1-1.10 VH. Underlined is the heavy chain constant region. Figure 2B. The amino acid sequence of H1-1.10 VL. The underlined is the light chain constant region.

Figure 3. Nucleotide and amino acid sequences of PSCA antibodies. Figure 3A. cDNA and amino acid sequence of H1-1.10 VH. Underlined is part of heavy chain constant region. Figure 3B. cDNA and amino acid sequence of H1-1.10 VL.

The underlined part is part of the light chain constant region.

Figure 4. Compilation of PSCA antibodies against germ cell V-D-J sequences. Figure 4A. Alignment of H1-1,10 VH with human VH4-31. Figure 4B.

Comparison of H1-1,10 VL with human O2

Figure 5. Expression of PSCA protein in recombinant 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 gene 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 FACS labeling with 1G8 anti-PSCA MAb (5 µg/ml). Shown is the FACS profile of each cell line demonstrating a fluorescent shift only in the PSCA-infected line indicative of cell surface PSCA expression. These lines are applicable in MAb development as immunogens, MAb screening reagents and in functional experiments.

Figure 6. Purification of PSCA protein from E.Coli. E. coli strain BL21 pLysS was transformed with pET-21b vector encoding amino acids 21-94 of PSCA cDNA. PSCA protein was expressed by induction of log phase cultures with IPTG and purified by affinity chromatography from either the soluble or insoluble fractions of the lysed bacteria. Shown are SDS-PAGE Coomasie blue-stained gels of the eluted fractions. This protein is useful as an MAb and pAb immunogen and as an antibody screening reagent.

Figure 7. Purification of recombinant glycosylated PSCA protein expressed from 293T cells. 293T cells were transfected with the psecTag2 vector containing a PSCA cDNA encoding amino acids 28-100. A stable recombinant PSCA-secreting cell line was generated by drug selection with hygromycin B. PSCA protein present in conditioned culture medium was purified by affinity chromatography using 1G8 MAb. Shown is a Coomasie blue labeled SDS-PAGE gel of the fractions eluted at low pH. The broad molecular weight "smear" of the protein shows glycosylation of recombinant PSCA protein as seen in endogenously expressed PSCA.

Figure 8. Purification of GST-PSCA protein from E.Coli. 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 protein was induced with isopropyl-beta-D-thiogalactopyranoside (IPTG) from log phase cultures and purified from lysed bacteria by glutathione agarose matrix affinity chromatography. An SDS-PAGE Coomasie blue-labeled gel of the glutathione-eluted fractions containing GST-PSCA is shown. Indicated are the intact GST-PSCA fusion protein and a minor degradation product containing GST. This protein is useful as an MAb and pAb immunogen and as an Ab screening reagent.

Figure 9. Screening for human PSCA antibodies by FACS.

Antibody concentration from supernatants was determined by ELISA. 50 µl/well of (neat) was added to 96-well FACS plates and serially diluted. PSCA-expressing cells were added (endogenous or recombinant, 50,000 cells/well) and the mixture incubated at 4ºC for two hours. After incubation, the cells were washed with FACS buffer and further incubated with 100ul detection antibody (anti-hIgG-PE) for 45 minutes at 4ºC. At the end of incubation, cells were washed with FACS buffer, fixed with formaldehyde and analyzed using FACScan. Data were analyzed using CellQuest Pro software. Solid histograms represent data from negative control antibody and open histograms indicate data from PSCA-positive cells.

Figure 10. Internalization of H1-1.10 in PC3-PSCA cells detected by FACS analysis. Internalization of H1-1.10 was studied using PC3-PSCA cells. The cells were first incubated at 4°C for 90 minutes to allow the antibodies to bind to the cell surface. Then the cells were divided into two groups and both incubated at 37°C to allow internalization of antibody or at 4°C as a control (no internalization). An acid wash after 37°C/4°C incubation was used to strip off H1-1.10 bound on the cell surface. A permeabilization stop was included to allow detection antibodies to bind internalized H1-1.10. After incubation with detection antibodies, cells were analyzed using FACS and mean fluorescence intensity was calculated. The results show that 40% of H1-1.10 was internalized after incubation at 37°C for two hours.

Figure 11. PC-3 cells engineered to express either the neomycin resistance gene (neo) or PSCA were plated in triplicate into 96-well tissue culture plates. After allowing the cells to adhere overnight, medium was removed and replaced with fresh medium containing the indicated concentrations of anti-PSCA MAb H1-1.10 or H1-1.10 together with a 3-fold excess of the indicated saporin-conjugated secondary antibody ( Advanced Targeting systems, San Diego, CA). The cells were incubated for 4 days and percent viability determined using an MTT assay (Promega Corp). The PSCA antibody H1-1.10 was specific as it did not mediate killing of cells not expressing PSCA (Panel A). H1-1.10 mediated cell killing when incubated with a secondary antibody recognizing the human Fc region but not with a secondary antibody recognizing the Fc region of a goat antibody (Panel B).

Figure 12. LNCap cells engineered to express either the neomycin resistance gene (neo) or PSCA were plated in triplicate in 96-well tissue culture plates. After allowing cells to adhere overnight, medium was removed and replaced with fresh medium containing the indicated concentrations of anti-PSCA MAb H1-1.10 or H1-1.10 together with a 3-fold excess of anti-human or anti-human saporin -conjugated secondary antibody (Advanced Targeting systems, San Diego, CA). The cells were incubated for 4 days and percent viability determined using an MTT assay (Promega Corp). The PSCA antibody H1-1.10 was specific as it did not mediate killing of LNCap cells that do not express PSCA.

Figure 13. Complement-mediated cytotoxicity of anti-H1-1.10 antibody. H1-1.10 (0–50 µg/ml) was diluted with RHB buffer (RPMI 1640, Gibco Life Technologies, 20 mM HEPES). B300.19-PSCA-expressing cells were washed in RHB buffer and resuspended at a density of 10<6 >cells/ml. In a typical assay, 50 µl of PSCA antibody, 50 µl of diluted rabbit complement serum (Cedarlan, Ontario, Can) and 50 µl of a cell suspension were added together into a flat-bottomed tissue culture 96-well plate. The mixture was incubated for 2 hours. at 37°C in a 5% CO2 incubator to facilitate complement-mediated cell lysis. 50 µl of Alamar Blue (Biosource Intl. Camarillo, CA) was added to each well and incubation continued for an additional 4-5 hours at 37°C. The 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 H1-1.10, a human Ig1, was able to mediate complement-dependent lysis of target cells while a control human IgG1 (H3-1.4) was not.

Figure 14. PSCA MAb H1-1.10 inhibits subcutaneous growth of human androgen-independent prostate cancer xenografts implanted in SCID mice. UGB-1, human androgen-independent prostate tumor cells (2.0 x 10 6 cells/mouse) were injected subcutaneously into male SCID mice. The mice were randomized into groups (n=10 mice in each group) and treatment initiated intraperitoneally (i.p.) at day 0 with H1-1.10, PBS or control human IgG1 MAb as indicated. Animals were treated twice weekly with a total of 5 doses until study day 16. Tumor growth was monitored using krumpasser measurements every 3 to 4 days as indicated. Tumor volume was calculated as Width2 x Length/2, where width is the smallest dimension and length is the largest. The results show that human anti-PSCA monoclonal antibody H1-1.10 significantly inhibited the growth of human prostate cancer xenografts implanted subcutaneously in SCID mice (p< 0.01, by Kruskal-Wallis test).

Figure 15. PSCA MAb H1-1,10 inhibits subcutaneous growth of established human androgen-independent prostate cancer xenografts in SCID mice. UGB-1, human androgen-independent human prostate tumor cells (1.5 x 10<6 >cells/mouse) were injected subcutaneously into male SCID mice. When tumors reached approximately 70 mm3, mice were randomized into groups (n=10 mice in each group) and treatment initiated intraperitoneally (i.p.) at day 0 with H1-1.10 or with constituent control as indicated. Animals were treated twice weekly with a total of 7 doses until study day 21. Tumor growth was monitored using krumpasser measurements every 3 to 4 days as indicated. Tumor volume was calculated as Width2 x Length/2, where width is the smallest dimension and length is the largest. The results show that human anti-PSCA monoclonal antibody H1-1.10 significantly inhibited the growth of established human prostate cancer xenografts implanted subcutaneously in SCID mice (p< 0.05, by Student's t-test).

Figure 16. PSCA MAb H1-1.10 inhibits subcutaneous growth of human androgen-dependent prostate cancer xenografts implanted in SCID mice. LAPC9-AD, human androgen-dependent tumor cells (2.0 x 10<6 >cells/mouse) were injected subcutaneously into male SCID mice. The mice were randomized into groups (n=10 mice in each group) and treatment initiated intraperitoneally (i.p.) at day 0 with H1-1.10 or controls as indicated. Animals were treated twice weekly with a total of 6 doses until study day 18. Tumor growth was monitored using krumpasser measurements every 3 to 4 days as indicated. Tumor volume was calculated as Width2 x Length/2, where width is the smallest dimension and length is the largest. The results show that human anti-PSCA monoclonal antibody H1-1.10 significantly inhibited the growth of human prostate cancer xenografts implanted subcutaneously in SCID mice (p< 0.01, by Kruskal-Wallis test).

Figure 17. PSCA MAb H1-1,10 inhibits subcutaneous growth of human pancreatic cancer xenografts implanted in SCID mice. HPAC, human pancreatic tumor cells (3.0 x 10 6 cells/mouse) were injected subcutaneously into male SCID mice. The mice were randomized into groups (n=10 mice in each group) and treatment initiated intraperitoneally (i.p.) at day 0 with H1-1.10 or control human IgG1 as indicated. Animals were treated twice weekly with a total of 5 doses until study day 18. Tumor growth was monitored using krumpasser measurements every 3 to 4 days as indicated. Tumor volume was calculated as Width2 x Length/2, where width is the smallest dimension and length is the largest. The results show that human anti-PCSA monoclonal antibody H1-1.10 inhibited the growth of human pancreatic cancer xenografts implanted subcutaneously in SCID mice (p< 0.05, by Student's t-test).

Figure 18. PSCA MAb H1-1.10 inhibits subcutaneous growth of human bladder cancer xenografts implanted in SCID mice. Human bladder cancer SW780 cells (2.0 x 10 6 cells/mouse) were injected subcutaneously into male SCID mice. The mice were randomized into groups (n=10 mice in each group) and treatment initiated intraperitoneally (i.p.) at day 0 with H1-1.10, PBS or control human IgG1 MAb as indicated. Animals became

treated twice weekly with a total of 6 doses until study day 18. Tumor growth was

monitored using caliper measurements every 3 to 4 days as indicated.

Tumor volume was calculated as Width2 x Length/2, where width is the smallest

the dimension and length are the largest. The results show that human anti-PCSA

monoclonal antibody H1-1.10 inhibited the growth of SW780 human bladder cancer

xenografts implanted subcutaneously in SCID mice (p< 0.05, by Dunnett test).

DETAILED DESCRIPTION OF THE INVENTION

Summary of paragraphs

I.) Definitions

II.) PSCA Polynucleotides

II.A.) Applications of PSCA Polynucleotides

II.A,1.) Monitoring of genetic abnormalities

II.A,2.) Antisense embodiments

II.A,3.) Primers and primer pairs

II.A,4.) Isolation of PSCA-encoding nucleic acid molecules II.A,5.) Recombinant nucleic acid molecules and host-vector systems III.) PSCA-related proteins

III.A.) Motif-bearing protein embodiments

III.B.) Expression of PSCA-related proteins

III.C.) Modifications of PSCA-related proteins

III.D.) Applications of PSCA-related proteins

IV.) PSCA Antibodies

V.) PSCA Cellular immune responses

VI.) PSCA Transgenic animals

VII.) Procedures for detection of PSCA

VIII.) Procedures for monitoring the status of PSCA-related genes and their products

IX.) Identification of molecules that interact with PSCA

X.) Therapeutic methods and mixtures

X.A.) Anti-cancer vaccines

X.B.) PSCA as a target for antibody-based therapy

X.C.) PSCA as a measure of cellular immune responses

X.C.1. Minigene vaccines

X.C.2. Combinations of CTL peptides with helper peptides X.C.3. Combinations of CTL peptides with T-cell priming agents X.C.4. Vaccine mixtures comprising DC pulsed with CTL and/or HTL peptides

X.D.) Adoptive immunotherapy

X.E.) Administration of vaccines for therapeutic or prophylactic purposes XI.) Diagnostic and prognostic embodiments of PSCA.

XII.) Inhibition of PSCA protein function

XII.A.) Inhibition of PSCA with intracellular antibodies XII.B.) Inhibition of PSCA with recombinant proteins

XII.C.) Inhibition of PSCA transcription or translation

XII.D.) General considerations for therapeutic strategies

XIII.) Identification, characterization and application of modulators for PSCA XIV.) RNAi and therapeutic application of small interfering RNAs (siRNAs) XV.) KIT/Products for manufacture

I.) Definitions:

Unless otherwise defined, all designations in the field, representations and other scientific designations or terminology used herein shall have the meanings commonly understood by professionals in the field to which the present invention belongs. In some cases, terms with commonly understood meanings are defined here for clarity and/or for clear reference and the inclusion of such definitions here should not necessarily be perceived as representing a significant difference from what is generally understood in the area. Many of the techniques and methods described or referenced herein are well understood and commonly used using conventional methodology by those skilled in the art, such as, for example, the commonly used molecular cloning methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate, procedures involving the use of commercially available kits and reagents are generally performed according to the manufacturer's defined protocols and/or parameters unless otherwise specified.

The terms “advanced prostate cancer”, “locally advanced prostate cancer”, “advanced disease” and “locally advanced disease” mean prostate cancer that has extended through the prostatic capsule and shall include stage C disease under the American Urological Association (AUA) system, stage C1 - C2 disease under the Whitmore-Jewett system and stage T3 - T4 and N+ disease under the TNM (tumour, node, metastasis) system. In general, surgery is not recommended for patients with locally advanced disease and these patients have mainly less favorable outcomes compared to patients who have clinically localized (organ-confined) prostate cancer. Locally advanced disease is clinically identified by palpable evidence of induration beyond the lateral side of the prostate or asymmetry or induration above the prostate base. Locally advanced prostate cancer is currently diagnosed pathologically after radical prostatectomy if the tumor invades or penetrates the prostatic capsule, extends into the surgical margin, or invades the seminal vesicles.

"Altering the native glycosylation pattern" is for purposes herein intended to mean the removal of one or more carbohydrate groups found in the native PSCA sequence (either by removing the underlying glycosylation site or by removing the glycosylation by chemical and/or enzymatic methods) and/or add one or more glycosylation sites not present in the native sequence of PSCA. In addition, the term includes qualitative changes in the glycosylation of the native proteins, which includes a change in the properties and proportions of the different carbohydrate groups present.

The term "analog" denotes a molecule that is structurally similar or has similar or equivalent properties to another molecule (eg, a PSCA-related protein). For example, an analog of a PSCA protein may be specifically bound by an antibody or T cell that specifically binds to PSCA.

The term “antibody” is used in the broadest sense unless clearly stated otherwise. Therefore, an "antibody" can be naturally occurring or artificial such as monoclonal antibodies produced by conventional hybridoma technology. Anti-PSCA antibodies include monoclonal and polyclonal antibodies as well as fragments containing the antigen-binding domains and/or one or more complementarity determining regions of these antibodies. As used herein, the term "antibody" refers to any form of antibody or fragment thereof that specifically binds PSCA and/or exhibits the desired biological activity and specifically includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., e.g., bispecific antibodies) and antibody fragments as long as they specifically bind PSCA and/or display the desired biological activity. Any specific antibody may be used in the methods and compositions provided herein. Accordingly, the term "antibody" includes, in one embodiment, 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 forms 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.

The antibodies useful in the present methods and compositions can be produced in cell culture, in subjects or in various animals, including but not limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, great apes, chimpanzees, monkeys . Therefore, in one embodiment, an antibody of the present invention is a mammalian antibody. Phage techniques can be used to isolate an initial antibody or to generate variants with altered specificity or avidity characteristics. Such techniques are routine and well known in the art. In one embodiment, the antibody is produced by recombinant methods 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 VL and one VH region into the host cell. Examples of descriptions of recombinant methods for the preparation and production of 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, latest edition). An antibody according to the present invention can be modified by recombinant methods to promote higher efficiency of the antibody with regard to mediating the desired function. Consequently, it is within the scope of the invention that antibodies can be modified by substitutions using recombinant methods. Typically, the substitutions will be conservative substitutions. For example, at least one amino acid in the constant region of the antibody can be replaced with a different residue. See, e.g., U.S. U.S. Patent No. 5,624,821 Patent No. 6,194,551, Application No. WO 9958572; and Angal, et al., Mol. Immunol. 30: 105-08 (1993). Modification in amino acids includes deletions, additions, substitutions of amino acids. In some cases, such changes are made to reduce unwanted activities, eg, complement-dependent cytotoxicity. Often the antibodies are labeled by linking, either covalently or non-covalently, a substance that gives a detectable signal. A number of brands and conjugation techniques are known and are widely disclosed in both the scientific literature and the patent literature. These antibodies can be screened for binding to normal or defective PSCA. See, e.g., ANTIBODY ENGINEERING: A PRACTICAL APPROACH (Oxford University Press, 1996). Suitable antibodies with the desired biological activities can be identified by the following in vitro assays including but not limited to: proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, transport, signal transduction and the following in vivo the experiments such as inhibition of tumor growth. The antibodies provided herein may also be useful in diagnostic applications. As binding or non-neutralizing antibodies, they can be screened for the ability to bind to the specific antigen without inhibiting the receptor binding 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 PSCA or its receptor.

An "antibody fragment" is defined as at least part of the variable region of the immunoglobulin molecule that binds to its target, i.e., the antigen-binding region. In one embodiment, it specifically comprises single anti-PSCA antibodies and clones thereof (including agonist, antagonist and neutralizing antibodies) and anti-PSCA antibody mixtures with poly-epitope specificity. The antibody according to the present methods and mixtures can be monoclonal or polyclonal. An antibody can be in the form of an antigen-binding antibody fragment comprising a Fab fragment, F(ab')2 fragment, a single chain variable region and the like. Fragments of intact molecules can be formed using methods well known in the art and include enzymatic cleavage and recombinant methods.

As used herein, any form of the "antigen" can be used to generate an antibody specific for PSCA. Accordingly, the eliciting antigen may be a single epitope, multiple epitopes or whole protein alone or in combination with one or more immunogenicity-promoting agents known in the art. The eliciting antigen can be an isolated full-length protein, a cell surface protein (eg, immunization with cells transfected with at least a portion of the antigen), or a soluble protein (eg, immunization with only the extracellular domain portion of the protein). The antigen can be produced in a genetically modified cell. DNA encoding the antigen may be genomic or non-genomic (eg, cDNA) and encode at least a portion of the extracellular domain. As used herein, the term "part" refers to the smallest number of amino acids or nucleic acids, as appropriate, that constitute an immunogenic epitope of the antigen of interest. Any genetic vectors suitable for transformation of the cells of interest may be used, 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 at least a portion of the extracellular domain of the PSCA of interest.

The antibodies or antigen-binding fragments thereof provided herein may be conjugated to a “bioactive agent.” As used herein, the term "bioactive agent" refers to any synthetic or naturally occurring compound that binds the antigen and/or enhances or mediates a desired biological effect to enhance cytotoxic toxins.

In one embodiment, the binding fragments useful in the present invention are biologically active fragments. As used herein, the term "biologically active" refers to an antibody or antibody fragment capable of binding the desired antigenic 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 are also useful in the present methods and compositions. As used herein, the term "bispecific antibody" refers to an antibody, typically a monoclonal antibody, which has 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 producing bispecific antibodies are known in the field. For example, bispecific antibodies can be produced recombinantly using co-expression of two immunoglobulin heavy chain/light chain pairs. See, e.g., Milstein et al., Nature 305:537-39 (1983). Alternatively, bispecific antibodies can be prepared using chemical binding. See, e.g., Brennan, et al., Science 229:81 (1985). Bispecific antibodies include bispecific antibody fragments. See, e.g., Hollinger, et al., Proc. Natl. Acad. Sci. U.S.A. 90:6444-48 (1993), Gruber, et al., J. Immunol. 152:5368 (1994).

The monoclonal antibodies herein include specifically "chimeric" antibodies where part of the heavy and/or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the rest of the chain ( e) is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they specifically bind the target antigen and/or display the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)).

The term "chemotherapeutic agent" 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 alkylsulfonates; antimetabolites; for example folic acid, purine or pyrimidine antagonists; mitotic inhibitors; for example, vinca alkaloids and derivatives of podophyllotoxin, cytotoxic antibiotics, compounds that damage or disrupt DNA expression and growth factor receptor antagonists. In addition, chemotherapeutic agents include cytotoxic agents (as defined herein), antibodies, biological molecules and small molecules.

The term "codon-optimized sequences" denotes nucleotide sequences that are optimized for a particular host species by replacing any codons that have a usage frequency of less than about 20%. Nucleotide sequences optimized for expression in a given host species by elimination of spurious polyadenylation sequences, elimination of exon/intron splicing signals, elimination of transposon-like repeats, and/or optimization of GC content in addition to codon optimization are referred to herein as a “ expression-enhanced sequences.”

A "combinatorial library" is a collection of different chemical compounds formed by either chemical synthesis or biological synthesis by combining several chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library, such as a polypeptide (eg, mutein) library, is made by combining a set of chemical building blocks designated amino acids in every possible way for a given length of a compound (ie, number of amino acids in a polypeptide compound). A variety of chemical compounds are synthesized through such combinatorial mixing of chemical building blocks (Gallop et al., J. Med. Chem. 37(9): 1233-1251 (1994)).

Preparation and screening of combinatorial libraries is well known to those skilled in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., 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), encoded peptides (PCT Publication WO 93/20242), random bio -oligomers (PCT Publication WO 92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 ( 1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), non-peptide peptidomimetics with a Beta-D-Glucose shell (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), analog organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho, et al ., Science 261:1303 (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, e.g., Stratagene, Corp.), peptide nucleic acid libraries (see, e.g., U.S. Patent 5,539,083), antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology 14(3): 309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science 274:1520-1522 (1996) and U.S. Patent No. 5,593,853) and small organic molecule libraries (see, e.g., benzodiazepines, Baum, C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent No. 5,569,588; thiazolidinones and metathiazanones, U.S. Patent No. 5,549,974; pyrrolidines, U.S. Patent No. 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent Nos. 5,506, 337; benzodiazepines, U.S. Patent No. 5,288,514; and the like).

Devices for making combinatorial libraries are commercially available (see, e.g., 357 NIPS, 390 NIPS, Advanced Chem Tech, Louisville KY; Symphony, Rainin, Woburn, MA; 433A, Applied Biosystems, Foster City, CA;

9050, Plus, Millipore, Bedford, NIA). Several well-known robotic systems have also been developed for solution-phase chemistries. These systems include automated workstations such as the automated synthesizer developed by Takeda chemical Industries, LTD. (Osaka, Japan) and many robotic systems using robotic arms (Zymat H, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.), which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) to enable them to function as described herein will be apparent to those skilled in the relevant art. In addition, a number of combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, NJ; Asinex, Moscow, RU; Tripos, Inc., St. Louis, MO; ChemStar, Ltd, Moscow, RU; 3D Pharmaceuticals, Exton, PA; Martek Biosciences, Columbia, MD; etc.).

As used herein, the term "conservative substitution" refers to substitutions of amino acids known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art recognize that single amino acid substitutions in nonessential regions of a polypeptide generally do not substantially alter biological activity (see, e.g., Watson, et al., MOLECULAR BIOLOGY OF THE GENE, The Benjamin/Cummings Pub. Co ., p.224 (4th Edition 1987)). Such examples of substitutions are preferably obtained according to those indicated in Table(s) III(a-b). For example, such changes include substituting any of isoleucine (I), valine (V) and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa. Other substitutions 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) can often be interchanged, as can alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can often be replaced by leucine and isoleucine and sometimes by valine. Lysine (K) and arginine (R) often alternate at locations where the significant feature of the amino acid residue is its charge and the deviant pK's for these two amino acid residues are not significant. Still other changes may be considered "conservative" in particular environments (see e.g. Table III(a) here; pages 13-15 “Biochemistry” 2nd ED. Lubert Stryer ed (Stanford University); Henikoff et al., PNAS 1992 Vol 8910915-10919;Lei et al., J Biol Chem 1995 May 19;

270(20):11882-6). Other substitutions are also permitted and can be determined empirically or in accordance with known conservative substitutions.

The term "cytotoxic agent" denotes a substance that inhibits or prevents the expression activity of cells, the function of cells and/or causes the destruction of cells. The term shall include radioactive isotopes, chemotherapeutic agents and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof. Examples of cytotoxic agents include, but are not limited to auristatins, auristatin e, auromycins, maytansinoids, yttrium, bismuth, ricin, ricin A chain, combrestatin, duocarmycins, dolostatins, doxorubicin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin , etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracine dione, actinomycin, diphtheriatoxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Sapaonaria officinalis inhibitor and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes such as At<211>, I<131>, I<125>, Y<90>, Re<186>, Re<188>, Sm<153>, Bi<212 >or <213>, P<32 >and radioactive isotopes of Lu including Lu<177>. Antibodies may also be conjugated to an anticancer prodrug-activating enzyme capable of converting the prodrug to its active form.

As used herein, the term "dimers" ("diabodies") refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL) . 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 form two antigen binding sites. Dimers are described more fully in, e.g., EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993). The "gene product" is used here to indicate a peptide/protein or mRNA. For example, a “gene product of the invention” is sometimes referred to herein as a “cancer amino acid sequence”, “cancer protein”, “cancer protein listed in Table I”, a “cancer mRNA”, “cancer mRNA listed in Table I", etc. In one embodiment, the cancer protein is encoded for by a nucleic acid according to Figure 1. The cancer protein can be a fragment or alternatively, be the full-length protein encoded for by nucleic acids according to Figure 1. In one embodiment, a cancer amino acid sequence used to determine sequence identity or similarity. In another embodiment, the sequences are naturally occurring allelic variants of a protein coded for by a nucleic acid according to Figure 1. In another embodiment, the sequences are sequence variants as further described here.

"Heteroconjugate" antibodies are useful in the present methods and compositions. As used herein, the term "heteroconjugate antibody" refers to two covalently linked antibodies. Such antibodies can be prepared using known methods in synthetic protein chemistry, including the use of cross-linking agents. See, e.g., U.S. Patent No. 4,676,980.

"High-throughput screening" analysis for the presence, absence, quantification or other properties of particular nucleic acids or protein products is well known to those skilled in the art. Correspondingly, binding experiments and reporter gene analyzes are correspondingly well known. Accordingly, for example, the U.S. Patent No. 5,559,410 high-throughput screening methods for proteins; U.S. Patent No. 5,585,639 describes high-throughput screening methods for nucleic acid binding (ie, in arrays); while the U.S. Patent nos. 5,576,220 and 5,541,061 describe high-throughput methods for screening for ligand/antibody binding.

In addition, high-throughput screening systems are commercially available (see, e.g., 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 entire procedures, including all sample and reagent pipetting, liquid dispensing, controlled incubations, and final readings of the microplate in the detector(s) appropriate for the assay. These configurable systems offer high capacity and fast start-up as well as a high degree of flexibility and individual customization. The manufacturers of such systems provide detailed protocols for various high-capacity systems. Accordingly, providers, e.g., Zymark Corp. technical bulletins describing screening systems for detecting modulation of gene transcription, ligand binding and the like.

The term "homologous" denotes a molecule that exhibits homology with another molecule, for example by having sequences of chemical residues that are the same or similar at corresponding positions.

In one embodiment, the antibody provided herein is a “human antibody.” As used herein, the term "human antibody" refers to an antibody in which substantially the entire light chain and heavy chain sequences, including the complementarity determining regions (CDRs), are from human genes. In one embodiment, human monoclonal antibodies are produced by the trioma technique, the human B-cell technique (see, e.g., Kozbor, et al., Immunol. Today 4: 72 (1983) ), the EBV transformation technique (see, e.g., , Cole et al. MONOCLONAL ANTIBODIES AND CANCER THERAPY 77-96 (1985)) or using phage display (see, e.g., Marks et al., J. Mol. Biol. 222:581 (1991)). In a specific embodiment, the human antibody is produced in a transgenic mouse. Techniques for producing such partially to fully human antibodies are known in the art and any of such techniques may be used. According to one particularly preferred embodiment, fully human antibody sequences are produced in a transgenic mouse engineered to express human heavy and light chain antibody genes An example description of the production of transgenic mice producing human antibodies found in Application No. WO 02/43478 and US Patent 6,657,103 (Abgenix) and its progeny .B cells from transgenic e mice that produce the desired antibody can then be fused to produce hybridoma cell lines for continuous production of the antibody. See, e.g., U.S. Patents No. 5,569,825;

5,625,126; 5,633,425; 5,661,016; and 5,545,806; and Jakobovits, Adv. Drug Del. Fox. 31:33-42 (1998); Green, et al., J. Exp. Med. 188:483-95 (1998).

“Human leukocyte antigen” or “HLA” is a human class I or class II Major Histocompatibility Complex (MHC) protein (see, e.g., Stites, et al., IMMUNOLOGY, 8TH ED., Lange Publishing, Los Altos, CA (1994).

As used herein, the term "humanized antibody" refers to forms of antibodies that contain sequences from non-human (eg, murine) antibodies as well as human antibodies. Such antibodies are chimeric antibodies containing 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, where 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 will optionally also comprise at least part of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, e.g., Cabilly U.S. Patent No. 4,816,567; Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033; and ANTIBODY ENGINEERING: A PRACTICAL APPROACH (Oxford University Press 1996). The terms "hybridize", "hybridize", "hybridize" and the like, used in the context of polynucleotides, are meant to refer to conventional hybridization conditions, preferably such as hybridization in 50% formamide/6XSSC/0.1% SDS/100 µg/ml ssDNA , where temperatures for hybridization are above 37 degrees C and temperatures for washing in 0.1XSSC/0.1% SDS are above 55 degrees C.

The terms "isolated" or "biologically pure" refer to material that is substantially or essentially free of components that normally accompany the material as found in its native state. Accordingly, isolated peptides according to the invention preferably do not contain materials normally associated with the peptides in their in situ environment. For example, a polynucleotide is said to be "isolated" when it is substantially separated from contaminating polynucleotides that correspond to or are complementary to genes other than the PSCA genes or that encode polypeptides other than the PSCA gene product or fragments thereof. Those skilled in the art can readily employ nucleic acid isolation procedures to obtain an isolated PSCA polynucleotide. A protein is said to be "isolated," for example, when physical, mechanical, or chemical methods are used to remove the PSCA proteins from cellular constituents normally associated with the protein. Those 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 methods.

Suitable "labels" include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent groups, chemiluminescent groups, magnetic particles and the like. Patents describing the use of such marks include U.S. Pat. Patents No. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;

4,275,149; and 4,366,241. Additionally, the antibodies provided herein may be useful as the antigen-binding component of fluorobodies. See, e.g., Zeytun et al., Nat. Biotechnol. 21:1473-79 (2003).

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

The terms "metastatic prostate cancer" and "metastatic disease" mean prostate cancer that has spread to regional lymph nodes or to distant sites and shall include stage D disease under the AUA system and stage TxNxM+ under the TNM system. As is the case with locally advanced prostate cancer, surgery is generally not indicated for patients with metastatic disease, and hormonal (androgen ablation) therapy is a preferred form of treatment. Patients with metastatic prostate cancer eventually develop an androgen-refractory state within 12 to 18 months of starting treatment. Approximately half of these androgen-refractory patients die within 6 months of developing this condition. The most common site for metastases from prostate cancer is bone.

Prostate cancer bone metastases are often osteoblastic rather than osteolytic (ie, resulting in net bone formation). Bone metastases are most often found in the spine, followed by the femur, pelvis, ribcage, skull and humerus. Other common sites of metastasis include the lymph nodes, lung, liver and brain.

Metastatic prostate cancer is typically diagnosed by open or laparoscopic pelvic lymphadenectomy, whole-body radionuclide scan, skeletal radiography and/or bone lesion biopsy.

The term "modulator" or "test compound" or "drug candidate" or grammatical equivalents as used herein describes any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., to be tested for the capacity to directly or indirectly alter the cancer phenotype or expression of a cancer sequence, e.g., a nucleic acid or protein sequence or effects of the cancer sequence (e.g., signaling, gene expression, protein interaction, etc.) I in one aspect, a modulator will neutralize the effect of the cancer protein according to the invention. By "neutralize" is meant that an activity of a protein is inhibited or blocked, together with the subsequent effect on the cell. In another aspect, a modulator will neutralize the effect of a gene and its corresponding protein, according to the invention by normalizing levels of said protein. In preferred embodiments, modulators alter expression profiles or expression profile nucleic acids or proteins provided herein or downstream effector pathways. In one embodiment, the modulator suppresses a cancer phenotype, e.g. to a normal tissue fingerprint. In another embodiment, a modulator induced a cancer phenotype. Generally, a number of assay mixtures are run in parallel with different concentrations of agent to obtain a differential response to the different concentrations. Typically, one of these concentrations serves as a negative control, ie, at zero concentration or below the level of detection.

Modulators, drug candidates or test compounds encompass a number of chemical classes, although they are typically organic molecules, preferably small organic compounds having a molecular weight of greater than 100 and less than about

2,500 Daltons. Preferred small molecules are less than 2000 or less than 1500 or less than 1000 or less than 500 D. Candidate agents include functional groups necessary for structural interaction with proteins, especially hydrogen bonding and typically include at least one amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.

The candidate agents often comprise cyclic carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Modulators also include biomolecules such as peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogues or combinations thereof. Particularly preferred are peptides. One class of modulators are peptides, for example of from approx. five to approx. 35 amino acids, where approx. five to approx. 20 amino acids are preferred and where from approx. 7 to approx.

15 is particularly preferred. Preferably, the cancer-modulating protein is soluble, comprises a non-transmembrane region and/or, has an N-terminal Cys to contribute to solubility. In one embodiment, the C-terminus of the fragment is kept as a free acid and the N-terminus is a free amine to aid binding, i.e., to cysteine. In one embodiment, the cancer protein according to the invention is conjugated to an immunogenic agent as described here. In one embodiment, the cancer protein is conjugated to BSA. The peptides according to the invention, for example, with preferred lengths, can be linked to each other or to other amino acids to form a longer peptide/protein. The modulating peptides can be cleavage wings of naturally occurring proteins described above, random peptides or "trending" random peptides. In a preferred embodiment, peptide/protein-based modulators are antibodies and fragments thereof, as defined herein.

Modulators of cancer can also be nucleic acids. Nucleic acid modulating agents can be naturally occurring nucleic acids, random nucleic acids or "biased" random nucleic acids. For example, cleavage of prokaryotic or eukaryotic genomes can be used in a method analogous to that described above for proteins.

The term "monoclonal antibody", 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 minor amounts. Monoclonal antibodies are highly specific, and are directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically comprise a plurality of antibodies directed against (or specific for) different epitopes. In one embodiment, the polyclonal antibody contains a variety of monoclonal antibodies with different epitope specificities, affinities or avidities within a single antigen containing multiple antigenic epitopes. The modifier "monoclonal" indicates the nature of the antibody being obtained from a substantially homogeneous population of antibodies and should not be construed to require production of the antibody by any particular method. For example, the monoclonal antibodies to be used according to the present invention can be prepared by the hybridoma method first described by Kohler et al., Nature 256: 495 (1975) or can be prepared by recombinant DNA methods (see, for example, U.S. Pat. No. 4,816,567). The "monoclonal antibodies" 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 typically bind with at least a Kd of about 1 μM, more commonly 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 better, usually determined by ELISA.

A "motif", as in biological motif of a PSCA-related protein, denotes any combination of amino acids forming part of the primary sequence of a protein, which is associated with a particular function (e.g. protein-protein interaction , protein-DNA interaction, etc.) or modification (e.g. phosphorylated, glycosylated or amidated) or localization (e.g. secretory sequence, nuclear localization sequence, etc.) or a sequence that is correlated with being immunogenic , either humoral or cellular. A motif may be either contiguous or capable of being juxtaposed with certain positions that are generally correlated with a particular function or property. In the context of HLA motifs, "motif" refers to the combination of residues in a peptide of defined length, usually a peptide of from about 8 to about 13 amino acids for a class I HLA motif and from about 6 to about 25 amino acids for a class II HLA motif, which is recognized by a special HLA molecule Peptide motifs for HLA binding are typically different for each protein encoded by each human HLA allele and differ in the combination of the primary and secondary anchor- Frequently occurring motifs are listed in Table V.

A "pharmaceutical additive" includes a material such as an adjuvant, a carrier, pH-regulating and buffering agents, tonicity-regulating agents, wetting agents, preservatives and the like.

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

The term "polynucleotide" means a polymeric form of nucleotides with at least 10 bases or base pairs in length, either ribonucleotides or deoxynucleotides or a modified form of one or the other type of nucleotide and shall include single and double-stranded forms of DNA and/or RNA. In the field, this term is often used interchangeably with "oligonucleotide". A polynucleotide can comprise a nucleotide sequence described here where thymidine (T), as shown for example in Figure 1, can also be uracil (U); this definition refers to the differences between the chemical structures of DNA and RNA, particularly the observation that one of the four main bases in RNA is uracil (U) instead of thymidine (T).

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

An HLA "primary anchor residue" is an amino acid at a specific position along a peptide sequence that is understood 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 generally define a “motif” for an immunogenic peptide. These residues are understood to fit in close contact with the peptide binding "pit" of an HLA molecule, with their side chains buried in specific pockets of the binding pit. In one embodiment, for example, the primary anchor residues of an HLA class I molecule are located at position 2 (from the amino-terminal position) and at the carboxy-terminal position of an 8, 9, 10, 11 or 12 residue peptide epitope according to to the invention. Alternatively, in another embodiment, the primary anchor residues of a peptide that binds an HLA class II molecule are spaced relative to each other, rather than to the terminal ends of a peptide, where the peptide generally has a length of at least 9 amino acids. The primary anchor positions for each motif and supermotif are listed in Table IV(a). For example, analogous peptides can be formed by altering the presence or absence of particular residues in the primary and/or secondary anchor positions shown in Table IV. Such analogs are used to modulate the binding affinity and/or population coverage for a peptide comprising a particular HLA motif or supermotif.

“Radioisotopes” include, but are not limited to the following (non-limiting examples of applications are also listed in Table IV(I)).

By "randomized" or grammatical equivalents as used herein for nucleic acids and proteins is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. These random peptides (or nucleic acids, described herein) may incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the entire length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.

In one embodiment, a library is "completely randomized," with no sequence preferences or constants at any position. In another embodiment, the library is a "biased random" library. This means that some positions within the sequence are either kept constant or are selected from a limited number of possibilities. For example, the nucleotides or amino acid residues are randomized within a defined class, e.g., of hydrophobic amino acids, hydrophilic residues, sterically one-sided (either small or large) residues, towards the formation of nucleic acid-binding domains, the formation of cysteines, for cross-linking, prolines for SH -3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc. or to purines, etc.

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

As used herein, the term "single chain Fv" or "scFv" or "single chain" antibody refers to antibody fragments comprising the VH and VL domains of antibody, where these domains are present in a single polypeptide chain. In general, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For an overview of sFv, see Pluckthun, THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES, vol.113, Rosenburg and Moore eds. Springer-Verlag, New York, p.

269-315 (1994).

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

As used herein, the term "specific" refers to selective binding of the antibody to the target antigen epitope. Antibodies can be tested for specificity of binding by comparing binding to the 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 antigen mixture, it is considered specific. In one embodiment, a specific antibody is one that only binds the PSCA antigen, but does not bind to the irrelevant antigen. In another embodiment, a specific antibody is one that binds human PSCA antigen but does not bind a non-human PSCA antigen by 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher amino acid homology with the PSCA antigen. In another embodiment, a specific antibody is one that binds human PSCA antigen and binds murine PSCA antigen, but with a higher degree of binding of the human antigen. In another embodiment, a specific antibody is one that binds human PSCA antigen and binds primate PSCA antigen, but with a higher degree of binding of the human antigen. In another embodiment, the specific antibody binds to human PSCA antigen and any non-human PSCA antigen, but with a higher degree of binding of the human antigen or any combination thereof.

The "stringency" of hybridization reactions can be easily determined by those skilled in the art and is generally an empirical calculation depending on probe length, wash temperature and salt concentration. In general, longer probes require higher temperatures for proper hybridization, while shorter probes require lower temperatures. Hybridization generally depends on the ability of 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 make the reaction conditions more stringent, while lower temperatures less so. For further details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

“Stringent conditions” or “high stringency conditions”, as defined herein, are identified by, but not limited to, those that: (1) use low ionic strength and high temperature in washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) use a denaturant 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) using 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, ultrasonicated salmon sperm DNA (50 µg/ml), 0.1% SDS and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed of a high stringency wash consisting of 0.1 x SSC containing EDTA at 55°C.

"Moderately stringent conditions" are described by, but not limited to, those in Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989 and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent than those described above. An example of moderately stringent conditions is overnight incubation at 65°C in a solution comprising: 1% bovine serum albumin, 0.5M sodium phosphate pH7.5, 1.25mM EDTA and 7% SDS 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), followed by washing the filters in 2 x SSC/1% SDS at 50°C and 0.2 x SSC/0.1% SDS at 50°C. Those skilled in the art will understand how to regulate temperature, ionic strength, etc. as necessary to adjust factors such as probe length and the like.

An HLA “supermotif” is a peptide binding specificity common to HLA molecules encoded by two or more HLA alleles. Total phenotypic frequencies of HLA supertypes in different ethnic populations are indicated in Table IV (f). The non-limiting constituents of various supertypes are as follows:

A2: A*0201, A*0202, A*0203, A*0204, A* 0205, A*0206, A*6802, A*6901, A*0207

A3: A3, A11, 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*3701, B*4402, B*4403, B*60 (B*4001), B61 (B*4006)

A1: A*0102, A*2604, A*3601, A*4301, A*8001

A24: 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*3901, B*3902, B*3903-04, B*4801-02, B*7301, B*2701-08

B58: B*1516, B*1517, B*5701, B*5702, B58

B62: B*4601, B52, B*1501 (B62), B*1502 (B75), B*1513 (B77)

Estimated population coverage given by different HLA-supertype combinations is indicated in Table IV(g).

As used herein, "treating" or "therapeutic" and grammatically related terms refer to any improvement of any consequence of disease, such as prolonged survival, less disease, and/or a reduction of side effects that are the byproducts of an alternative therapeutic form; as is simply understood in the field, complete eradication of disease is a preference if not a requirement for a treatment measure.

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

As used herein, an HLA or cellular immune response "vaccine" is a mixture containing or encoding one or more peptides according to the invention. There are a number of embodiments of such vaccines, such as a cocktail of one or more individual peptides; one or more peptides according to the invention comprised of a polyepitopic peptide; or nucleic acids encoding such individual peptides or polypeptides, eg, a minigene encoding a polyepitopic peptide. The “one or more peptides” may comprise any whole unit integer from 1-150 or more, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150 or more peptides according to the invention.

The peptides or polypeptides may optionally be modified, such as by lipidation, addition of targeting or other sequences. HLA class I peptides according to the invention can be mixed with or bound to, HLA class II peptides, to facilitate activation of both cytotoxic T-lymphocytes and helper T-lymphocytes. HLA vaccines may also comprise peptide-pulsed antigen-presenting cells, eg, dendritic cells.

The term "variant" denotes a molecule that exhibits a variation from a described type or norm, such as a protein that has one or more different amino acid residues in the corresponding position(s) of a specifically described protein (e.g. PSCA -the protein shown in Figure 1.) An analogue is an example of a variant protein. Splice isoforms and single nucleotide polymorphisms (SNPs) are further examples of variants.

The "PSCA-related proteins" according to the invention include those specifically identified here, as well as allelic variants, conservative substitution variants, analogues and homologues that can be isolated/formed and characterized without unnecessary experimentation by following the methods described here or readily available in the field. Fusion proteins combining parts of different PSCA proteins or fragments thereof, as well as fusion proteins of a PSCA protein and a heterologous polypeptide are also encompassed. Such PSCA proteins are collectively referred to as the PSCA-related proteins, the proteins of the invention or PSCA. The term “PSCA-related protein” denotes a polypeptide fragment or a PSCA protein sequence with 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 or more than 25 amino acids; or, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150 , 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 330, 335, 339 or more amino acids.

II.) PSCA Polynucleotides

One aspect according to the invention provides polynucleotides corresponding or complementary to all or part of a PSCA gene, mRNA and/or coding sequence, preferably in isolated form, comprising polynucleotides encoding a PSCA-related protein and fragments thereof, DNA, RNA, DNA /RNA hybrid and related molecules, polynucleotides or oligonucleotides complementary to a PSCA gene or mRNA sequence or part thereof and polynucleotides or oligonucleotides that hybridize to a PSCA gene, mRNA or to a PSCA-encoding polynucleotide (collectively, “PSCA -polynucleotides”). In all cases, T, when referred to in this section, can also be U in Figure 1.

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

Polynucleotides which code for relatively long portions of a PSCA protein are also within the scope of the invention. For example, polynucleotides that code for from approx. 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 "or variant" shown in Figure 1 or Figure 3 is prepared by a variety of techniques well known in the art . These polynucleotide fragments may comprise any part of the PSCA sequence as shown in Figure 1.

II.A.) Applications of PSCA polynucleotides

II.A.1. Monitoring of Genetic Abnormalities The polynucleotides of the preceding paragraphs have several different specific applications. The human PSCA gene has been localized to the chromosomal site indicated in the example labeled “Chromosomal Mapping of PSCA.” For example, because the PSCA gene is mapped to this chromosome, polynucleotides encoding different regions of the PSCA proteins are used to cytogenetically characterize abnormalities of this chromosomal locus, such as abnormalities identified as being associated with various cancers. In certain genes, a number of chromosomal abnormalities involving rearrangements have been identified as frequent cytogenetic abnormalities in several different cancers (see, e.g., Krajinovic et al., Mutat. Res. 382(3-4): 81-83 (1998); Johansson et al., Blood 86(10): 3905-3914 (1995) and Finger et al., P.N.A.S. 85(23): 9158-9162 (1988)). Consequently, polynucleotides encoding specific regions of the PSCA proteins provide new tools that can be used to delineate, with greater precision than 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 field to expand the sensitivity of chromosomal screening to identify more subtle and less common chromosomal abnormalities (see, e.g., Evans et al., Am. J. Obstet. Gynecol 171(4): 1055-1057 (1994)).

Furthermore, as PSCA was shown to be highly expressed in prostate and other cancers, PSCA polynucleotides are used in methods to 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 abnormalities (such as deletions, insertions, point mutations or changes resulting in a loss of an antigen, etc.) in specific regions of the PSCA gene, such as regions containing one or more motifs. Examples of assays include both RT-PCR assay as well as single strand conformation polymorphism (SSCP) assay (see, e.g., Marrogi et al., J. Cutan. Patol. 26(8): 369-378 (1999), both of which use polynucleotides encoding specific regions of a protein to probe those regions within the protein.

II.A.2. Antisense embodiments

Other specifically encompassed nucleic acid-related embodiments of the invention described herein are genomic DNA, cDNAs, ribozymes and antisense molecules, as well as nucleic acid molecules based on an alternative backbone or comprising alternative bases, whether derived from natural sources or synthesized and include molecules capable of inhibiting RNA or protein expression of PSCA. For example, antisense molecules can be RNAs or other molecules, including peptide nucleic acids (PNAs) or non-nucleic acid molecules such as phosphorothioate derivatives that specifically bind DNA or RNA in a base pair-dependent manner. Those skilled in the art can readily obtain these classes of nucleic acid molecules using the PSCA polynucleotides and polynucleotide sequences described herein.

Antisense technology involves the administration of exogenous oligonucleotides that bind to a target polynucleotide located in the cells. The term "antisense" refers to the fact that such oligonucleotides are complementary to their intracellular target, eg, 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 according to the present invention comprise derivatives such as S-oligonucleotides (phosphorothioate derivatives or S-oligos, see, Jack Cohen, supra), which exhibit improved cancer cell growth inhibitory activity. S-oligos (nucleoside phosphorothioates) are isoelectronic analogues of an oligonucleotide (O-oligo) where a non-bridging oxygen atom of the phosphate group is replaced by a sulfur atom. The S-oligos according to the present invention can be prepared by treating the corresponding O-oligos with 3H-1,2-benzodithiol-3-one-1,1-dioxide, which is a sulfur transfer reagent. See, e.g., Iyer, R.P. et al., J. Org. Chem. 55:4693-4698 (1990); and Lyer, R.P. et al., J. Am. Chem. Soc.

112:1253-1254 (1990). Additional PSCA antisense oligonucleotides according to the present invention include morpholino antisense oligonucleotides known in the art (see, e.g., Partridge et al., 1996, Antisense & Nucleic Acid Drug Development 6: 169-175).

The PSCA antisense oligonucleotides according to the present invention can typically be RNA or DNA that are complementary to and stably hybridize with the first 1005' codons or the last 1003' codons of a PSCA genomic sequence or the corresponding mRNA. Absolute complementarity is not required, although a high degree of complementarity is preferred. Use of an oligonucleotide complementary to this region allows selective hybridization to PSCA mRNA and not to mRNA specifying other protein kinase regulatory subunits. In one embodiment, PSCA antisense oligonucleotides of the present invention are 15 to 30-mer fragments of the antisense DNA molecule that have a sequence that hybridizes to PSCA mRNA. Optionally, the PSCA antisense oligonucleotide is a 30-mer oligonucleotide that is complementary to a region in the first 105' codons or the last 103' codons of PSCA. Alternatively, the antisense molecules are modified to use ribozymes in the inhibition of PSCA expression, see, e.g., L. A. Couture & D. T. Stinchcomb; Trends Genet 12: 510-515 (1996).

II.A.3. Primers and primer pairs

Further specific embodiments of these nucleotides according to the invention include primers and primer pairs, which allow specific amplification of polynucleotides according to the invention or of any specific parts thereof and probes which selectively or specifically hybridize to nucleic acid molecules according to the invention or to any part thereof. Probes may be labeled with a detectable marker, such as, for example, a radioisotope, fluorescent compound, bioluminescent compound, 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 of detecting 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 appreciated by those skilled in the art, a wide variety of primers and probes can be prepared based on the sequences provided herein and effectively used to amplify and/or detect a PSCA mRNA.

PSCA polynucleotides according to the invention are useful for a number of purposes, including but not limited to their use as probes and primers for amplification and/or 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 expression of PSCA polypeptides; as tools to modulate or inhibit expression of the PSCA gene(s) and/or translation of the PSCA transcript(s); and as therapeutic agents.

The present invention encompasses 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 or other mammals, as well as the isolated nucleic acid sequence per se, which would include any or most of the sequences found in the probe used.

II.A.4. Isolation of PSCA Encoding Nucleic Acid Molecules The PSCA cDNA sequences described herein enable the isolation of other polynucleotides encoding PSCA gene product(s), as well as the isolation of polynucleotides encoding PSCA gene product homologues, alternatively spliced isoforms, allelic variants and mutant forms of a PSCA gene product as well as polynucleotides encoding analogs of PSCA-related proteins.

Various molecular cloning methods that can be used to isolate full-length cDNAs encoding a PSCA gene are well known (see, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2d ed., 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 methods can be conveniently employed, using commercially available cloning systems (eg, Lambda ZAP Express, Stratagene). Phage clones containing PSCA gene cDNAs can be identified by probing with a labeled PSCA cDNA or a fragment thereof. For example, in one embodiment, a PSCA cDNA (eg, Figure 1) or a portion thereof can be synthesized and used as a probe to recover overlapping and full-length cDNAs corresponding to a PSCA gene. A PSCA gene can itself be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BACs), yeast artificial chromosome libraries (YACs) and the like, with PSCA DNA probes or primers.

II.A.5. Recombinant nucleic acid molecules and host-vector systems

The invention also provides recombinant DNA or RNA molecules containing a PSCA polynucleotide, a fragment, analog or homolog thereof, including but not limited to phages, plasmids, phagemids, cosmids, YACs, BACs, as well as various viral and non-viral vectors well known in the art and cells transformed or transfected with such recombinant DNA or RNA molecules. Methods for making such molecules are well known (see, for example, Sambrook et al., 1989, supra).

The invention further provides a host-vector system comprising a recombinant DNA molecule containing a PSCA polynucleotide, fragment, analog or homolog 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 (eg, a baculovirus infectable cell such as an Sf9 or HighFive cell). Examples of suitable mammalian cells include various prostate cancer cell lines such as DU145 and TsuPr1, other transfectable or transducable prostate cancer cell lines, primary cells (PrEC), as well as several mammalian cells routinely used for expression of recombinant proteins (eg, COS, CHO, 293, 293T -cells).

More specifically, a polynucleotide comprising the coding sequence of PSCA or a fragment, analog or homolog thereof can be used to produce PSCA proteins or fragments thereof using any number of host-vector systems routinely used and commonly known in the art.

A wide range of host-vector systems suitable for 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 mammalian expression include but are not limited to pcDNA 3,1 myc-His-tag (Invitrogen) and the retroviral vector pSR□ tkneo (Muller et al., 1991, MCB 11:1785). Using these expression vectors, PSCA can be expressed in many prostate cancer and non-prostate cell lines, including, for example, 293, 293T, rat-1, NIH 3T3 and TsuPr1. The host-vector systems according to the invention are applicable for the production of a PSCA protein or fragment thereof. Such host-vector systems can be used to study the functional properties of PSCA and PSCA mutations or analogues.

Recombinant human PSCA protein or an analog or homologue or fragment thereof can be produced from 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 fragment, analog, or homolog 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). In another embodiment, a PSCA coding sequence is subcloned into the retroviral vector pSRαMSVtkneo and used to infect various mammalian cell lines, such as NIH 3T3, TsuPr1, 293 and rat-1 to establish PSCA expressing cell lines. Various other expression systems well known in the art can also be used. Expression constructs that encode a leader peptide bound in reading frame to a PSCA coding sequence can be used to produce a secreted form of recombinant PSCA protein.

As described here, redundancy in the genetic code allows variation in PSCA gene sequences. In particular, it is known in the field that specific host species often have specific codon preferences and consequently one can adapt the described sequence as preferred for a desired host. For example, preferred analogous codon sequences typically have rare codons (ie, codons having a usage frequency of less than about 20% in known sequences of the desired host) replaced with higher frequency codons. Codon preferences for a specific species are calculated, for example, using tables of codon usage available on the INTERNET such as URL dna.affrc.go.jp/~nakamura/codon.html.

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

III.) PSCA-related proteins

Another aspect of the present invention provides PSCA-related proteins. Specific embodiments of PSCA proteins comprise a polypeptide having all or part of the amino acid sequence of human PSCA as shown in Figure 1, preferably Figure 1A. Alternatively, embodiments of PSCA proteins comprise variant, homologous or analogous polypeptides having changes in the amino acid sequence of PSCA shown in Figure 1.

Embodiments of a PSCA polypeptide include: a PCSA polypeptide having a sequence shown in Figure 1, a peptide sequence of a PSCA as shown in Figure 1 where T is U; at least 10 consecutive nucleotides of a polypeptide having the sequence shown in Figure 1; or, at least 10 consecutive peptides of a polypeptide having the sequence as shown in Figure 1 where T is U.

Amino acid abbreviations are given in Table II. Conservative amino acid substitutions can often be carried out in a protein without changing either the conformation or the function of the protein. Proteins according to the invention may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 conservative substitutions.

Embodiments according to the invention described herein include a number of species-accepted variants or analogs of PSCA proteins such as polypeptides having amino acid insertions, deletions and substitutions. PSCA variants can be produced using methods known in the art such as site-directed mutagenesis, alanine scanning and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)) or other known techniques can be performed on the cloned DNA to produce the PSCA variant DNA.

Scanning amino acid analysis can also be used to identify one or more amino acids along a contiguous sequence that is involved in a specific biological activity such as a protein-protein interaction. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine and cysteine. Alanine is typically a preferred scanning amino acid in this group because it removes the side chain beyond the beta carbon and is unlikely to change the main chain conformation of the variant. Alanine is also typically preferred because it is the most common amino acid. Furthermore, it is often found in both buried and exposed positions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chotia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does not provide sufficient amounts of variant, an isosteric amino acid can be used.

As defined herein, PSCA variants, analogs or homologues, have the distinguishing characteristic of having at least one epitope that is "cross-reactive" with a PSCA protein having an amino acid sequence according to Figure 1. As used in this sentence, "cross-reactive" means that a antibody or T cell that specifically binds to a PSCA variant also specifically binds to a PSCA protein having an amino acid sequence shown in Figure 1. A polypeptide ceases to be a variant of a protein shown in Figure 1 when it no longer contains any epitope capable of being recognized by an antibody or T cell that specifically binds to the parent PSCA protein. Those skilled in the art understand that antibodies that recognize proteins bind to epitopes of varying size and a cluster on the order of about four or five amino acids, consecutive or not, is considered a typical number of amino acids in a minimal epitope. See, e.g., Nair et al., J. Immunol 2000165(12): 6949-6955; Hebbes et al., Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Immunol (1985) 135(4):2598-608.

Other classes of PSCA-related protein variants have 70%, 75%, 80%, 85%, 90%, 95% or more similarity to an amino acid sequence according to Figure 1 or a fragment thereof. Another specific class of PSCA protein variants or analogs comprises one or more of the PSCA biological motifs described herein or currently known in the art. The scope of the present invention is therefore analogues of PSCA fragments (nucleic acid or amino acid) which have changed functional (e.g. immunogenic) properties in relation to the starting fragment. It should be understood that motifs that are now or will become part of the state of the art are to be used for nucleic acid or amino acid sequences according to Figure 1.

As described herein, embodiments of the present invention include polypeptides containing less than the complete amino acid sequence of a PSCA protein shown in Figure 1. For example, representative embodiments of the invention include peptides/proteins having any 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive amino acids of a PSCA protein shown in Figure 1.

PSCA-related proteins are prepared using standard peptide synthesis technology or using methods of chemical cleavage well known in the art. Alternatively, recombinant methods can be used to produce nucleic acid molecules that encode a PSCA-related protein. In one embodiment, nucleic acid molecules provide a means to prepare defined fragments of a PSCA protein (or variants, homologues or analogs thereof).

III.A.) Motif-bearing protein embodiments

Further illustrative embodiments according to the invention described here comprise PSCA polypeptides comprising the amino acid residues of one or more of the biological motifs contained within a PSCA polypeptide sequence indicated in Figure 1. Various motifs are known in the field and a protein can be evaluated for the presence of such motifs by several publicly available Internet sites (see, e.g., URL addresses: pfam.wustl.edu/; searchlauncher.bcm.tmc.edu/seqsearch/struc-predict.html; psort.ims.u-tokyo.ac.jp/ ; cbs.dtu.dk/; ebi.ac.uk/interpro/scan.html; expasy.ch/tools/scnpsit1.html; Epimatrix™ and Epimer™, Brown University, brown.edu/Research/TB-HIV_Lab/epimatrix /epimatrix.html; and BIMAS, bimas.dcrt.nih.gov/.).

Motif-bearing subsequences of all PSCA variant proteins are listed and identified in Tables V-XVIII and XXII-LI.

Table IV(h) shows many frequently occurring motifs based on a pfam search (see URL address pfam.wustl.edu/). The columns in Table IV(h) list (1) motif name abbreviation, (2) percent identity found among the various members of the motif family, (3) motif name or description, and (4) most common function; localization information is included if the subject is relevant for localization.

Polypeptides comprising one or more of the PSCA motifs described above are useful in elucidating the specific characteristics of a malignant phenotype in view of the observation that the PSCA motifs described above are associated with dysregulation of growth and because PSCA is overexpressed in certain cancers (see e.g., Table I). Casein kinase II, cAMP and cAMP-dependent protein kinase and Protein kinase C, for example, are enzymes known to be associated with the development of the malignant phenotype (see, e.g., Chen et al., Lab Invest., 78(2): 165- 174 (1998); Gaiddon et al., Endocrinology 136(10): 4331-4338 (1995); Hall et al., Nucleic Acids Research 24(6): 1119-1126 (1996); Peterziel et al., Oncogene 18 (46): 6322-6329 (1999) and O'Brian, Oncol. Rep. 5(2): 305-309 (1998)). Furthermore, both glycosylation and myristoylation are protein modifications also associated with cancer and cancer progression (see, e.g., Dennis et al., Biochem. Biophys. Acta 1473(1):21-34 (1999); Raju et al., Exp. Cell Res .235(1): 145-154 (1997)).

Amidation is another protein modification also associated with cancer and cancer progression (see, e.g., Treston et al., J. Natl. Cancer Inst. Monogr. (13): 169-175 (1992)).

In another embodiment, proteins according to the invention comprise one or more of the immunoreactive epitopes identified according to species-accepted methods, such as the peptides previously indicated in Tables V-XVIII and XXII-LI. CTL epitopes can be determined using specific algorithms to identify peptides within a PSCA protein that are able to bind optimally to designated HLA alleles (eg, Table IV; Epimatrix™ and Epimer™, Brown University, URL brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS, URL bimas.dcrt.nih.gov/.) Furthermore, processes to identify peptides that have sufficient binding affinity for HLA molecules and that are correlated with being immunogenic epitopes, well known in the art and are performed without unnecessary experimentation. In addition, processes for identifying peptides that are immunogenic epitopes are well known in the art and are performed without unnecessary experimentation either in vitro or in vivo.

Also known in the art are principles for making analogs of such epitopes to modulate immunogenicity. For example, one starts with an epitope carrying a CTL or HTL motif (see, e.g., HLA Class I and HLA Class II motifs/supermotifs according to Table IV). The epitope is made analogous by replacing an amino acid at one of the indicated positions and replacing it with another amino acid indicated for this position. For example, on the basis of the residues defined in Table IV, one can substitute a deleterious residue in favor of any other residue, such as a preferred residue; replacing a less preferred residue with a preferred residue; or replace an originally occurring preferred residue with another preferred residue. Substitutions can occur at primary anchor positions or at other positions in a peptide; see, e.g., Table IV.

A number of references reflect the state of the art regarding the identification and preparation of epitopes in a protein of interest as well as analogs thereof. See, for example, WO 97/33602 by Chesnut et al.; Sette, Immunogenetics 199950(3-4): 201-212; Sette et al., J. Immunol. 2001 166(2): 1389-1397; Sidney et al., Hum. Immunol. 1997 58(1): 12-20; Kondo et al., Immunogenetics 199745(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 19941(9): 751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92.

Related embodiments according to the invention comprise polypeptides comprising combinations of the various motifs indicated in Table(s) IV(a), IV(b), IV(c), IV(d) and IV(h) and/or, one or more of the putative CTL epitopes according to Tables V-XVIII and XXII-LI and/or, one or more of the predicted HTL epitopes according to Tables XLVIII-LI and/or, one or more of the T-cell binding motifs known in the art. Preferred embodiments contain no insertions, deletions or substitutions either within the motifs or within the intervening sequences of the polypeptides. In addition, embodiments that include several either N-terminal and/or C-terminal amino acid residues on both sides of these motifs may be desirable (to, for example, include a larger part of the polypeptide architecture where the motif is located). Typically, the number of N-terminal and/or C-terminal amino acid residues on one or the other side of a motif is between about 1 to about 100 amino acid residues, preferably 5 to about 50 amino acid residues.

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

The invention also provides PSCA proteins comprising biologically active fragments of a PSCA amino acid sequence shown in Figure 1. Such proteins exhibit properties of the parent PSCA protein, such as the ability to elicit the formation of antibodies that specifically bind an epitope associated with the parent PSCA -the protein; to be bound by such antibodies; to induce activation of HTL or CTL; and/or, to be recognized by HTL or CTL that also specifically bind to the starting protein.

PSCA-related polypeptides containing structures of particular interest can be predicted and/or identified using various analytical techniques well known in the art, including, for example, the methods of Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson- Wolf assay or based on immunogenicity. Fragments containing such structures are particularly useful in the generation of subunit-specific anti-PSCA antibodies or T cells or in the identification of cellular factors that bind to PSCA. For example, hydrophilicity profiles can be generated and immunogenic peptide fragments identified, using the method of Hopp, T.P. and Woods, K.R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828. Hydropathicity profiles can be generated and immunogenic peptide fragments identified, using the method of Kyte, J. and Doolittle, R.F., 1982, J. Mol. Biol. 157:105-132. Percent (%) Available Residue profiles can be generated and immunogenic peptide fragments identified, using the method of Janin J., 1979, Nature 277:491-492. Average flexibility profiles can be generated and immunogenic peptide fragments identified, using the method of Bhaskaran R., Ponnuswamy P.K., 1988, Int. J. Pept. Protein Res.

32:242-255. Beta-turn profiles can be generated and immunogenic peptide fragments identified, using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294.

CTL epitopes can be determined using specific algorithms to identify peptides within a PSCA protein capable of optimal binding to specified HLA alleles eg, using the SYFPEITHI site at the World Wide Web URL syfpeithi.bmi -heidelberg.com/; the lists in Table IV(A)-(E);

Epimatrix™ and Epimer™, Brown University, URL (brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and BIMAS, URL bimas.dcrt.nih.gov/). To illustrate this, peptide epitopes from PSCA that are presented in the context of human MHC Class I molecules, e.g., HLA-A1, A2, A3, A11, A24, B7, and B35 were predicted (see, e.g., Tables V-XVIII, XXII-LI). Specifically, the complete amino acid sequence of the PSCA protein and relevant parts of other variants, i.e., for HLA Class I predictions 9 flanking residues on one or the other side of a point mutation or exon splicing and for HLA Class II predictions 14 flanking residues on one or the other side of a point mutation or exon splicing corresponding to this variant, entered into the HLA peptide motif search algorithm found in the Bioinformatics and Molecular Analysis Section (BIMAS) web site listed above; in addition to the area SYFPEITHI, at the URL syfpeithi.bmi-heidelberg.com/.

The HLA peptide motif search algorithm was developed by Dr. Ken Parker based on the binding of specific peptide sequences in the pit of HLA Class I molecules, particularly HLA-A2 (see, e.g., Falk et al., Nature 351: 290-6 ( 1991); Hunt et al., Science 255:1261-3 (1992); Parker et al., J. Immunol. 149:3580-7 (1992); Parker et al., J. Immunol. 152:163-75 (1994)). This algorithm allows the localization and ranking of 8-mer, 9-mer and 10-mer peptides from a complete protein sequence for predicted binding to HLA-A2 as well as a variety of other HLA Class I molecules. Many HLA class I binding peptides are 8-, 9-, 10-, or 11-mers. For Class I HLA-A2, for example, the epitopes preferably contain a leucine (L) or methionine (M) at position 2 and a valine (V) or leucine (L) at the C-terminal end (see, e.g., Parker et al., J. Immunol. 149:3580-7 (1992)). Selected results of PSCA predicted binding peptides are shown in Tables V-XVIII and XXII-LI herein. In Tables V-XVIII and XXII-XLVIII, selected candidates, 9-mers and 10-mers, for each family member are shown along with their localization, the amino acid sequence of each specific peptide and a calculated binding score. In Tables XLVIII-LI, selected candidates, 15-mers, for each family member are shown along with their localization, the amino acid sequence of each specific peptide and a calculated binding score. The binding score corresponds to the calculated half hour for dissociation of complexes containing the peptide at 37°C at pH 6.5. Peptides with the highest binding score are predicted to be the most tightly bound to HLA Class I on the cell surface for the longest period of time and consequently represent the best immunogenic targets for T-cell recognition.

Actual binding of peptides to an HLA allele can be evaluated by stabilizing HLA expression on the antigen-processing defective cell line T2 (see, e.g., Xue et al., Prostate 30:73-8 (1997) and Peshwa et al. ., Prostate 36:129-38 (1998)). Immunogenicity of specific peptides can be evaluated in vitro by stimulation of CD8+ cytotoxic T lymphocytes (CTL) in the presence of antigen presenting cells such as dendritic cells.

It is to be understood that each epitope predicted by the BIMAS site, Epimer™ and Epimatrix™ sites or specified by HLA class I or class II motifs available in the field or becoming part of the state of the art as set forth in Table IV (or determined by application of World Wide Web site URL syfpeithi.bmi-heidelberg.com/ or BIMAS, bimas.dcrt.nih.gov/) shall "use" a PSCA protein according to the invention. As used in this context, "using" means that a PSCA protein is evaluated, e.g., visually or by computer-based pattern-searching methods, as understood by those skilled in the relevant art. Each subsequence of a PSCA protein of 8, 9, 10 or 11 amino acid residues carrying an HLA Class I motif or a subsequence of 9 or more amino acid residues carrying an HLA Class II motif is within the scope of the invention.

III.B.) Expression of PSCA-related proteins

In one embodiment described in the examples that follow, PSCA can conveniently be expressed in cells (such as 293T cells) transfected with a commercially available expression vector such as a CMV-driven expression vector encoding PSCA with a C-terminal 6XHis and MYC tag ( pcDNA3,1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, Nashville TN). The Tag5 vector provides an IgGK secretion signal that can be used to facilitate production of a secreted PSCA protein in transfected cells. The secreted HIS-tagged PSCA in the culture media can be purified, for example, using a nickel column using standard techniques.

III.C.) Modifications of PSCA-related proteins

Modifications of PSCA-related proteins such as covalent modifications are included within the scope of the present invention. One type of covalent modification involves reacting the targeted amino acid residues of a PSCA polypeptide with an organic derivatizing agent capable of reacting with selected side chains or the N- or C-terminal residues of a PSCA protein. Another type of covalent modification of a PSCA polypeptide encompassed within the scope of the present invention comprises changing the native glycosylation pattern of a protein according to the invention. Another type of covalent modification of PSCA involves attaching a PSCA polypeptide to one of a variety of non-proteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Patents No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The PSCA-related proteins according to the present invention can also be modified to form a chimeric molecule comprising PSCA fused to another, heterologous polypeptide or amino acid sequence. Such a chimeric molecule can be synthesized chemically or recombinantly. A chimeric molecule can be a protein according to the invention fused to another tumor-associated antigen or fragment thereof. Alternatively, a protein according to the invention may comprise a fusion of fragments of a PSCA sequence (amino or nucleic acid) so that a molecule is formed which is not, throughout its length, directly homologous to the amino or nucleic acid sequences shown in Figure 1. A such 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 can selectively bind, with cytokines or with growth factors. The epitope tag is generally located at the amino or carboxy terminal ends of a PSCA protein. In an alternative embodiment, 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 an "immunoadhesin"), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain-deleted or inactivated) form of a PSCA polypeptide in place of at least one variable region within an Ig molecule. In a preferred embodiment, the immunoglobulin fusion comprises the hinge, CH2 and CH3 or hinge, CH1, CH2 and CH3 regions of an IgGI molecule. For the preparation of immunoglobulin fusions see, e.g., U.S. Pat. Patent No. 5,428,130 granted June 27, 1995.

III.D.) Applications of PSCA-related proteins

The proteins according to the invention have several different specific applications. As PSCA is highly expressed in prostate and other cancers, PSCA-related proteins are used in methods that determine the status of PSCA gene products in normal versus cancerous tissues, thereby elucidating the malignant phenotype. Typically, polypeptides from specific regions of a PSCA protein are used to assess the presence of abnormalities (such as deletions, insertions, point mutations, etc.) in those regions (such as regions containing one or more motifs). Examples of experiments use antibodies or T cells that target PSCA-related proteins comprising the amino acid residues of one or more of the biological motifs contained within a PSCA polypeptide sequence to evaluate the properties of this region in normal versus cancerous tissues or to elicit an immune response against the epitope . Alternatively, PSCA-related proteins containing the amino acid residues of one or more of the biological motifs in a PSCA protein are used to screen for factors that interact with this region of PSCA.

PSCA protein fragments/subsequences are particularly useful for generating and characterizing domain-specific antibodies (eg, antibodies that recognize an extracellular or intracellular epitope of a PSCA protein), to identify agents or cellular factors that bind to PSCA or a particular structural domain thereof and in various therapeutic and diagnostic contexts, including but not limited to diagnostic tests, cancer vaccines and methods for producing such vaccines.

Proteins encoded by the PSCA genes or by analogs, homologues or fragments thereof have a variety of applications, including but not limited to the production of antibodies and in methods for identifying ligands and other agents and cellular constituents that bind to a PSCA gene product. Antibodies raised against a PSCA protein or fragment thereof are useful in diagnostic and prognostic analyzes and imaging methods in the treatment of human cancer characterized by expression of PSCA protein, such as those listed in Table I. Such antibodies can be expressed intracellularly and used in methods for the treatment of patients with such cancer. PSCA-related nucleic acids or proteins have also been used in generating HTL or CTL responses.

Various immunological assays applicable to the detection of PSCA proteins are used, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA), immunocytochemical methods and the like. Antibodies can be labeled and used as immunological imaging reagents capable of detecting PSCA-expressing cells (eg, in radioscintigraphic imaging methods). PSCA proteins are also particularly useful in the production of cancer vaccines, as further described herein.

IV.) PSCA antibodies

Another aspect of the invention provides antibodies that bind to PSCA-related proteins. Preferred antibodies specifically bind to a PSCA-related protein and do not bind (or bind weakly) 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) through 4); these reactions preferably take place at pH 7.5, alternatively in a range of pH 7.0 to 8.0 or alternatively in a range of pH 6.5 to 8.5; likewise, these reactions take place at a temperature of between 4ºC and 37ºC. For example, antibodies that bind PSCA can bind PSCA-related proteins such as their homologues or analogs.

PSCA antibodies according to the invention are particularly applicable in cancer (see, for example, Table I) diagnostic and prognostic analyzes and imaging methods. Correspondingly, such antibodies are applicable in the treatment, diagnosis and/or prognosis of prostate and other forms of cancer, to the extent that PSCA is also expressed or overexpressed in these other forms of cancer. Furthermore, intracellularly expressed antibodies (eg, single chain antibodies) are therapeutically useful for the treatment of cancers in which expression of PSCA is involved, such as advanced or metastatic prostate cancer or other advanced or metastatic cancers.

The invention also provides various immunological assays useful for the detection and quantification of PSCA and mutant PSCA-related proteins. Such assays may include one or more PSCA antibodies capable of recognizing and binding a PSCA-related protein, as appropriate. These tests are carried out within different immunological analysis formats well known in the field, including but not limited to different types of radioimmunoassays, enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunofluorescent tests (ELIFA) and the like.

Immunological non-antibody assays according to the invention also include T-cell immunogenicity assay (inhibitory or stimulating) as well as major histocompatibility complex (MHC) binding assays.

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

PSCA antibodies have also been used in methods for purification of a PSCA-related protein and for isolating PSCA homologues and related molecules. For example, a method for purifying a PSCA-related protein comprises incubating a PSCA antibody, which is coupled to a solid matrix, with a lysate or other solution containing a PSCA-related protein under conditions that allow the PSCA antibody to bind to the PSCA-related protein; washing the solid matrix to remove impurities; and eluting the PSCA-related protein from the coupled antibody. Other uses of PSCA antibodies according to the invention include the generation of anti-idiotypic antibodies that mimic a PSCA protein.

Various methods for producing antibodies are well known in the field. For example, antibodies can 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 and Lane (1988); Harlow , Antibodies, Cold Spring Harbor Press, NY (1989)). In addition, fusion proteins of PSCA can also be used, such as a PSCA-GST fusion protein. In a particular embodiment, a GST fusion protein comprising all or most of the amino acid sequence of Figure 1 is prepared, then used as an immunogen to raise 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 purified PSCA-related protein or PSCA-expressing cells) to elicit 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 for specific regions of the PSCA protein for generating antibodies. For example, hydrophobicity and hydrophilicity analyzes of a PSCA amino acid sequence are used to identify hydrophilic regions of the PSCA structure. Regions of a PSCA protein exhibiting 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 analysis.

Hydrophilicity profiles can be produced using the method of Hopp, T.P. and Woods, K.R., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828.

Hydropathicity profiles can be produced using the method of Kyte, J. and Doolittle, R.F., 1982, J. Mol. Biol. 157:105-132. Percent (%) available residue profiles can be generated using the method of Janin J., 1979, Nature 277:491-492. Average flexibility profiles can be produced using the method of Bhaskaran R., Ponnuswamy P.K., 1988, Int. J.

pept. Protein Res.32:242-255. Beta-turn profiles can be generated using the method of Deleage, G., Roux B., 1987, Protein Engineering 1:289-294.

Accordingly, each region identified by any of these programs or methods is within the scope of the present invention. Preferred methods for generating PSCA antibodies are further illustrated by the examples provided herein. Methods for preparing a protein or polypeptide for use as an immunogen are well known in the art. Also well known in the field are methods for producing immunogenic conjugates of a protein with a carrier, such as BSA, KLH or other carrier protein. In some cases, direct conjugation using, for example, carbodiimide reagents is used; in other cases, binding reagents such as those supplied by Pierce Chemical Co., Rockford, IL, are effective. Administration of a PSCA immunogen is often accomplished by injection over a suitable period of time and with the use of a suitable adjuvant, as is understood in the art. During the course of the immunization schedule, titers of antibodies may be taken to determine adequacy of antibody formation.

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

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

Methods for producing fully human monoclonal antibodies include phage display and transgenic methods (for review, see Vaughan et al., 1998, Nature Biotechnology 16: 535-539). Fully human PSCA monoclonal antibodies can be generated using cloning technologies using large human Ig gene combinatorial libraries (ie, phage display) (Griffiths and Hoogenboom, Building an in vitro immune system: human antibodies from phage display libraries. I: Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man, Clark, M. (Ed.), Nottingham Academic, pp 45-64 (1993); Burton and Barbas, Human Antibodies from combinatorial libraries. Id., pp 65-82 ). Fully human PSCA monoclonal antibodies can also be produced using transgenic mice engineered to contain human immunoglobulin gene loci as described in PCT Patent Application WO98/24893, Kucherlapati and Jakobovits et al., published December 3, 1997 (see also, Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4): 607-614; U.S. Patents 6,162,963 issued December 19, 2000; 6,150,584 issued November 12, 2000; and, 6,114598 issued September 5, 2000). This method avoids the in vitro manipulation required with phage display technology and effectively provides high-affinity authentic human antibodies.

Reactivity of PSCA antibodies with a PSCA-related protein can be established by several well-known methods, including Western blot, immunoprecipitation, ELISA and FACS assays using, as appropriate, PSCA-related proteins, PSCA-expressing cells or extracts thereof. A PSCA antibody or fragment thereof may be labeled with a detectable marker or conjugated to a second molecule. Suitable detectable markers include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, chemiluminescent compound, a metal chelator, or an enzyme. Furthermore, bispecific antibodies specific for two or more PSCA epitopes are prepared using methods generally known in the art. Homodimeric antibodies can also be prepared by cross-linking techniques known in the art (eg, Wolff et al., Cancer Res. 53: 2560-2565).

In one embodiment, the invention provides monoclonal antibodies identified as H1-1.10 which were sent (via Federal Express) to the American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108 on May 4, 2005 and designated Accession No. PTA-6697.

V.) PSCA cellular immune responses

The mechanism by which T cells recognize antigens has been described.

Effective peptide-epitope vaccine mixtures according to the invention induce a therapeutic or prophylactic immune response in very large parts of the global population. For an understanding of the value and effectiveness of compositions of the invention in inducing cellular immune responses, a brief overview of immunology-related technology is provided.

A complex of an HLA molecule and a peptide antigen serves as the ligand recognized by HLA-restricted T cells (Buus, S. et al., Cell 47:1071, 1986; Babbitt, B. P. et al., Nature 317:359, 1985 ; Townsend, A. and Bodmer, H., Annu.Rev.

Immunol. 7:601, 1989; Germain, R.N., Annu. Fox. Immunol. 11:403, 1993).

Through study of single amino acid-substituted antigen analogues and sequencing of endogenously bound, naturally processed peptides, critical residues corresponding to motifs required for specific binding to HLA antigen molecules have been identified and are listed in Table IV (see also, e.g. ., Southwood, et al., J. Immunol. 160:3363, 1998; Rammensee, et al., Immunogenetics 41:178, 1995; Rammensee et al., SYFPEITHI, accessed via the World Wide Web at URL (134.2, 96,221/scripts.hlaserver.dll/home.htm); Sette, A. and Sidney, J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, V. H., Curr. Opin. Immunol. 6:13, 1994; Sette, A. and Grey, H. M., Curr. Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J. Curr. Biol. 6:52, 1994; Ruppert et al., Cell 74:929- 937, 1993; Kondo et al., J.

Immunol. 155:4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490, 1996; Sidney et al., Human Immunol. 45:79-93, 1996; Sette, A. and Sidney, J.

Immunogenetics 1999 Nov; 50(3-4):201-12, Overview).

Furthermore, X-ray crystallographic analyzes of HLA-peptide complexes have revealed pockets within the peptide-binding cleft/pit of HLA molecules that accommodate, in an allele-specific manner, residues carried by peptide ligands; these residues in turn determine the HLA binding capacity of the peptides in which they are present. (see, e.g., Madden, D.R. Annu. Rev. Immunol. 13:587, 1995; Smith, et al., Immunity 4:203, 1996; Fremont et al., Immunity 8:305, 1998; Stern et al. ., Structure 2:245, 1994; Jones, E. Y. Curr. Opin. Immunol. 9:75, 1997; Brown, J. H. et al., Nature 364:33, 1993; Guo, H. C. et al., Proc. Natl. Acad. .Sci. USA 90:8053, 1993; Guo, H. C. et al., Nature 360:364, 1992; Silver, M. L. et al., Nature 360:367, 1992; Matsumura, M. et al., Science 257:927 , 1992; Madden et al., Cell 70:1035, 1992; Fremont, D. H. et al., Science 257:919, 1992; Saper, M. A. , Bjorkman, P. J. and Wiley, D. C., J. Mol. Biol. 219:277 , 1991.)

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

Accordingly, by an HLA motif identification method, candidates for epitope-based vaccines have been identified; such candidates can be further evaluated by HLA peptide binding assays to determine binding affinity and/or the time period of association of the epitope and its corresponding HLA molecule. Further confirmatory work can be performed to select, among these vaccine candidates, epitopes with preferred properties in terms of population coverage and/or immunogenicity.

Different strategies can be used to assess cellular immunogenicity, including:

1) Evaluation of primary T cell cultures from normal individuals (see, e.g., Wentworth, P. A. et al., Mol. Immunol. 32:603, 1995; Celis, E. et al., Proc. Natl. Acad. Sci .USA 91:2105, 1994; Tsai, V. et al., J. Immunol. 158:1796, 1997;

Kawashima, I. et al., Human Immunol. 59:1, 1998). This procedure involves stimulation of peripheral blood lymphocytes (PBL) from normal subjects with a test peptide in the presence of antigen-presenting cells in vitro over a period of many weeks. T cells specific for the peptide are activated during this time 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, e.g., Wentworth, P. A. et al., J. Immunol. 26:97, 1996; Wentworth, P. A. et al., Int. Immunol. 8:651, 1996;

Alexander, J. et al., J. Immunol. 159:4753, 1997). For example, in such methods peptides in incomplete Freund's adjuvant are administered subcutaneously to HLA transgenic mice. Several weeks after immunization, splenocytes are removed and cultured in vitro in the presence of test peptide for approximately one week. Peptide-specific T cells are detected using, for example, a 51 Cr release assay involving peptide-sensitized target cells and target cells expressing endogenously produced antigen.

3) Demonstration of recall T-cell responses from immune individuals who have either been effectively vaccinated and/or from chronically ill patients (see, e.g., Rehermann, B. et al., J. Exp. Med. 181:1047 , 1995; Doolan, D. L. et al., Immunity 7:97, 1997; Bertoni, R. et al., J. Clin. Invest. 100:503, 1997; Threlkeld, S. C. et al., J. Immunol. 159: 1648, 1997; Diepolder, H.M. et al., J. Virol. 71:6011, 1997).

Consequently, recall responses are detected by culturing PBL from subjects who have been exposed to the antigen due to illness and have thus formed an immune response "naturally" or from patients who were vaccinated against the antigen. PBL from subjects are cultured in vitro for 1-2 weeks in the presence of test peptide plus antigen presenting cells (APC) to allow activation of "memory" T cells, compared to "naive" T cells. At the end of the culture period, T cell activity is detected using assays including 51 Cr release involving peptide sensitized targets, T cell proliferation or lymphokine release.

VI.) PSCA transgenic animals

Nucleic acids encoding a PSCA-related protein can also be used to generate either transgenic animals or "knock out" animals which, in turn, are useful in the development and screening of therapeutically useful reagents. According to established techniques, cDNA encoding PSCA can be used to clone genomic DNA encoding PSCA. The cloned genomic sequences can then be used to generate transgenic animals containing cells expressing DNA encoding PSCA. Methods for the production of transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Pat. Patent No. 4,736,866 granted April 12, 1988 and 4,870,009 granted September 26, 1989. Typically, specific cells would be targeted for PSCA transgene incorporation with tissue-specific enhancers.

Transgenic animals comprising one copy of a transgene encoding PSCA can be used to investigate the effect of increased expression of DNA encoding PSCA. Such animals can be used as test animals for reagents believed to provide protection against, for example, pathological disorders associated with its overexpression. According to this aspect of the invention, an animal is treated with a reagent and a reduced incidence of a pathological disorder, compared to untreated animals carrying the transgene, would indicate a potential therapeutic intervention for the pathological disorder.

Alternatively, non-human homologs of PSCA can be used to construct a PSCA "knock out" animal that has a defective or altered gene encoding PSCA as a result of homologous recombination between the endogenous gene encoding PSCA and altered genomic DNA that codes for PSCA introduced into an embryonic cell from the animal. For example, cDNA encoding PSCA can be used to clone genomic DNA encoding PSCA according to established techniques. A portion of genomic DNA encoding PSCA can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration. Typically, many kilobases of unchanged flanking DNA (at both the 5' and 3' ends) are included in the vector (see, e.g., Thomas and Capecchi, Cell, 51:503 (1987) for a description of vectors for homologous recombination) . The vector is introduced into an embryonic stem cell line (eg, by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected (see, eg, Li et al., Cell, 69:915 (1992 )). The selected cells are then injected into a blastocyst from an animal (eg, a mouse or rat) to form aggregation chimeras (see, eg, Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal. Progeny containing the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all the cells of the animal contain the homologously recombined DNA. Knock out animals can be characterized, for example, for their ability to defend against certain pathological conditions or for their development of pathological disorders due to the absence of a PSCA polypeptide.

VII.) Methods for detection of PSCA

Another aspect of the present invention relates to methods for detecting 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 status of PSCA gene products provides information useful for predicting a variety of factors including susceptibility to advanced stage disease, rate of progression and/or tumor aggressiveness. As described in detail herein, the status of PSCA gene products in patient samples can be analyzed by many different methods well known in the art including immunohistochemical analysis, the variety of Northern blotting techniques including in situ hybridization, RT-PCR analysis (eg, on laser binding micro-dissected samples), Western blot analysis and tissue matrix analysis.

More specifically, the invention 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, alternative splice variant PSCA mRNAs and recombinant DNA or RNA molecules containing a PSCA polynucleotide. Several methods for amplifying and/or detecting the presence of PSCA polynucleotides are well known in the field and can be used when practicing this aspect according to the invention.

In one embodiment, a method for detecting a PSCA mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA thus prepared using a PSCA polynucleotides as sense and antisense primers to amplify PSCA cDNAs therein; and detecting presence of the amplified PSCA cDNA. Optionally, the sequence of the amplified PSCA cDNA can be determined.

In another embodiment, a method for detecting a PSCA gene in a biological sample first comprises isolating genomic DNA from the sample; amplifying the isolated genomic DNA using PSCA polynucleotides as sense and antisense primers; and detecting the presence of the amplified PSCA gene. Any number of suitable sense and antisense probe combinations can be designed from a PSCA nucleotide sequence (see, e.g., Figure 1) and used for this purpose.

The invention also provides tests 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 detecting 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-reactive fragment thereof, or a recombinant protein containing an antigen-binding region of a PSCA antibody ; and then detect the binding of PSCA-related protein in the sample.

Methods of identifying a cell expressing PSCA are also within the scope of the invention. In one embodiment, an assay to identify a cell expressing a PSCA gene comprises detecting the presence of PSCA mRNA in the cell. Methods for the detection of particular mRNAs in cells are well known and include, for example, hybridization experiments using complementary DNA probes (such as in situ hybridization using labeled PSCA riboprobes, Northern blot and related techniques) and various nucleic acid amplification assays (such as such as RT-PCR using complementary primers specific for PSCA and other amplification type detection methods, such as for example branched DNA, SISBA, TMA and the like). Alternatively, an attempt 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 used for the detection of PSCA-related proteins and cell PSCA expression analysis is also useful as a tool to identify and evaluate agents that modulate PSCA gene expression. For example, PSCA expression is significantly upregulated in prostate cancer and is expressed in cancer in the tissues listed in Table I. Identification of a molecule or biological agent that inhibits PSCA expression or over-expression in cancer 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 PSCA-related genes and their products

Oncogenesis is known to be a multistep process in which cellular growth becomes progressively misregulated and cells progress from a normal physiological state to a precancerous and then cancerous state (see, e.g., Alers et al., Lab Invest.77(5): 437 -438 (1997) and Isaacs et al., Cancer Surv. 23: 19-32 (1995)). In this context, examination of a biological sample for evidence of dysregulated cell growth (such as aberrant PSCA expression in cancer) allows early detection of such aberrant physiology, before a pathological condition such as cancer has progressed to a stage where therapeutic options are more limited and or the prognosis is worse. In such investigations, the status of PSCA in a biological sample of interest can be compared, for example, with the status of PSCA in a corresponding normal sample (e.g. a sample from this individual or alternatively another individual not affected by a pathology). A change in the status of PSCA in the biological sample (compared to the normal sample) provides evidence for misregulation of cellular growth. In addition to using a biological sample that is not affected by a pathology as a normal sample, one can also use a predetermined normative value such as a predetermined normal level of mRNA expression (see, e.g., Grever et al., J. Comp. Neurol. 1996 Dec 9; 376(2): 306-14 and U.S. Patent No. 5,837,501) to compare PSCA status in a sample.

The term "status" in this context is used according to its accepted meaning in the field and denotes the state or status of a gene and its products. Typically, professionals use several parameters to judge the condition or status of a gene and its products. These include, but are not limited to localization of expressed gene products (including localization of PSCA-expressing cells) as well as the level and biological activity of expressed gene products (such as PSCA mRNA, polynucleotides and polypeptides). Typically, a change in the status of PSCA includes a change in the localization of PSCA and/or PSCA-expressing cells and/or an increase in PSCA mRNA and/or protein expression.

PSCA status in a sample can be analyzed by several methods well known in the field, including without limitation, immunohistochemical analysis, in situ hybridization, RT-PCR analysis of "laser capture micro-dissected" samples, Western blot analysis and tissue array analysis. Typical procedures for evaluating the status 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). Accordingly, the status of PSCA in a biological sample is evaluated by various methods employed by those skilled in the art including, but not limited to, genomic Southern analysis (to examine, for example, abnormalities in a PSCA gene), Northern analysis and/or PCR analysis of PSCA mRNA (to examine, for example, changes in the polynucleotide sequences or expression levels of PSCA mRNAs) and, Western and/or immunohistochemical analysis (to examine, for example, changes in polypeptide sequences, changes in polypeptide localization within a sample, changes in expression levels of PSCA proteins and/or associations of PSCA proteins with polypeptide binding partners). Detectable PSCA polynucleotides include, for example, a PSCA gene or fragment thereof, PSCA mRNA, alternative splice variants, PSCA mRNAs and recombinant DNA or RNA molecules containing a PSCA polynucleotide.

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

Expression status of PSCA provides information including the presence, stage and localization of dysplastic, precancerous and cancerous cells, predicts susceptibility to different stages of disease and/or for measurement of tumor aggressiveness. Furthermore, the expression profile makes it useful as an imaging reagent for metastatic disease. Accordingly, one aspect of the invention relates to the various molecular prognostic and diagnostic methods for investigating the status of PSCA in biological samples such as those from individuals suffering from or suspected of suffering from a pathology characterized by dysregulated cellular growth, such as cancer.

As described above, the status of PSCA in a biological sample can be investigated by several well-known methods in the field. For example, the status of PSCA 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 (eg, those expressing PSCA mRNAs or proteins). This examination may provide evidence of dysregulated cellular growth, for example when PSCA-expressing cells are found in a biological sample that does not normally contain such cells (such as a lymph node), because such changes in the status of PSCA in a biological sample are often associated with misregulated cellular growth. Specifically, one indicator of dysregulated cellular growth is the metastases of cancer cells from an organ of origin (such as the prostate) to a different area of the body (such as a lymph node). In this context, evidence for misregulated cellular growth is important, for example, because occult lymph node metastases can be detected in a significant proportion of patients with prostate cancer and such metastases are associated with known predictors of disease progression (see, e.g., Murphy et al., Prostate 42 (4): 315-317 (2000); Su et al., Semin. Surg. Oncol. 18(1): 17-28 (2000) and Freeman et al., J Urol 1995 Aug 154(2 Pt 1): 474-8).

In one aspect, the invention provides methods for monitoring PSCA gene products by determining the status of PSCA gene products expressed by cells from an individual suspected of having a disease associated with dysregulated cell growth (such as hyperplasia or cancer) and then comparing the status determined to this way with the status of PSCA gene products in a corresponding normal sample.

The presence of deviant PSCA gene products in the test sample compared to the normal sample gives an indication of the presence of misregulated cell growth in the cells from the individual.

In another aspect, the invention provides assays useful for determining the presence of cancer in an individual, comprising detecting a significant increase in PSCA mRNA or protein expression in a test cell or tissue sample relative to expression levels in the corresponding normal cell or tissue. For example, presence of PSCA mRNA can be evaluated in tissues including but not limited to those listed in Table I. Presence of significant PSCA expression in any of these tissues is useful to indicate the occurrence, presence and/or severity of cancer , since the corresponding normal tissues do not express PSCA mRNA or express it at lower levels.

In a related embodiment, 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 cells in a test tissue sample and comparing the level determined in this way with the level of PSCA expressed in a corresponding normal sample. In one embodiment, the presence of PSCA protein is evaluated, for example, using immunohistochemical methods. PSCA antibodies 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.

In a further embodiment, one can evaluate the status of PSCA nucleotide and amino acid sequences in a biological sample to identify abnormalities in the structure of these molecules. These deviations may include insertions, deletions, substitutions and the like. Such evaluations are useful because deviations in nucleotide and amino acid sequence have been observed in a large number of proteins associated with a growth-misregulated phenotype (see, e.g., Marrogi et al., 1999, J. Cutan. Pathol.

26(8):369-378). For example, a mutation in the sequence of PSCA may be indicative of the presence or promotion of a tumor. Such analyzes therefore have diagnostic and predictive value where a mutation in PSCA indicates a potential loss of function or increase in tumor growth.

A number of analyzes to observe deviations in nucleotide and amino acid sequences are well known in the field. For example, size and structure of nucleic acid or amino acid sequences of PSCA gene products are observed by Northern, Southern, Western, PCR and DNA sequencing methods described herein. In addition, other methods for observing deviations in nucleotide and amino acid sequences such as single strand conformation polymorphism analysis are well known in the field (see, for example, U.S. Patent No. 5,382,510 granted 7 September 1999 and 5,952,170 granted 17 January 1995).

In addition, one can investigate the methylation status of a PSCA gene in a biological sample. Aberrant demethylation and/or hypermethylation of CpG islands in gene 5' regulatory regions often occurs in immortalized and transformed cells and can result in altered expression of various genes. For example, promoter hypermethylation of pi-class glutathione S-transferase (a protein expressed in normal prostate but not expressed in >90% of prostate carcinomas) appears to permanently silence transcription of this gene and is the most frequently detected genomic alteration in prostate carcinomas ( De Marzo et al., Am. J. Pathol. 155(6): 1985-1992 (1999)). In addition, this change is present in at least 70% of cases of high-grade prostatic intraepithelial neoplasia (PIN) (Brooks et al., Cancer Epidemiol.

Biomarkers Prev., 1998, 7:531-536). In another example, expression of the LAGE-I tumor specific gene (which is not expressed in normal prostate but is expressed in 25-50% of prostate cancer) is induced by deoxy-azacytidine in lymphoblastoid cells, indicating that tumor expression is due to demethylation (Lethe et al., Int. J. Cancer 76(6): 903-908 (1998)). A number of assays for examining the methylation status of a gene are well known in the art. For example, in Southern hybridization methods, methylation-sensitive restriction enzymes that cannot cleave sequences containing methylated CpG sites can be used to assess the methylation status of CpG islands. In addition, MSP (methylation-specific PCR) can rapidly profile the methylation status of all CpG sites present in a CpG island of a given gene. This procedure involves initial modification of DNA by sodium bisulfite (which will convert all unmethylated cytosines to uracil) followed by amplification using primers specific for methylated versus unmethylated DNA.

Methods involving methylation interference can also be found, for example, in Current Protocols in Molecular Biology, Unit 12, Frederick M.

Ausubel et al. eds., 1995.

Gene amplification is an additional method to assess the status of PSCA. Gene amplification is measured in a sample directly, for example by conventional Southern blotting or Northern blotting to quantify the transcription of mRNA (Thomas, 1980, Proc. Natl. Acad. Sci. USA, 77:52015205), dot blotting (DNA- assay) or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies are used which recognize specific duplexes, including DNA duplexes, RNA duplexes and DNA RNA hybrid duplexes or DNA protein duplexes. The antibodies are in turn labeled and the experiment carried out where the duplex is bound to a surface, so that when the duplex is formed on the surface, the presence of antibody bound to the duplex can be detected.

Biopsy tissue or peripheral blood can conveniently be examined for the presence of cancer cells using, for example, Northern, dot blot or RT-PCR analysis to detect PSCA expression. Presence of RT-PCR amplifiable PSCA mRNA provides an indication of the presence of cancer. RT-PCR analysis is well known in the art. RT-PCR detection assays for tumor cells in peripheral blood are currently being evaluated for use in the diagnosis and treatment of several human solid tumors. In the field of prostate cancer, these include RT-PCR assays for the detection of cells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res.25:373-384; Ghossein et al., 1995, J. Clin. Oncol .

13:1195-2000; Heston et al., 1995, Clin. Chem. 41:1687-1688).

A further aspect according to the invention is an assessment of the susceptibility that an individual has to developing cancer. In one embodiment, a method of predicting susceptibility to cancer comprises detecting PSCA mRNA or PSCA protein in a tissue sample, where its presence indicates susceptibility to cancer, wherein the degree of PSCA mRNA expression correlates with the degree of susceptibility. In a specific embodiment, the presence of PSCA in the prostate or other tissues is examined, where the presence of PSCA in the sample provides an indication of susceptibility to prostate cancer (or the appearance or existence of a prostate tumor). Similarly, one can evaluate the integrity of PSCA nucleotide and amino acid sequences in a biological sample, to identify deviations in the structure of these molecules such as insertions, deletions, substitutions and the like. The presence of one or more abnormalities in PSCA gene products in the sample is indicative of susceptibility to cancer (or the appearance or existence of a tumor).

The invention also includes methods for measuring tumor aggressiveness. In one embodiment, a method for measuring aggressiveness of a tumor comprises determining the level of PSCA mRNA or PSCA protein expressed by tumor cells, comparing the level thus determined with the level of PSCA mRNA or PSCA protein expressed in a corresponding normal tissue taken from the same individual or a reference sample from normal tissue, where the degree of PSCA mRNA or PSCA protein expression in the tumor sample compared to the normal sample indicates the degree of aggressiveness. In a specific embodiment, aggressiveness of a tumor is evaluated by determining the degree to which PSCA is expressed in the tumor cells, with higher expression levels indicating more aggressive tumors. Another embodiment is the evaluation of the integrity of PSCA nucleotide and amino acid sequences in a biological sample, to identify deviations in the structure of these molecules such as insertions, deletions, substitutions and the like. Presence of one or more abnormalities indicates more aggressive tumors.

Another embodiment according to the invention relates to methods for observing the progression of a malignant disease in an individual over time. In one embodiment, methods of observing the progression of a malignant disease in an individual over time comprise determining the level of PSCA mRNA or PSCA protein expressed by cells in a sample of the tumor, comparing the level so determined to the level of PSCA- mRNA or PSCA protein expressed in an equivalent tissue sample taken from the same individual at a different time, where the degree of PSCA mRNA or PSCA protein expression in the tumor sample over time provides information about the progression of the cancer. In a specific embodiment, the progression of cancer is evaluated by determining PSCA expression in the tumor cells over time, where increased expression over time indicates a progression of the cancer. Likewise, one can evaluate the integrity of PSCA nucleotide and amino acid sequences in a biological sample to identify abnormalities in the structure of these molecules such as insertions, deletions, substitutions and the like, where the presence of one or more abnormalities indicates a progression of the cancer.

The above diagnostic methods can be combined with any of a number of prognostic and diagnostic protocols known in the art. For example, another embodiment according to the invention relates to methods for observing a coincidence between expression of PSCA gene and PSCA gene products (or abnormalities in PSCA gene and PSCA gene products) and a factor associated with malignant disease, as a method of to diagnose and prognosticate the status of a tissue sample. A number of factors associated with malignant disease can be used, such as expression of genes associated with malignant disease (eg PSA, PSCA and PSM expression for prostate cancer, etc.) as well as gross cytological observations (see, eg, Bocking et al., 1984, Anal. Quant. Cytol. 6(2):74-88; Epstein, 1995, Hum. Patol. 26(2):223-9; Thorson et al., 1998, Mod. Patol. 11( 6):543-51; Baisden et al., 1999, Am. J. Surg. Pathol. 23(8):918-24).

Methods for observing a coincidence between expression of PSCA gene and PSCA gene products (or abnormalities in PSCA gene and PSCA gene products) and another factor associated with malignant disease are useful, for example, because the presence of a set of specific factors that coincide with disease provide information important for diagnosis and prognostication of the status of a tissue sample.

In one embodiment, methods for observing a correspondence between expression of PSCA gene and PSCA gene products (or discrepancy in PSCA gene and PSCA gene products) and another factor associated with malignant disease include detecting the overexpression of PSCA mRNA or protein in a tissue sample, detecting the overexpression of PSA mRNA or protein in a tissue sample (or PSCA or PSM expression) and observing a correspondence between PSCA mRNA or protein and PSA mRNA or protein overexpression (or PSCA or PSM expression). In a specific embodiment, expression of PSCA and PSA mRNA in prostate tissue is examined, where agreement between PSCA and PSA mRNA overexpression in the sample indicates the existence of prostate cancer, prostate cancer susceptibility, or the appearance or status of a prostate tumor.

Methods for detecting and quantifying expression of PSCA mRNA or protein are described herein and standard nucleic acid and protein detection and quantification technologies are well known in the art. Standard methods for the detection and quantification of PSCA mRNA include in situ hybridization using labeled PSCA riboprobes, Northern blot and related techniques using PSCA polynucleotide probes, RT-PCR analysis using primers specific for PSCA and other amplification-type detection methods, such as such as branched DNA, SISBA, TMA and the like. In a specific embodiment, semi-quantitative RT-PCR is used to detect and quantify PSCA mRNA expression. Any number of primers capable of amplifying PSCA can be used for this purpose, including but not limited to the various primer sets specifically described herein. In a specific embodiment, polyclonal or monoclonal antibodies specifically reactive with the wild-type PSCA protein can be used in an immunohistochemical analysis of biopsy tissue.

IX.) Identification of molecules that interact with PSCA

The PSCA protein and nucleic acid sequences described herein allow those skilled in the art to identify proteins, small molecules, and other agents that interact with PSCA, as well as pathways activated by PSCA through any of a number of previously known methods. For example, one of the so-called interaction trap systems (also referred to as the "two-hybrid analysis") can be used. In such systems, molecules interact and reconstitute a transcription factor that directs expression of a reporter gene, after which expression of the reporter gene is analyzed. Other systems identify protein-protein interactions in vivo through reconstitution of a eukaryotic transcriptional activator, see, e.g., U.S. Pat. Patent No. 5,955,280 granted 21 September 1999, 5,925,523 published 20 July 1999, 5,846,722 granted 8 December 1998 and 6,004,746 granted 21 December 1999. Algorithms are also available in the area of genome-based predictions of protein function (see e.g., Marcotte, et al. , Nature 402: 4 November 1999, 83-86).

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

Consequently, peptides that have a variety of applications, such as therapeutic, prognostic or diagnostic 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 described, for example, in U.S. Pat. Patent no. 5,723,286 granted 3 March 1998 and 5,733,731 granted 31 March 1998.

Alternatively, cell lines expressing PSCA are used to identify protein-protein interactions mediated by PSCA. Such interactions can be examined using immunoprecipitation techniques (see, e.g., Hamilton B.J., et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51). PSCA protein can be immunoprecipitated from PSCA-expressing cell lines using anti-PSCA antibodies. Alternatively, antibodies against 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 by methods such as Western blotting, <35>S-methionine labeling of proteins, protein microsequencing, silver labeling and two-dimensional gel electrophoresis.

Small molecules and ligands that interact with PSCA can be identified through related embodiments of such screening assays. For example, small molecules that interfere with protein function can be identified, including molecules that interfere with PSCA's ability to mediate phosphorylation and dephosphorylation, interaction with DNA or RNA molecules as an indication of cell cycle regulation, second messenger signaling or tumorigenesis. Similarly, small molecules that modulate PSCA-related ion channel, protein pump, or cell communication functions have been identified and used to treat patients who have cancers that express PSCA (see, e.g., Hille, B., Ionic Channels of Excitable Membranes 2nd Ed., Sinauer Assoc. , Sunderland, MA, 1992). Furthermore, ligands that regulate PSCA function can be identified based on their ability to bind PSCA and activate a reporter construct. Typical methods are described, for example, in U.S. Pat. Patent no. 5,928,868 granted 27 July 1999 and includes methods for forming hybrid ligands where at least one ligand is a small molecule. In an illustrative embodiment, cells engineered to express a fusion protein of PSCA and a DNA binding protein are used to co-express a fusion protein of a hybrid ligand/small molecule and a cDNA library transcriptional activator protein. The cells also 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 that occurs only if the hybrid ligand binds to target sites on both hybrid proteins. The cells that express the reporter gene are selected and the unknown small molecule or the unknown ligand is identified. This method provides a method for the identification of modulators, which activate or inhibit PSCA.

One embodiment of the invention comprises a method of screening for a molecule that interacts with a PSCA amino acid sequence shown in Figure 1, comprising the steps of contacting a population of molecules with a PSCA amino acid sequence, allowing the population of molecules and the PSCA amino acid sequence interacting under conditions that facilitate an interaction, determining the presence of a molecule that interacts with the PSCA amino acid sequence and then separating molecules that do not interact with the PSCA amino acid sequence from molecules that do. In a specific embodiment, the five-step method further comprises purification, characterization and identification of a molecule that interacts with the PSCA amino acid sequence. The identified molecule can be used to modulate a function performed by PSCA. In a preferred embodiment, the PSCA amino acid sequence is contacted with a library of peptides.

X.) Therapeutic methods and mixtures

Identification of PSCA as a protein that is normally expressed in a limited collection of tissues, but is also expressed in cancers such as those listed in Table I, opens several therapeutic methods for the treatment of such cancers.

It is worth noting that targeted antitumor therapies are applicable even when the targeted protein is expressed in normal tissues, even vital tissues from normal organs. A vital organ is one that is necessary to sustain life, such as the heart or 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 the ovary, breast and prostate.

For example, Herceptin® is an FDA-approved pharmaceutical consisting of an antibody that is immunoreactive with the protein alternately 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 nearly $400 million in 2002. Herceptin® is a treatment for HER2-positive metastatic breast cancer. However, expression of HER2 is not limited to such tumors. The same protein is expressed in several normal tissues. In particular, it is known that HER2/neu is present in normal kidney and heart, thus these tissues are present in all human recipients of Herceptin. The presence of HER2/neu in normal kidney has also been confirmed by Latif, Z., et al., B.J.U. 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 protein and mRNA are produced in benign kidney tissue. In particular, HER2/neu protein was highly overexpressed in benign kidney tissue.

Despite the fact that HER2/neu is expressed in such vital tissues as the heart and kidney, Herceptin is a highly applicable, FDA-approved, and commercially successful drug. The effect of Herceptin on heart tissue, i.e., “cardiotoxicity,” has only been a side effect of treatment. When patients were treated with Herceptin alone, significant cardiotoxicity occurred in a very low percentage of patients. To limit cardiotoxicity, there are stricter access requirements for treatment with HER2/neu. Factors such as predisposition to heart disease are evaluated before treatment can take place.

Of particular note is that, although kidney tissue is indicated to show normal expression, possibly even higher expression than heart tissue, kidney has absolutely no noticeable Herceptin side effect. Furthermore, of the various collections of normal tissue where HER2 is expressed, there is very little incidence of any side effect whatsoever. Only heart tissue has manifested any noticeable side effect at all. A tissue such as the kidney, where HER2/neu expression is particularly noticeable, has not been the starting point for any side effects whatsoever.

Furthermore, beneficial therapeutic effects have been found for antitumor treatments targeting the epidermal growth factor receptor (EGFR); Erbitux (ImClon). EGFR is also expressed in a variety of normal tissues. There have been very limited side effects in normal tissue following the use of anti-EGFR therapeutic agents. A common side effect that occurs with EGFR treatment is a severe skin rash observed in 100% of patients undergoing treatment.

Accordingly, expression of a target protein in normal tissue, even vital normal tissue, does not negate the utility of an agent targeting 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 dispensable, such as the prostate and ovary, can be removed without affecting mortality. Finally, some vital organs are not affected by normal organ expression due to immune privileging. Immunoprivileged organs are organs that are protected from blood by a blood-organ barrier and are therefore not available for immunotherapy. Examples of immune-privileged organs are the brain and the testicles.

Accordingly, therapeutic methods that inhibit the activity of a PSCA protein are useful for patients suffering from cancers that express PSCA. These therapeutic methods generally fall into three classes. The first class modulates PSCA function as it relates to tumor cell growth leading to inhibition or retardation of tumor cell growth or induces its killing. The second class comprises different methods for inhibiting binding or association of a PSCA protein with its binding partner or with other proteins. The third class comprises a number of methods to inhibit transcription of a PSCA gene or translation of PSCA mRNA.

X.A.) Anti-cancer vaccines

The invention provides cancer vaccines comprising a PSCA-related protein or PSCA-related nucleic acid. Given the expression of PSCA, cancer vaccines prevent and/or treat PSCA-expressing cancers with minimal or no effects on non-target tissues. The use of a tumor antigen in a vaccine that generates cell-mediated humoral immune responses as anticancer therapy is well known in the art and has been used 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 using a PSCA-related protein or a PSCA-encoding nucleic acid molecule and recombinant vectors capable of expressing and presenting the PSCA immunogen (typically comprising multiple T-cell epitopes or antibodies). Those skilled in the art will appreciate that a variety of vaccine systems for delivery of immunoreactive epitopes are known in the art (see, e.g., Heryln et al., Ann Med 1999 Feb 31(1):66-78; Maruyama et al., Cancer Immunol Immunother 2000 Jun 49(3):123-32) Briefly, such methods of producing an immune response (eg, cell-mediated and/or humoral) in a mammal comprise the steps: exposing the mammalian immune system to an immunoreactive epitope (eg, a epitope present in a PSCA protein shown in Figure 1 or analog or homolog thereof) such that the mammal generates an immune response specific for this epitope (eg, produces antibodies that specifically recognize this epitope).

The entire PSCA protein, immunogenic regions or epitopes thereof can be combined and delivered by various methods. Such vaccine compositions may include, for example, lipopeptides (eg, Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), peptide compositions encapsulated in poly(DL-lactide-co-glycolide) (“PLG ”) microspheres (see, e.g., Eldridge, et al., Molec. Immunol. 28:287-294, 1991: Alonso et al., Vaccine 12:299-306, 1994; Jones et al., Vaccine 13: 675-681, 1995), peptide mixtures contained in immune-stimulatory complexes (ISCOMS) (see, e.g., Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin Exp Immunol. 113:235 -243, 1998), multiple antigen peptide systems (MAPs) (see, e.g., Tam, J.P., Proc. Natl. Acad. Sci. U.S.A.85:5409-5413, 1988; Tam, J.P., J. Immunol. Methods 196 :17-32, 1996), peptides formulated as multivalent peptides; peptides for use in ballistic delivery systems, typically crystallized peptides, viral delivery vectors (Perkus, M. E. et al., In: Concepts in vaccine development, Kaufmann, S. H. E., ed., p.379, 1996; Chakrabarti, S. et al., Nature 320:535, 1986; Hu, S. L. et al., Nature 320:537, 1986; Kieny, M.-P. et al., AIDS Bio/Technology 4:790, 1986; Top, F. H. et al., J. Infect. Dis. 124:148, 1971;

Chanda, P. K. et al., Virology 175:535, 1990), particles of viral or synthetic origin (eg, Kofler, N. et al., J. Immunol. Methods. 192:25, 1996; Eldridge, J. H. et al., Sem. Hematol. 30:16, 1993; Falo, L. D., Jr. et al., Nature Med. 7:649, 1995), adjuvantia (Warren, H. S., Vogel, F. R. and Chedid, L. A. Annu. Rev .

Immunol. 4:369, 1986; Gupta, R. K. et al., Vaccine 11:293, 1993), liposomes (Reddy, R. et al., J. Immunol. 148:1585, 1992; Rock, K. L., Immunol. Today 17:131, 1996), or , naked or particle-absorbed cDNA (Ulmer, J. B. et al., Science 259:1745, 1993; Robinson, H. L., Hunt, L. A. and Webster, R. G., Vaccine 11:957, 1993; Shiver, J. W. et al., I: Concepts in vaccine development, Kaufmann, S. H. E., ed., p.423, 1996; Cease, K. B. and Berzofsky, J. A., Annu. Rev. Immunol.

12:923, 1994 and Eldridge, J.H. et al., Sem. Hematol. 30:16, 1993). Toxin-targeted delivery technologies also known as receptor-mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Massachusetts) may also be used.

In patients with PSCA-associated cancer, the vaccine compositions according to the invention can also be used together with other treatments used for cancer, e.g., surgery, chemotherapy, drug treatments, radiation therapies, etc. comprehensive use in combination with immune adjuvants such as IL-2, IL-12, GM-CSF and the like.

Cellular vaccines:

CTL epitopes can be determined using specific algorithms to identify peptides within PSCA protein that bind corresponding HLA alleles (see, e.g., Table IV; Epimer™ and Epimatrix™, Brown University (URL brown.edu/Research/ TB-HIV_Lab/epimatrix/epimatrix.html); and, BIMAS, (URL bimas.dcrt.nih.gov/; SYFPEITHI at URL syfpeithi.bmi-heidelberg.com/). In a preferred embodiment, a PSCA immunogen contains one or multiple amino acid sequences identified using techniques well known in the art, such as the sequences shown in Tables V-XVIII and XXII-LI or a peptide of 8, 9, 10 or 11 amino acids specified by an HLA Class I motif/supermotif (e.g. , Table IV (A), Table IV (D) or Table IV (E)) and/or a peptide of at least 9 amino acids comprising an HLA Class II motif/supermotif (eg, Table IV (B) or Table IV (C)).As understood in the art, the HLA Class I binding cleft is essentially closed at one end so that peptides of only a particular s drying region can fit into the pit and be bound, generally HLA Class I epitopes are 8, 9, 10 or 11 amino acids long. In contrast, the HLA Class II binding cleft is predominantly open at one end; therefore, a peptide of about 9 or more amino acids can be bound by an HLA Class II molecule. Due to binding pocket differences between HLA Class I and II, HLA Class I motifs are length-specific, ie, position two of a Class I motif is the second amino acid in an amino to carboxyl direction of the peptide. The amino acid positions in a Class II motif are relative only to each other, not the total peptide, ie, additional amino acids may be attached to the amino and/or carboxyl termini of a motif-bearing sequence. HLA Class II epitopes are often 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids long or longer than 25 amino acids.

A number of methods for producing an immune response in a mammal are known in the art (eg as the first step in the formation of hybridomas).

Methods of producing an immune response in a mammal comprise exposing the mammalian immune system to an immunogenic epitope on a protein (eg, a PSCA protein) such that an immune response is generated. A typical embodiment comprises a method of eliciting an immune response against PSCA in a host by contacting the host with a sufficient amount of at least one PSCA B-cell or cytotoxic T-cell epitope or analog thereof; and at least one periodic interval thereafter contacting the host again with the PSCA B cell or cytotoxic T cell epitope or analog thereof. Presented herein is a method of eliciting an immune response against a PSCA-related protein or an artificial multi-epitope peptide comprising: administering a PSCA immunogen (eg, a PSCA protein or peptide fragment thereof, a PSCA fusion protein or analog, etc.) in a vaccine preparation for a human or other mammal. Typically, such vaccine preparations further contain a suitable adjuvant (see, e.g., U.S. Patent No.

6,146,635) or a universal helper epitope such as a PADRETM peptide (Epimmune Inc., San Diego, CA; see, e.g., Alexander et al., J. Immunol. 2000 164(3); 164(3): 1625 -1633; Alexander et al., Immunity 19941(9): 751-761 and Alexander et al., Immunol. Res. 1998 18(2): 79-92). An alternative method comprises producing an immune response in a subject against a PSCA immunogen by: administering in vivo to muscle or skin of the subject's body a DNA molecule comprising a DNA sequence encoding a PSCA immunogen, wherein the DNA sequence is operably linked to regulatory sequences that control expression of the DNA sequence; where the DNA molecule is taken up by cells, the DNA sequence is expressed in the cells and an immune response is generated against the immunogen (see, e.g., U.S. Patent No. 5,962,428). Optionally, a genetic vaccine facilitator such as anionic lipids; saponins; lectins; estrogenic compounds; hydroxylated lower alkyls; dimethyl sulfoxide; and urea also administered. In addition, an anti-idiotypic antibody that mimics PSCA may be administered to elicit a response to the target antigen.

Nucleic acid vaccines:

Vaccine mixtures according to the invention comprise nucleic acid-mediated forms. DNA or RNA encoding protein(s) according to the invention can be administered to a patient. Genetic immunization methods can be used to generate prophylactic or therapeutic humoral and cellular immune responses directed against PSCA-expressing cancer cells. Constructs comprising DNA encoding a PSCA-related protein/immunogen and appropriate regulatory sequences can be injected directly into the muscle or skin of an individual so that the cells in the muscle or skin take up the construct and express the encoded PSCA protein/immunogen. Alternatively, a vaccine comprises a PSCA-related protein. Expression of the PSCA-related protein immunogen results in the generation of prophylactic or therapeutic humoral and cellular immunity against cells containing a PSCA protein. Various prophylactic and therapeutic genetic immunization techniques known in the art can be used (for an overview, see information and references published at 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. Patents No. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720. Examples of DNA-based delivery technologies include "naked DNA", facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated ("gene gun") or pressure-mediated delivery (see, e.g., U.S. Patent No. 5,922,687).

For therapeutic or prophylactic immunization purposes, proteins according to the invention can be expressed via viral or bacterial vectors. Various viral gene delivery systems that can be used in the practice of the invention include, but are not limited to, vaccinia, fowlpox, canarypox, adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus and sindbis virus (see, e.g., Restifo, 1996, Curr. Opin. Immunol. 8:658-663; Tsang et al. J. Natl. Cancer Inst. 87:982-990 (1995)). Non-viral delivery systems can also be used by introducing naked DNA encoding a PSCA-related protein into the patient (eg, intramuscularly or intradermally) to induce an anti-tumor response.

Vaccinia virus is used, for example, as a vector to express nucleotide sequences that code for the peptides according to the invention. Upon introduction into a host, the recombinant vaccinia virus expresses the protein immunogenic peptide and thereby elicits a host's immune response. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al., Nature 351:456-460 (1991). A number of other vectors useful for therapeutic administration or immunization of the peptides of the invention, e.g. adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled in the art from the description herein.

Accordingly, gene delivery systems are used to deliver a PSCA-related nucleic acid molecule. In one embodiment, full-length human PSCA cDNA is used. In another embodiment, PSCA nucleic acid molecules encoding specific cytotoxic T lymphocyte (CTL) and/or antibody epitopes are used.

Ex Vivo Vaccines

Various ex vivo strategies can also be used to produce an immune response. One method involves using antigen presenting cells (APCs) such as dendritic cells (DC) to present PSCA antigen to a patient's immune system. Dendritic cells express MHC class I and II molecules, B7 co-stimulator and IL-12 and are consequently highly specialized antigen-presenting cells. In prostate cancer, autologous dendritic cells pulsed with peptides of prostate-specific membrane antigen (PSMA) are being used in a Phase I clinical trial to stimulate immune systems in prostate cancer patients (Tjoa et al., 1996, Prostate 28:65-69; Murphy et al., 1996, Prostate 29:371-380). Accordingly, dendritic cells can be used to present PSCA peptides to T cells in the context of MHC class I or II molecules. In one embodiment, autologous dendritic cells are pulsed with PSCA peptides capable of binding to MHC class I and/or class II molecules. In another embodiment, dendritic cells are pulsed with the full-length PSCA protein. Yet another embodiment involves engineering overexpression of a PSCA gene in dendritic cells using various implementing vectors known in the art, such as adenovirus (Arthur et al., 1997, Cancer Gen Ther.4:17-25), retrovirus ( Henderson et al., 1996, Cancer Res.56:3763-3770), lentivirus, adeno-associated virus, DNA transfection (Ribas et al., 1997, Cancer Res.57:2865-2869) or tumor-derived RNA transfection ( Ashley et al., 1997, J. Exp. Med. 186:1177-1182). Cells expressing PSCA can also be engineered to express immunomodulators, such as GM-CSF, and used as immunizing agents.

X.B.) PSCA as a target for antibody-based therapy

PSCA is an attractive target for antibody-based therapeutic strategies. Several antibody strategies are known in the art for targeting both extracellular and intracellular molecules (see, e.g., complement and ADCC-mediated killing as well as the use of intrabodies). Because PSCA is expressed by cancer cells of different lineages relative to corresponding normal cells, systemic administration of PSCA immunoreactive compositions is prepared that exhibits excellent sensitivity without toxic, nonspecific, and/or off-target effects caused by binding of the immunoreactive composition to non- target organs and tissues. Antibodies specifically reactive with domains of PSCA are useful for treating PSCA-expressing cancers systemically, either as conjugates with a toxin or therapeutic agent or as naked antibodies capable of inhibiting cell proliferation or function.

PSCA antibodies can be introduced into a patient such that the antibody binds to the PSCA and modulates a function, such as an interaction with a binding partner and consequently mediates destruction of the tumor cells and/or inhibits the growth of the tumor cells. Mechanisms by which such antibodies exert a therapeutic effect 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-Hodgkin's Lymphoma, Herceptin® for metastatic breast cancer and Erbitux® for colorectal cancer.

Those skilled in the art understand that antibodies can be used to specifically target and bind immunogenic molecules such as an immunogenic region of a PSCA sequence shown in Figure 1. In addition, those skilled in the art understand that it is routine to conjugate antibodies to cytotoxic agents (see e.g. ., Slevers et al Blood 93:113678-3684 (June 1, 1999)). When cytotoxic and/or therapeutic agents are delivered directly to cells, such as by conjugating them to antibodies specific for a molecule expressed by that cell (eg, PSCA), the cytotoxic agent will exert its known biological effect (ie, cytotoxicity ) on these cells.

A variety of compositions and methods for using antibody-cytotoxic agent conjugates to kill cells are known in the art. In the context of cancer, typical methods comprise administering to an animal having a tumor a biologically effective amount of a conjugate comprising a selected cytotoxic and/or therapeutic agent bound to a targeting agent (eg, an anti-PSCA antibody) that binds to a marker (eg PSCA) expressed, available for binding or located on cell surfaces. A typical embodiment is a method for delivering a cytotoxic and/or therapeutic agent to a cell expressing PSCA, comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to a PSCA epitope and exposing the cell to the antibody-agent conjugate. Also presented is a method for treating an individual suspected of suffering from metastatic cancer, comprising a step of parenterally administering to said individual a pharmaceutical composition comprising a therapeutically effective amount of an antibody conjugated to a cytotoxic and/or therapeutic agent.

Cancer immunotherapy using anti-PSCA antibodies can be performed according to various methods that have been successfully used in the treatment of other types of cancer, including but not limited to colon cancer (Arlen et al., 1998, Crit. Rev.

Immunol. 18:133-138), multiple myeloma (Ozaki et al., 1997, Blood 90:3179-3186, Tsunenari et al., 1997, Blood 90:2437-2444), gastric cancer (Kasprzyk et al., 1992, Cancer Res.52:2771-2776), B-cell lymphoma (Funakoshi et al., 1996, J. Immunother. Emphasis Tumor Immunol.19:93-101), leukemia (Zhong et al., 1996, Leuk. Res.20 :581-589), colorectal cancer (Moun et al., 1994, Cancer Res.54:6160-6166; Velders et al., 1995, Cancer Res.55:4398-4403) and breast cancer (Shepard et al., 1991 , J. Clin. Immunol. 11:117-127). Some therapeutic methods involve conjugation of naked antibody to a toxin or radioisotope, such as conjugation of Y<91 >or I<131 >to anti-CD20 antibodies (eg, ZevalinTM, IDEC Pharmaceuticals corp. or Bexxar™, Coulter Pharmaceuticals) respectively, while others involve co-administration of antibodies and other therapeutic agents, such as HerceptinTM (trastuzu MAb) with paclitaxel (Genentech, Inc.). The antibodies may be conjugated to a therapeutic agent. To treat prostate cancer, for example, PSCA antibodies can be administered together with radiation, chemotherapy or hormone ablation. Likewise, antibodies may be conjugated to a toxin such as calicheamicin (eg, Mylotarg™, Wyeth-Ayerst, Madison, NJ, a recombinant humanized IgG4 kappa antibody conjugated to the antitumor antibiotic calicheamicin) or a maytansinoid (eg, taxane-based Tumor-Activated Prodrug, TAP, platform, ImmunoGen, Cambridge, MA see also, e.g., US Patent 5,416,064) or Auristatin E (Nat Biotechnol. 2003 Jul; 21(7):778-84. (Seattle Genetics)) .

Although PSCA antibody therapy is applicable to all stages of cancer, antibody therapy may be particularly appropriate in advanced or metastatic cancer. Treatment with the antibody therapy presented herein is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy is combined with a chemotherapy or radiation regimen for patients who have not received chemotherapy treatment. In addition, antibody therapy may enable the use of reduced doses of concomitant chemotherapy, especially for patients who do not tolerate the toxicity of the chemotherapeutic agent very well. Fan et al. (Cancer Res. 53:4637-4642, 1993), Prewett et al. (International J. of Onco. 9:217-224, 1996) and Hancock et al. (Cancer Res. 51:4575-4580, 1991) describes the use of various antibodies together with chemotherapeutic agents.

Although PSCA antibody therapy is applicable to all stages of cancer, antibody therapy may be particularly appropriate for advanced or metastatic cancer. Treatment with the antibody therapy presented herein is indicated for patients who have received one or more rounds of chemotherapy. Alternatively, antibody therapy as presented herein is combined with a chemotherapeutic or radiation regimen for patients who have not received chemotherapeutic treatment. In addition, antibody therapy may enable the use of reduced doses of concomitant chemotherapy, especially for patients who do not tolerate the toxicity of the chemotherapeutic agent very well.

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

Anti-PSCA monoclonal antibodies that treat prostate and other cancers include those that initiate a strong immune response against the tumor or those that are directly cytotoxic. In this regard, anti-PSCA monoclonal antibodies (MAbs) can induce tumor cell lysis by either complement-mediated or antibody-dependent cell cytotoxicity (ADCC) mechanisms, both of which require an intact Fc portion of the immunoglobulin molecule for interaction with effector cell Fc receptor sites on complement proteins. In addition, anti-PSCA MAbs that exert a direct biological effect on tumor growth are useful for treating PSCA-expressing cancers. Mechanisms by which direct cytotoxic MAbs act include: inhibition of cell growth, modulation of cellular differentiation, modulation of tumor angiogenesis factor profiles and induction of apoptosis. The mechanism(s) by which a particular anti-PSCA MAb exerts an anti-tumor effect is evaluated using any number of in vitro assays that evaluate cell death such as ADCC, ADMMC, complement-mediated cell lysis, and so on, which are generally known in the area.

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

Therapeutic methods include the administration of single anti-PSCA MAbs as well as combinations, or cocktails, of different MAbs (ie, PSCA MAbs or Mabs that bind another protein). Such MAb cocktails may have certain advantages as they contain MAbs that target different epitopes, exploit different effector mechanisms, or combine directly cytotoxic MAbs with MAbs that rely on immune effector functionality. Such MAbs can in combination show synergistic therapeutic effects. In addition, PSCA MAbs may be co-administered with other therapeutic modalities, including but not limited to various chemotherapeutic and biological agents, androgen blockers, immunomodulators (eg, IL-2, GM-CSF), surgery, or radiation. PSCA MAbs are administered in their “naked” or unconjugated form or may have a therapeutic agent(s) conjugated to them.

Anti-PSCA antibody formulations are administered via any route capable of delivering the antibodies to a tumor cell. Methods of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumor, intradermal, and the like. Treatment generally comprises repeated administration of the anti-PSCA antibody preparation, through an acceptable route of administration such as intravenous injection (IV), typically in a dose in the range of approx.

0,1, ,2, ,3, ,4, ,5, ,6, ,7, ,8, ,9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 mg/kg body weight. In general, doses are in the range of 10-1000 mg MAb per week effectively and well tolerated.

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

Optionally, patients should be evaluated for the levels of PSCA in a given sample (eg, the levels of circulating PSCA antigen and/or PSCA-expressing cells) to aid in the determination of the most effective dosing regimen, etc. Such evaluations are also used for monitoring purposes throughout the treatment and is applicable for measuring therapeutic success in combination with evaluation of other parameters (for example urine cytology and/or ImmunoCyt levels in bladder cancer treatment or analogously, serum PSA levels in prostate cancer therapy).

Anti-idiotypic anti-PSCA antibodies can also be used in anti-cancer therapy as a vaccine to induce an immune response against cells expressing a PSCA-related protein. In particular, generation of anti-idiotypic antibodies is well known in the art; this methodology can be easily adapted to generate antiidiotypic anti-PSCA antibodies that mimic 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 anti-idiotypic antibody can be used in cancer vaccine strategies.

An object of the present invention is to provide PSCA antibodies, which inhibit or delay the growth of tumor cells expressing PSCA. Further presented is the provision of methods for inhibiting angiogenesis and other biological functions and thereby reducing tumor growth in mammals, preferably humans, using such PSCA antibodies and particularly using such PSCA antibodies combined with radiation and chemotherapy or both.

In one embodiment, there is synergy when tumors, including human tumors, are treated with PSCA antibodies together with chemotherapeutic agents or radiation or combinations thereof. In other words, inhibition of tumor growth by a PSCA antibody is enhanced more than expected when combined with chemotherapeutic agents or radiation or combinations thereof. Synergy can be shown, for example, by greater inhibition of tumor growth with combined treatment than would be expected from a treatment with PSCA antibodies alone or the additive effect of treatment with a PSCA antibody and a chemotherapeutic agent or radiation. Preferably, synergy is demonstrated by remission of the cancer where remission is not expected from treatment either from 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 a PSCA antibody and a combination of chemotherapy or radiation or both comprises administering the PSCA antibody before, during or after the initiation of chemotherapy or radiation therapy, as well as any combination thereof (i.e. before and during, before and after, during and after or before, during and after starting chemotherapy and/or radiation therapy). For example, PSCA antibody is typically administered between 1 and 60 days, preferably between 3 and 40 days, more preferably between 5 and 12 days before the initiation of radiation therapy and/or chemotherapy. However, depending on the treatment protocol and the specific needs of the patient, the procedure is performed in a way that will provide the most effective treatment and ultimately prolong the life of the patient.

Administration of chemotherapeutic agents can be carried out in a number of ways including systemically by the parenteral and enteral routes. In one embodiment, the PSCA antibody and the chemotherapeutic agent are administered as separate molecules. In another embodiment, PSCA antibody is linked, for example by conjugation, to a chemotherapeutic agent. (See the example entitled “Human clinical trials for the treatment and diagnosis of human carcinomas through the use of human anti-PSCA antibodies in vivo”) and (see the section entitled “PSCA as a target for antibody-based therapy”). Specific examples of chemotherapeutic agents or chemotherapy include cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide , methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere), aldesleukin, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon alfa, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil, taxol and combinations thereof.

The source of radiation, used in combination with a PSCA antibody, can be either external or internal to the patient being treated. When the source is external to the patient, the treatment is known as external beam radiation therapy (EBRT). When the source of radiation is inside 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 the AECL Theratron and Varian Clinac. The dose of radiation depends on a number of factors well known in the art. Such factors include the organ being treated, the healthy organs in the radiation path that could inadvertently be adversely affected, the tolerance of the patient for radiation therapy and the area of the body in need of treatment. The dose will typically be between 1 and 100 Gy and more precisely between 2 and 80 Gy. Some doses that have been reported include 35 Gy to the spinal cord, 15 Gy to the kidney, 20 Gy to the liver, and 65-80 Gy to the prostate. However, it should be emphasized that the invention is not limited to any particular dose. The dose will be determined by the attending physician according to the particular factors in a given situation, including the factors mentioned above.

The distance between the source of the external radiation and the point of entry into the patient can be any distance that represents an acceptable balance between killing target cells and limiting side effects. Typically, the source of the external radiation is between 70 and 100 cm from the point of penetration into the patient.

Brachytherapy is generally performed by placing the source of radiation inside the patient. Typically, the source of radiation is placed approximately 0-3 cm from the tissue being treated. Known techniques include interstitial, intercavitary and surface brachytherapy. The 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 radium, cesium-137, and iridium-192. Some additional radioactive atoms that have been used in brachytherapy include americium-241 and gold-198. The dose of radiation for brachytherapy can be similar to that mentioned above for external beam radiation therapy. In addition to the factors mentioned above, in order to determine the dose of external beam in radiation therapy, the type of radioactive atom used is also included in the calculation for determining the dose of brachytherapy.

X.C.) PSCA as a measure of cellular immune responses

Vaccines and methods for producing vaccines containing an immunogenically effective amount of one or more HLA-binding peptides are also described herein. Furthermore, vaccines according to the invention comprise mixtures of one or more of the peptides according to the present invention. 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 different peptides. Polymers have the advantage of increased immunological response and, where different peptide epitopes are used to make up the polymer, the additional ability to induce antibodies and/or CTLs that react with different antigenic determinants of the pathogenic organism or tumor-related peptide targeted for an immune response. The mixture may be a naturally occurring region of an antigen or may be produced, eg, recombinantly or by chemical synthesis.

Carriers that can be used with vaccines according to the invention are well known in the art and include, for example, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B virus core protein and such. The vaccines may contain a physiologically tolerable (ie, acceptable) diluent such as water or saline, preferably phosphate buffered saline. The vaccines also typically include an adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide or alum are examples of materials well known in the art. In addition, as described herein, CTL responses can be primed by conjugation of peptides according to the invention to lipids, such as tripalmitoyl-S-glycerylcysteine-lyseryl-serine (P3CSS). Furthermore, an adjuvant such as a synthetic cytosine-phosphorothiolated-guanine-containing (CpG) oligonucleotides has been found to increase CTL responses 10- to 100-fold (see, e.g., Davila and Celis, J. Immunol. 165:539-547 ( 2000)).

Upon immunization with a peptide mixture according to the invention, via injection, aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal or other suitable routes, the immune system of the host responds to the vaccine by producing large amounts of CTLs and/or HTLs 'are specific for the desired antigen. Accordingly, the host becomes at least partially immune to later development of cells expressing or overexpressing PSCA antigen or at least achieves some therapeutic benefit when the antigen becomes tumor-associated.

In some embodiments, it may be desirable to combine the class I peptide components with components that induce or facilitate antibody neutralization and or assist T-cell responses directed against the target antigen. A preferred embodiment of such a mixture comprises class I and class II epitopes according to the invention. An alternative embodiment of such a mixture comprises a class I and/or class II epitope according to the invention, together with a cross-reactive HTL epitope such as the PADRE™ (Epimmune, San Diego, CA) molecule (described e.g. , in U.S. Patent No. 5,736,142).

A vaccine according to the invention may also comprise antigen-presenting cells (APC), such as dendritic cells (DC), as a constituent for presenting peptides according to the invention. Vaccine mixtures can be formed in vitro, after dendritic cell mobilization and harvesting, whereby loading of dendritic cells occurs in vitro. For example, 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. Vaccine mixtures, either DNA- or peptide-based, can also be administered in vivo in combination with dendritic cell mobilization whereby loading of dendritic cells takes place in vivo.

Preferably, the following principles are used in the selection of a number of epitopes for inclusion in a polyepitope mixture for use in a vaccine or to select distinct 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 perform the selection. The multiple epitopes to be incorporated into a given vaccine composition may, but need not be, consecutive in sequence in the native antigen from which the epitopes are derived.

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

2.) Epitopes are selected that have the desired binding affinity established to be correlated with immunogenicity: for HLA Class I an IC50 of 500 nM or less, often 200 nM or less; and for Class II an IC50 of 1000 nM or less.

3.) Sufficient supermotif-bearing peptides or a sufficient array of allele-specific motif-bearing peptides are selected, providing broad population coverage. For example, it is preferred to have at least 80% population coverage. A Monte Carlo analysis, a statistical evaluation known in the art, can be used to assess the breadth or redundancy of population coverage.

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

5.) Of particular relevance are epitopes referred to as “nested epitopes.” Nested epitopes occur where at least two epitopes overlap in a given peptide sequence. A nested peptide sequence may comprise B cell, HLA class I and/or HLA class II epitopes. When nested epitopes are provided, a general goal is to provide the largest number of epitopes per sequence. Accordingly, one aspect is to avoid providing a peptide that is somewhat longer than the amino-terminus of the amino-terminal epitope and the carboxyl-terminus of the carboxyl-terminal epitope in the peptide. When providing a multi-epitopic sequence, such as a sequence comprising nested epitopes, it is generally important to screen the sequence to ensure that it does not have pathological or other deleterious biological properties.

6.) If a polyepitope protein is produced or by forming a minigene, an aim is to produce the smallest peptide that includes the epitopes of interest. This principle is similar, if not the same, as that used in selecting a peptide comprising nested epitopes. However, with an artificial polyepitope peptide, the goal of minimizing size is balanced against the need to integrate any spacer sequences between epitopes in the polyepitopic protein. Spacer amino acid residues can, for example, be introduced to avoid junctional epitopes (an epitope recognized by the immune system, not present in the target antigen and only formed by the artificial assembly of epitopes) or to facilitate cleavage between epitopes and thereby promote epitope presentation. Joining epitopes should generally be avoided because the recipient may mount an immune response against the non-native epitope. Of particular importance is a junctional epitope that is a "dominant epitope." A dominant epitope can lead to such an avid response that immune responses against other epitopes are reduced or suppressed.

7.) Where the sequences of multiple variants of the same target protein are present, potential peptide epitopes can also be selected on the basis of their conservation. For example, a criterion for conservation may define that the entire sequence of an HLA class I binding peptide or the entire 9-mer core of a class II binding peptide shall be conserved in a specified percentage of the sequences evaluated for a specific protein antigen.

X.C.1. Minigen vaccines

Several different methods are available which allow simultaneous delivery of multiple epitopes. Nucleic acids that code for the peptides according to the invention are a particularly applicable embodiment according to the invention. Epitopes for inclusion in a minigene are preferably selected according to the guidelines set forth in the preceding paragraph. A preferred method for administering nucleic acids that encode the peptides according to the invention uses minigene constructs that encode a peptide comprising one or multiple epitopes according to the invention.

Use of multi-epitope minigenes is described below and in, Ishioka et al., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, J.L., J. Virol. 71:2292, 1997; Thomson, S.A. et al., J. Immunol. 157:822, 1996; Whitton, J.L. et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine 16:426, 1998. For example, a multi-epitope DNA plasmid encoding supermotif and/or motif-bearing epitopes derived from PSCA, PADRE™ universal helper T-cell epitope or multiple HTL epitopes from PSCA (see, e.g., Tables V-XVIII and XXII through LI), and an endoplasmic reticulum translocation signal sequence is constructed. A vaccine may also include epitopes 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 epitopes tested. Furthermore, the immunogenicity of DNA-encoded epitopes in vivo may be correlated with the in vitro responses of specific CTL lines against target cells transfected with the DNA plasmid. Consequently, these experiments can show that the minigene serves to both: 1.) generate a CTL response and 2.) that the induced CTLs recognized cells expressing the encoded epitopes.

For example, to create a DNA sequence encoding the selected epitopes (minigene) for expression in human cells, the amino acid sequences of the epitopes can be reverse translated. A table of human codon usage can be used to guide codon selection for each amino acid. These epitope-encoding DNA sequences may be directly contiguous, so that when translated, a continuous polypeptide sequence is formed. To optimize expression and/or immunogenicity, additional elements can be introduced into the minigene model.

Examples of amino acid sequences that can be reverse translated and included in the minigene sequence include: HLA class I epitopes, HLA class II epitopes, antibody epitopes, a ubiquitination signal sequence and/or an endoplasmic reticulum targeting signal. In addition, HLA presentation of CTL and HTL epitopes can be enhanced by including synthetic (eg, 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 to DNA by assembling oligonucleotides that code for the plus and minus strands of the minigene.

Overlapping oligonucleotides (30-100 bases long) can be synthesized, phosphorylated, purified and hybridized under appropriate conditions using well known techniques. The ends of the oligonucleotides can be connected, for example by using T4 DNA ligase. This synthetic minigene, which codes for the epitope polypeptide, can then be cloned into a desired expression vector.

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

Additional vector modifications may be desired to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression and one or more synthetic or naturally occurring introns could be incorporated into the transcribed region of the minigene. The inclusion of mRNA stabilization sequences and sequences for replication in mammalian cells can also be considered to increase minigene expression.

As soon as an expression vector is chosen, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is transformed into an appropriate E. coli strain and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as any other elements contained in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells containing the correct plasmid can be stored as a master cell bank and an operational cell bank.

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

In some embodiments, a bi-cistronic expression vector that allows production of both the minigene-encoded epitopes and a second protein (included to increase or decrease immunogenicity) may be used. Examples of proteins or polypeptides that could advantageously enhance the immune response if co-expressed include cytokines (eg, IL-2, IL-12, GM-CSF), cytokine-inducing molecules (eg, LeIF), costimulatory molecules or for HTL responses, pan-DR binding proteins (PADRE™, Epiimmune, San Diego, CA). Helper (HTL) epitopes can be bound to intracellular targeting signals and expressed separately from expressed CTL epitopes; this allows targeting of the HTL epitopes to a cell compartment different from that of the CTL epitopes. If necessary, this could facilitate more efficient introduction of HTL epitopes into the HLA class II response pathway, thereby enhancing HTL induction. In contrast to HTL or CTL induction, specific reduction of the immune response by co-expression of immunosuppressive molecules (eg TGF-β) may be beneficial in certain diseases.

Therapeutic amounts of plasmid DNA can be produced, for example, by fermentation in E. coli, followed by purification. Aliquots from the operating cell bank are used to inoculate growth medium and grown to saturation in shake flasks or a bioreactor according to well-known techniques. Plasmid DNA can be purified using standard bioseparation technologies such as solid phase anion exchange resins supplied by QIAGEN, Inc. (Valencia, California). If necessary, supercoiled DNA can be isolated from the open circular and linear forms using gel electrophoresis or other methods.

Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is the reconstitution of lyophilized DNA in sterile phosphate buffered saline (PBS). This method, known as "naked DNA," is currently being used for intramuscular (IM) administration in clinical trials.

To maximize the immunotherapeutic effects of minigene DNA vaccines, an alternative method of formulating purified plasmid DNA may be desirable. A number of methods are described and new techniques may become available. Cationic lipids, glycolipids and fusogenic liposomes can also be used in the formulation (see, e.g., as described by WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682 (1988); U.S. Pat. No. 5,279,833; WO 91/06309; and Felgner, et al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987). In addition, peptides and compounds referred to collectively as protective, interactive, non-condensing compounds (PINCs) could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion or trafficking to specific organs or cell types.

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

In vivo immunogenicity is a second method for functional testing of minigene DNA formulations. Transgenic mice expressing appropriate human HLA proteins are immunized with the DNA product. The dose and route of administration are formulation dependent (eg, IM for DNA in PBS, intraperitoneal (i.p.) for lipid complexed DNA). Twenty-one days after immunization, splenocytes are harvested and restimulated for one week in the presence of peptides encoding each epitope tested. Then, for CTL effector cells, assays are performed for cytolysis of peptide-loaded, <51>Cr-labeled target cells using standard techniques. Lysis of target cells sensitized by HLA loaded with peptide epitopes corresponding to minigene-encoded epitopes demonstrates DNA vaccine function for in vivo induction of CTLs. Immunogenicity of HTL epitopes is confirmed in transgenic mice in an analogous manner.

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

Minigenes can also be delivered using other bacterial or viral delivery systems well known in the art, for example, an expression construct encoding epitopes according to the invention can be introduced into a viral vector such as vaccinia.

X.C.2. Combinations of CTL peptides with helper peptides Vaccine compositions comprising CTL peptides according to the invention can be modified, e.g., analogously, to provide desired properties, such as improved serum half-life, wider population coverage or improved immunogenicity.

For example, the ability of a peptide to induce CTL activity can be enhanced by linking the peptide to a sequence containing at least one epitope capable of inducing a T helper cell response. Although a CTL peptide can be directly bound to a T-helper peptide, CTL-epitope/HTL-epitope conjugates are often bound by a spacer molecule. The spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are mainly uncharged under physiological conditions. The spacers are typically selected from, e.g., Ala, Gly or other neutral spacers of non-polar amino acids or neutral polar amino acids. It will be understood that the spacer that may be present need not be made up of the same residues and can thus be a hetero- or homo-oligomer. When present, the spacer will usually be at least one or two residues, more commonly three to six residues and sometimes 10 or more residues. The CTL peptide epitope can be linked to the T helper peptide epitope either directly or through a spacer either at the amino or carboxy terminal end of the CTL peptide. The amino terminus of either the immunogenic peptide or the T-helper peptide can be acylated.

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

X.C.3. Combinations of CTL peptides with T-cell priming agents

In some embodiments, it may be desirable to include in the pharmaceutical mixtures according to the invention at least one component that primes B-lymphocytes or T-lymphocytes. Lipids have been identified as agents capable of priming CTL in vivo. For example, palmitic acid residues can be attached to the ε- and α-amino groups of a lysine residue and then attached, for example, through one or more connecting 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, eg, incomplete Freund's adjuvant. In a preferred embodiment, a particularly effective immunogenic composition comprises palmitic acid bound to ε- and α- amino groups of Lys, which are linked through linkage, e.g., Ser-Ser, to the amino-terminal end of the immunogenic peptide.

As another example of lipid priming of CTL responses, E. coli lipoproteins, such as tripalmitoyl-S-glycerylcysteine lyseryl-serine (P3CSS) can be used to prime virus-specific CTL when covalently bound to an appropriate peptide (see e.g. ., Deres, et al., Nature 342:561, 1989). Peptides according to the invention can be linked to P3CSS, for example, and the lipopeptide administered to an individual to specifically prime an immune response against the target antigen. Furthermore, because induction of neutralizing antibodies can also be primed with P3CSS-conjugated epitopes, two such mixtures can be combined to more effectively elicit both humoral and cell-mediated responses.

X.C.4. Vaccine mixtures comprising DC pulsed with CTL and/or HTL peptides

An embodiment of a vaccine mixture according to the invention comprises the ex vivo administration of a cocktail of epitope-bearing peptides against PBMC or isolated DC therefrom, from the patient's blood. A pharmaceutical agent to facilitate harvest of DC can be used, such as Progenipoietin™ (Pharmacia-Monsanto, St. Louis, MO) or GM-CSF/IL-4. After pulsing DC with peptides and before reinfusion into patients, DC are washed to remove unbound peptides. In this embodiment, a vaccine comprises peptide-pulsed DCs that present the pulsed peptide epitopes complexed with HLA molecules on their surfaces.

DC can be pulsed ex vivo with a cocktail of peptides, some of which stimulate CTL responses against PSCA. Optionally, a helper T cell (HTL) peptide, such as a natural or artificial soluble bound HLA Class II peptide, may be included to facilitate the CTL response. Thus, a vaccine according to the invention is used to treat cancer that expresses or overexpresses PSCA.

X.D.) Adoptive immunotherapy

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

Transfected dendritic cells can also be used as antigen-presenting cells.

X.E.) Administration of vaccines for therapeutic or prophylactic purposes Pharmaceutical and vaccine compositions according to the invention are typically used to treat and/or prevent cancers that express or overexpress PSCA. In therapeutic applications, peptide and/or nucleic acid mixtures are administered to a patient in an amount sufficient to elicit an effective B-cell, CTL and/or HTL response against the antigen and to cure or at least partially stop or slow symptoms and/or or complications. An amount sufficient to achieve this is defined as a "therapeutically effective dose." Amounts effective for this use will depend on, e.g., the particular composition administered, the method of administration, the stage and severity of the disease being treated, the weight and general health of the patient, and the judgment of the attending physician.

For pharmaceutical compositions, the immunogenic peptides of the invention or DNA encoding them are generally administered to an individual already carrying a tumor expressing PSCA. The peptides or DNA encoding them can be administered individually or as fusions of one or more peptide sequences. Patients can be treated with the immunogenic peptides separately or in conjunction with other treatments, such as surgery, as appropriate.

For therapeutic use, administration should generally begin at the first diagnosis of PSCA-associated cancer. This is followed by booster doses until symptoms are mainly subdued and for a period thereafter.

The design of the vaccine preparation (ie, including but not limited to designs such as peptide cocktails, polyepitope polypeptides, minigenes or TAA-specific CTLs or pulsed dendritic cells) delivered to the patient may vary according to the stage of the disease or the state of health of the patient . For example, for a patient with a tumor expressing PSCA, a vaccine comprising PSCA-specific CTL may be more effective in killing tumor cells in the patient with advanced disease than alternative embodiments.

It is generally important to provide an amount of the peptide epitope delivered by a method of administration sufficient to effectively stimulate a cytotoxic T-cell response; compositions that stimulate helper T-cell responses may also be administered according to this embodiment of the invention.

The dosage for an initial therapeutic immunization generally occurs in a unit dose range where the lower value is about 1, 5, 50, 500 or 1,000 µg and the higher value is about 10,000; 20,000; 30,000; or 50,000 µg. Dose values for a human typically range from approx. 500 µg to approx. 50,000 µg per 70 kg. patient. Booster doses of between about 1.0 µg to about 50,000 µg of peptide according to a boosting regimen over weeks to months can be administered depending on the patient's response and condition as determined by measuring the specific activity of CTL and HTL obtained from the patient's blood. Administration should continue until at least clinical symptoms or laboratory tests indicate that the neoplasia has been removed or reduced and for a period thereafter. The doses, administration methods and dose regimens are regulated according to methods known in the field.

In certain embodiments, the peptides and mixtures according to the present invention are used in severe disease states, i.e. life-threatening or potentially life-threatening situations. In such cases, as a result of the minimal amounts of extraneous substances and the relatively non-toxic nature of the peptides in preferred compositions according to the invention, it is possible and may seem desirable to the attending physician to administer significant excesses of these peptide compositions in relation to these indicated dosage amounts.

The vaccine mixtures according to the invention can also be used simply as prophylactic agents. In general, the dose for an initial prophylactic immunization generally occurs in a unit dose range where the lower value is about 1, 5, 50, 500 or 1000 µg and the higher value is about 10,000; 20,000; 30,000; or 50,000 µg. Dose values for a human typically range from approx.

500 µg to approx. 50,000 µg per 70 kg. patient. This is followed by booster doses of between about 1.0 µg to about 50,000 µg of peptide administered at defined intervals from about four weeks to six months after the initial administration of vaccine. The immunogenicity of the vaccine can be assessed by measuring the specific activity of CTL and HTL obtained from a sample of the patient's blood.

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

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

The mixtures may contain pharmaceutically acceptable excipients as necessary for approximately physiological conditions, such as pH adjusting and buffering agents, tonicity regulating agents, wetting agents, preservatives and the like, for example sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate , etc.

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

A human unit dosage form of a composition is typically included in a pharmaceutical composition comprising a human unit dose of an acceptable carrier, in one embodiment an aqueous carrier and is administered in a volume/amount known to those skilled in the art to be used for the administration of such compositions in humans (see, e.g., Remington's Pharmaceutical sciences, 17th Edition, A. Gennaro, Editor, Mack Publishing Co., Easton, Pennsylvania, 1985). For example, a peptide dose for initial immunization can be from about 1 to about

50,000 µg, generally 100-5,000 µg, for a 70 kg patient. For example, for nucleic acids, an initial immunization can be performed using an expression vector in the form of naked nucleic acid administered IM (or SC or ID) in amounts of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 µg) can also be administered using a gene gun. After an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-10<7 > to 5x10<9 >pfu.

For antibodies, a treatment generally comprises repeated administration of the anti-PSCA antibody preparation, through an acceptable route of administration such as intravenous injection (IV), typically in a dose in the range of about 0.1 to about 10 mg/kg body weight. In general, doses are in the range of 10-500 mg MAb per week effective and well tolerated. Furthermore, an initial initial dose of approximately 4 mg/kg patient body weight IV, followed by weekly doses of approximately 2 mg/kg IV of the anti-PSCA MAb preparation represents an acceptable dosing regimen. As understood by those skilled in the art, various factors can influence the ideal dose in a particular case. Such factors include, for example, the half-life of a mixture, the binding affinity of an Ab, the immunogenicity of a substance, the degree of PSCA expression in the patient, the extent of circulating shed PSCA antigen, the desired steadystate concentration level, frequency of treatment and the influence of chemotherapeutic or other means used in combination with the treatment method according to the invention, as well as the state of health of a particular patient. Non-limiting preferred human unit doses are, for example, 500μg - 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, e.g., with subsequent weekly doses of 1-3 mg/kg; 0.5 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/kg body weight followed, for example, by two, three or four weeks of weekly doses; 0.5 - 10 mg/kg body weight, eg, followed by two, three or four weeks of weekly doses; 225, 250, 275, 300, 325, 350, 375, 400 mg m<2 >body surface weekly; 1-600 mg m<2 >body surface weekly; 225-400 mg m<2 >body surface 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, human unit dosage forms of polynucleotides comprise a suitable dosage range or effective amount that provides any therapeutic effect. As understood by those skilled in the art, a therapeutic effect depends on several factors, including the sequence of the polynucleotide, molecular weight of the polynucleotide, and route of administration. Doses are generally chosen by the doctor or other healthcare personnel according to a number of parameters known in the field, such as the severity of symptoms, history of the patient and the like. In general, for a polynucleotide of about 20 bases, a dose range can be selected from, for example, an independently selected lower limit such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 or 500 mg/kg up to an independently selected upper limit, higher than the lower limit, of about 60, 80, 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, a dose can be about any of the following: 0.1 to 100 mg/kg, 0.1 to 50 mg/kg, 0.1 to 25 mg/kg, 0.1 to 10 mg/kg, 1 to 500 mg/kg, 100 to 400 mg/kg, 200 to 300 mg/kg, 1 to 100 mg/kg, 100 to 200 mg/kg, 300 to 400 mg/kg, 400 to 500 mg/kg, 500 to 1000 mg/kg, 500 to 5000 mg/kg or 500 to 10,000 mg/kg. In general, parenteral administration methods may require higher doses of polynucleotide compared to more direct applications of the nucleotide to diseased tissue, such as polynucleotides of increasing length.

In one embodiment, human unit dosage forms of T cells comprise a suitable dose range or effective amount that provides any therapeutic effect. As understood by those skilled in the art, a therapeutic effect depends on several factors. Doses are generally chosen by the doctor or other healthcare personnel according to a number of parameters known in the field, such as the severity of symptoms, history of the patient and the like. A dose can be approx.10<4>cells to approx.10<6>cells, approx.10<6>cells to approx.10<8>cells, approx.10<8>to approx. 10<11 >cells or approx.10<8 >to approx.5 x 10<10 >cells. A dose can also approx.10<6 >cells/m<2 >to approx.10<10 >cells/m<2 >or approx.10<6 >cells/m<2 >to approx. 108 cells/m<2>.

Protein(s) according to the invention and/or nucleic acids encoding the protein(s) can also be administered via liposomes, which can also serve to: 1) target proteins to a particular tissue, such as lymphoid tissue; 2) target selectively to disease cells; or, 3) increase the half-life of the peptide mixture. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations, the peptide to be delivered is incorporated as part of a liposome, alone or together with a molecule that binds to a receptor widespread among lymphoid cells, such as monoclonal antibodies that bind to the CD45 antigen, or with other therapeutic or immunogenic mixtures. Thus, liposomes either filled or decorated with a desired peptide according to the invention can be directed to the seat of lymphoid cells, where the liposomes then deliver the peptide mixtures. Liposomes for use according to the invention are formed from standard vesicle-forming lipids, which generally comprise neutral and negatively charged phospholipids and a sterol, such as cholesterol. Selection of lipids is generally guided by consideration of, for example, liposome size, acid lability and stability of the liposomes in the bloodstream. A variety of methods are available for the preparation of liposomes, as described in, e.g., Szoka, et al., Ann. Fox. Biophys. Bioeng. 9:467 (1980) and U.S. Patents Nos. 4,235,871, 4,501,728, 4,837,028 and 5,019,369.

For targeting cells of the immune system, a ligand to be incorporated into the liposome may comprise, for example, antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells.

A liposome suspension containing a peptide can be administered intravenously, locally, topically, etc. in a dose that varies according to, inter alia, the method of administration, the peptide delivered and the stage of the disease being treated.

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

For aerosol administration, immunogenic peptides are preferably delivered in finely divided form together with a surfactant and propellant. Typical percentages of peptides are approx. 0.01%-20% by weight, preferably about 1%-10%. The surfactant must, of course, be non-toxic and preferably soluble in the propellant.

Representative such agents are the esters or partial esters of fatty acids containing from about 6 to 22 carbon atoms, such as caproic acid, octanoic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides can be used. The surfactant can make up about 0.1%-20% by weight of the mixture, preferably about 0.25-5%. The predominant part of the mixture is usually propellant. A carrier may also be included, as desired, as with, for example, lecithin for intranasal delivery.

XI.) Diagnostic and prognostic embodiments of PSCA.

As described herein, PSCA polynucleotides, polypeptides, reactive cytotoxic T cells (CTL), reactive helper T cells (HTL), and anti-polypeptide antibodies are used in well-known diagnostic, prognostic, and therapeutic assays investigating disorders associated with dysregulated cell growth such as cancer, particularly the cancers listed in Table I (see, e.g., both its specific pattern of tissue expression as well as its overexpression in certain cancers as described, for example, in the example entitled “Expression Analysis of PSCA in Normal Tissue and Patient Samples”).

PSCA can be analogized to a prostate-associated antigen PSA, the archetypal marker that has been used by medical doctors for years to identify and monitor the presence of prostate cancer (see, e.g., Merrill et al., J. Urol.163(2 ): 503-5120 (2000); Polascik et al., J. Urol. Aug; 162(2):293-306 (1999) and Fortier et al., J. Nat. Cancer Inst. 91(19): 1635 -1640(1999)). A number of other diagnostic markers are also used in similar contexts including p53 and K-ras (see, e.g., Tulchinsky et al., Int J Mol Med 1999 Jul 4(1):99-102 and Minimoto et al., Cancer Detect Prev 2000;24(1):1-12). Therefore, the present description of PSCA polynucleotides and polypeptides (as well as PSCA polynucleotide probes and anti-PSCA antibodies used to identify the presence of these molecules) and their properties allow those skilled in the art to use these molecules by methods analogous to those used, for example in a number of diagnostic tests aimed at investigating disorders associated with cancer.

Typical embodiments of diagnostic methods using the PSCA polynucleotides, polypeptides, reactive T cells and antibodies are analogous to the methods of well-established diagnostic assays, which use, for example, PSA polynucleotides, polypeptides, reactive T cells and antibodies. For example, just as PSA polynucleotides are used as probes (for example, in Northern analysis, see, e.g., Sharief et al., Biochem. Mol. Biol. Int. 33(3):567-74(1994)) and primers (eg, in PCR analysis, see, e.g., Okegawa et al., J. Urol. 163(4): 1189-1190 (2000)) to observe the presence and/or level of PSA mRNA' are in methods for monitoring PSA overexpression or metastasis of prostate cancer, the PSCA polynucleotides described here can be used in the same way to detect PSCA overexpression or metastasis of prostate and other cancers that express this gene. Alternatively, just as PSA polypeptides are used to generate antibodies specific for PSA which can then be used to observe the presence and/or level of PSA proteins by methods to monitor PSA protein overexpression (see, e.g., Stephan et al., Urology 55(4):560-3 (2000)) or metastasis of prostate cells (see, e.g., Alanen et al., Patol. Res. Pract. 192(3):233-7 (1996 )), the PSCA polypeptides described herein can be used to generate antibodies for use in detecting PSCA overexpression or metastasis of prostate cells and cells of other cancers expressing this gene.

Specifically, because 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), experiments examining a biological sample for the presence of cells expressing PSCA polynucleotides and/or or polypeptides are used to provide evidence of metastasis. For example, when a biological sample from tissue that does not normally contain PSCA-expressing cells (lymph node) is found to contain PSCA-expressing cells such as the PSCA expression seen in LAPC4 and LAPC9, xenografts isolated from lymph node and bone metastasis, respectively, this finding is indicative of metastasis.

Alternatively, PSCA polynucleotides and/or polypeptides can be used to provide evidence of cancer, for example when cells in a biological sample that normally do not express PSCA or express PSCA at a different level are found to express PSCA or have an increased expression of PSCA ( see, e.g., PSCA expression in the cancers listed in Table I and in patient samples, etc. shown in the accompanying Figures). In such trials, technicians may further wish to provide 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, e.g., Alanen et al. , Patol. Res. Pract. 192(3): 233-237 (1996)).

The use of immunohistochemistry to identify the presence of a PSCA polypeptide in a tissue section may indicate an altered status of certain cells within that tissue. It is well known in the art that the ability of an antibody to localize to a polypeptide expressed in cancer cells is a means of diagnosing the presence of disease, disease stage, progression and/or tumor aggressiveness. Such an antibody can also detect a changed distribution of the polypeptide within the cancer cells, compared to corresponding non-malignant tissue.

The PCSA polypeptide and immunogenic compositions are also useful with respect to the phenomena of altered subcellular protein localization in disease states. The change of cells from a normal to a diseased state causes changes in cellular morphology and is often associated with changes in subcellular protein localization/distribution. For example, cell membrane proteins that are expressed in a polarized manner in normal cells can be altered in disease, resulting in the distribution of the protein in a non-polar manner over the entire cell surface.

The phenomenon of altered subcellular protein localization in a disease state has been demonstrated with MUC1 and Her2 protein expression using immunohistochemical methods. Normal epithelial cells have a typical apical distribution of MUC1, in addition to some supranuclear localization of the glycoprotein, while malignant lesions often show an apolar labeling 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 either negative for Her2 protein or exhibits only a basolateral distribution while malignant cells may express the protein over the entire cell surface (De Potter, et al, International Journal of Cancer, 44; 969-974 (1989): McCormick, et al, 117;935-943 (2002)).

Alternatively, distribution of the protein may change from a localization only on the surface to include diffuse cytoplasmic expression in the disease state. One such example can be seen with MUC1 (Diaz, et al, The Breast Journal, 7: 40-45 (2001)).

Changes in the localization/distribution of a protein in the cell, as detected by immunohistochemical methods, can also provide valuable information regarding the benefits of certain forms of treatment. This last point is illustrated by a situation where a protein can be intracellular in normal tissue, but at the cell surface in malignant cells; cell surface localization makes the cells advantageously amenable to antibody-based diagnostic and treatment regimens. When such a change in protein localization occurs for PSCA, the PSCA protein and immune responses related to it are highly applicable. Accordingly, the ability to determine whether alteration of subcellular protein localization occurred for 24P4C12 makes the PSCA protein and immune responses related thereto very useful. Use of the PSCA mixtures allows those skilled in the art to make important diagnostic and therapeutic decisions.

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

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

Just as PSA polynucleotide fragments and polynucleotide variants are used by those skilled in the art for use in methods of monitoring PSA, PSCA polynucleotide fragments and polynucleotide variants are used in an analogous manner.

Typical PSA polynucleotides used in methods for monitoring PSA are probes or primers consisting of fragments of the PSA cDNA sequence. To illustrate this, primers used to PCR amplify a PSA polynucleotide must encompass less than the entire PSA sequence to function in polymerase chain reaction. In the context of such PCR reactions, those skilled in the art generally generate a variety of different polynucleotide fragments that can be used as primers to amplify different parts of a polynucleotide of interest or to optimize amplification reactions (see, e.g., Caetano-Anolles, G.

Biotechniques 25(3): 472-476, 478-480 (1998); Robertson et al., Methods Mol. Biol. 98:121-154 (1998)). A further illustration of the use of such fragments is given in the example entitled "Expression analysis of PSCA in normal tissue and patient samples," 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, e.g., Sawai et al., Fetal Diagn. Ther.

1996 Nov-Dec 11(6):407-13 and Current Protocols in Molecular Biology, Volume 2, Unit 2, Frederick M. Ausubel et al. eds., 1995)). Polynucleotide fragments and variants are useful in this context where they are capable of binding to a target polynucleotide sequence (eg, a PSCA polynucleotide shown in Figure 1 or variant thereof) under conditions of high stringency.

Furthermore, PSA polypeptides containing an epitope that can be recognized by an antibody or T cell that specifically binds to this epitope are used in methods for monitoring PSA. PCSA polypeptide fragments and polypeptide analogs or variants can also be used in an analogous manner. This practice of using polypeptide fragments or polypeptide variants to form antibodies (such as anti-PSA antibodies or T cells) is typical in the field with a variety of systems such as fusion proteins used by practitioners (see, e.g., Current Protocols in Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubel et al. eds., 1995). In this context, each epitope(s) functions to provide the architecture with which an antibody or T cell is reactive. Typically, those skilled in the art form a variety of different polypeptide fragments that can be used to generate immune responses specific to different parts of a polypeptide of interest (see, e.g., U.S. Patent No. 5,840,501 and U.S. Patent No. 5,939,533). For example, it may be preferred to use a polypeptide comprising one of the PSCA biological motifs described herein or a motif-bearing subsequence that is readily identified by those skilled in the art based on motifs available in the art. Polypeptide fragments, variants, or analogs are typically useful in this context as long as they comprise an epitope capable of generating an antibody or T cell specific for a target polypeptide sequence (eg, a PCSA polypeptide shown in Figure 1 ).

As shown herein, the PSCA polynucleotides and polypeptides (as well as the 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 as listed in Table I. Diagnostic assays that measure the presence of PSCA gene products, to evaluate the presence or onset of a disease state described herein, such as prostate cancer, are used to identify patients for preventive measurement or further monitoring, which has been successfully performed with PSA . Furthermore, these materials satisfy a need in the field for molecules that have similar or complementary characteristics to PSA in situations where, for example, an unequivocal diagnosis of metastasis of prostate origin cannot be made on the basis of a test for PSA alone (see, e.g., , Alanen et al., Patol. Res. Pract. 192(3): 233-237 (1996)) and, accordingly, materials such as PSCA polynucleotides and polypeptides (as well as the PSCA polynucleotide probes and anti-PSCA antibodies) must be used to identify the presence of these molecules) are used to confirm metastases of prostatic origin.

Finally, in addition to their use in diagnostic assays, the PSCA polynucleotides described herein have several other uses such as their use in the identification of oncogenetically associated chromosomal abnormalities in the chromosomal region to which the PSCA gene is localized (see the example labeled “Chromosomal mapping of PSCA” below). Furthermore, in addition to their use in diagnostic assays, the PSCA-related proteins and polynucleotides described herein have other uses such as their use in forensic analysis of tissue of unknown origin (see, e.g., Takahama K Forensic Sci Int 1996 Jun 28;80(1-2): 63-9).

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

XII.) Inhibition of PSCA protein function

The invention encompasses 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 PSCA function.

XII.A.) Inhibition of PSCA with intracellular antibodies

In one method, a recombinant vector encoding single-chain antibodies that specifically bind to PSCA is introduced into PSCA-expressing cells through gene transfer technologies. Accordingly, the encoded single-chain anti-PSCA antibody is expressed intracellularly, binds to PSCA protein, and thereby inhibits its function. Methods for the construction of such intracellular single chain antibodies are well known. Such intracellular antibodies, also known as "intrabodies", are specifically targeted at a specific compartment within the cell, which gives control over where the inhibitory activity of the treatment is focused. This technology has been successfully applied in the field (for overview, see Richardson and Marasco, 1995, TIBTECH vol.

13). Intrabodies have been shown to virtually eliminate the expression of otherwise abundant cell surface receptors (see, e.g., Richardson et al., 1995, Proc. Natl. Acad. Sci. USA 92: 3137-3141; Beerli et al., 1994, J. Biol. Chem. 289: 23931-23936; Deshan et al., 1994, Gen Ther. 1: 332-337).

Single chain antibodies comprise the heavy and light chain variable domains linked 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 linked to the light chain constant region. Well-known intracellular trafficking signals are engineered into recombinant polynucleotide vectors that encode such single-chain antibodies to precisely target the intrabody to the desired intracellular compartment. For example, intrabodies targeted to the endoplasmic reticulum (ER) are engineered to incorporate a leader peptide and, optionally, a C-terminal ER retention signal, such as the KDEL amino acid motif. Intrabodies intended to exert activity in the nucleus are engineered to include a nuclear localization signal. Lipid groups are attached to intrabodies to bind the intrabody to the cytosolic side of the plasma membrane. Intrabodies can also be targeted to exert function in the cytosol. For example, cytosolic intrabodies are used to sequester factors within the cytosol, thereby preventing them from being transported to their natural cellular destination.

In one embodiment, intrabodies are used to bind PSCA in the nucleus, thereby preventing its activity in the nucleus. Nuclear targeting signals are engineered into such PSCA intrabodies to achieve the desired targeting. Such PSCA intrabodies are designed to bind specifically to a particular PSCA domain. In another embodiment, cytosolic intrabodies that specifically bind to a PSCA protein are used to prevent PSCA from accessing the nucleus, thereby preventing it from exerting any biological activity in the nucleus (eg, preventing PSCA from forming transcription complexes with other factors).

To specifically direct expression of such intrabodies to particular cells, the transcription of the intrabody is placed under the regulatory control of an appropriate tumor-specific promoter and/or enhancer. To target intrabody expression specifically to the prostate, for example, the PSA promoter and/or promoter/enhancer can be used (see, for example, U.S. Patent No. 5,919,652 granted July 6, 1999).

XII.B.) Inhibition of PSCA by recombinant proteins

In another method, recombinant molecules bind to PSCA and thereby inhibit PSCA function. For example, these recombinant molecules prevent or inhibit PSCA from accessing/binding to its binding partner(s) or associating with other protein(s). Such recombinant molecules may, for example, contain the reactive part(s) of a PSCA-specific antibody molecule. In a particular embodiment, the PSCA-binding domain of a PSCA binding partner is engineered into a dimeric fusion protein, whereby the fusion protein comprises two PSCA ligand-binding domains bound to the Fc portion of a human IgG, such as human IgG1. Such IgG portion 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 cancers associated with expression of PSCA, whereby the dimeric fusion protein specifically binds to PSCA and blocks PSCA interaction with a binding partner. Such dimeric fusion proteins are further combined into multimeric proteins using known antibody binding technologies.

XII.C.) Inhibition of PSCA transcription or translation

The present invention also includes various methods and compositions for inhibiting the transcription of the PSCA gene. Correspondingly, the invention also provides methods and compositions for inhibiting translation of PSCA mRNA into protein.

In one method, a method of inhibiting the transcription of the PSCA gene comprises contacting the PSCA gene with a PSCA antisense polynucleotide. In another method, a method of inhibiting PSCA mRNA translation comprises contacting a PSCA mRNA with an antisense polynucleotide. In another method, a PSCA-specific ribozyme is used to cleave a PSCA messenger, thereby inhibiting translation. Such antisense and ribozyme-based methods can also target the regulatory regions of the PSCA gene, such as PSCA promoter and/or enhancer elements. Similarly, proteins capable of inhibiting a PSCA gene transcription factor are used to inhibit PSCA mRNA transcription. The various polynucleotides and mixtures useful in the above methods are described above. The use of antisense and ribozyme molecules to inhibit transcription and translation is well known in the art.

Other factors that inhibit transcription of PSCA by affecting PSCA transcriptional activation are also useful for treating cancers that express PSCA. Similarly, factors that interfere with PSCA processing are useful for treating cancers that express PSCA. Cancer treatment methods using such factors are also within the scope of the invention.

XII.D.) General Considerations for Therapeutic Strategies Gene transfer and gene therapy technologies can be used to deliver therapeutic polynucleotide molecules to tumor cells that synthesize PSCA (ie, antisense, ribozyme, polynucleotides encoding intrabodies and other PSCA-inhibiting molecules). Several gene therapy methods are known in the field.

Recombinant vectors encoding PSCA antisense polynucleotides, ribozymes, factors capable of affecting PSCA transcription, and so on, can be delivered to target tumor cells using such gene therapy methods.

The above therapeutic methods can be combined with any of a variety of surgical, chemotherapy or radiation therapy regimens. The therapeutic methods according to the invention can enable the use of reduced doses of chemotherapy (or other therapies) and/or less frequent administration, an advantage for all patients and especially for those who do not tolerate the toxicity of the chemotherapeutic agent very well.

The antitumor activity of a particular compound (eg, antisense, ribozyme, intrabody) or a combination of such compounds can be evaluated using various in vitro and in vivo assay systems. In vitro assays evaluating therapeutic activity include cell growth assays, soft agar assays and other assays indicative of tumor-promoting activity, binding assays capable of determining the extent to which a therapeutic compound will inhibit the binding of PSCA to a binding partner, etc.

In vivo, the effect of a PSCA therapeutic mixture can be evaluated in a suitable animal model. For example, xenogeneic prostate cancer models can be used, where human prostate cancer explants or passaged xenograft tissues are introduced into immunocompromised animals, such as nude or SCID mice (Klein et al., 1997, Nature Medicine 3: 402-408). For example, PCT Patent Application WO98/16628 and U.S. Pat. Patent 6,107,540 various xenograft models of human prostate cancer capable of recapitulating the development of primary tumors, micrometastasis and the formation of osteoblastic metastases characteristic of late-stage disease. Effectiveness can be predicted by using tests that measure inhibition of tumor formation, tumor regression or metastasis and the like.

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

The therapeutic compositions used in the practice of the foregoing methods can be formulated into pharmaceutical compositions comprising a carrier suitable for the desired method of delivery. Suitable carriers include any material which when combined with the therapeutic composition retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Examples include, but are not limited to, any of several 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).

Therapeutic formulations can be solubilized and administered through any route capable of delivering the therapeutic composition to the tumor site. Potentially effective methods of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumor, intradermal, intraorgan, orthotopic, and the like. A preferred formulation for intravenous injection comprises the therapeutic mixture in a solution of preserved bacteriostatic water, sterile unpreserved water and/or diluted in polyvinyl chloride or polyethylene bags containing 0.9% sterile Sodium Chloride for Injection, USP. Therapeutic protein preparations can be lyophilized and stored as sterile powders, preferably under vacuum, and then reconstituted in bacteriostatic water (containing, for example, benzyl alcohol preservative) or in sterile water before injection.

Doses and administration protocols for treating cancer using the foregoing methods will vary with the method and the target cancer and will generally depend on several other factors understood in the art.

XIII.) Identification, characterization and application of modulators by PSCA Methods for identifying and applying modulators

In one embodiment, screening is performed to identify modulators that induce or suppress a particular expression profile, suppress or induce specific pathways, thereby preferentially producing the associated phenotype. In another embodiment, after identifying differentially expressed genes important in a particular condition; screenings are performed to identify modulators that alter the expression of individual genes, either increasing or decreasing. In another embodiment, screening is performed to identify modulators that alter a biological function of the expression product of a differentially expressed gene. Again, after identifying the importance of a gene in a particular condition, screens are performed to identify agents that bind and/or modulate the biological activity of the gene product.

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

Modulator-related identification and screening trials:

Gene expression-related analyses

Proteins, nucleic acids and antibodies according to the invention are used in screening experiments. The cancer-associated proteins, antibodies, nucleic acids, modified proteins and cells containing these sequences are used in screening experiments, such as evaluating the effect of drug candidates on a "gene expression profile," expression profile of polypeptides or alteration of biological function. In one embodiment, the expression profiles used, preferably in conjunction with high-throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent (eg, Davis, GF, et al, J Biol Screen 7:69 (2002); Zlokarnik, et al ., Science 279:84-8 (1998); Heid, Genom Res 6:986-94,1996).

The cancer proteins, antibodies, nucleic acids, modified proteins and cells containing the native or modified cancer proteins or genes are used in screening experiments. Thus, the present invention includes methods for screening for compounds that modulate the cancer phenotype or a physiological function of a cancer protein according to the invention. This is done on a gene itself or by assessing the effect of drug candidates on a "gene expression profile" or biological function. In one embodiment, expression profiles are used, preferably in conjunction with high-throughput screening techniques to allow monitoring after treatment with a candidate agent, see Zlokamik, supra.

A number of tests have been carried out targeting the genes and proteins according to the invention. Experiments are carried out at an individual nucleic acid or protein level. That is, when a particular gene has been identified as upregulated in cancer, test compounds are screened for the ability to modulate gene expression or for binding to the cancer protein according to the invention. "Modulation" in this context includes an increase or a decrease in gene expression. The preferred amount of modulation will depend on the initial change in gene expression in normal versus tissue undergoing cancer, with changes of at least 10%, preferably 50%, more preferably 100-300% and in some embodiments 300-1000% or more . If a gene exhibits a 4-fold increase in cancer tissue compared to normal tissue, a reduction of approximately four-fold is therefore often desired; similarly, a 10-fold reduction in cancer tissue compared to normal tissue is a target value of a 10-fold increase in expression of the test compound often desired. Modulators that worsen the type of gene expression seen in cancer are also applicable, e.g., as an up-regulated target in further analyses.

The amount of gene expression is monitored using nucleic acid probes and quantification of gene expression levels, or, alternatively, a gene product itself is monitored, for example, through the use of antibodies against the cancer protein and standard immunoassays. Proteomics and separation techniques also allow quantification of expression.

Expression monitoring to identify compounds that modify gene expression

In one embodiment, gene expression monitoring, ie, an expression profile is monitored simultaneously for multiple units. Such profiles will typically involve one or more of the genes according to Figure 1. In this embodiment, for example, cancer nucleic acid probes are bound to biochips to detect and quantify cancer sequences in a particular cell. Alternatively, PCR can be used. Thus, a number of, e.g., wells of a microtiter plate can be used with supplied primers in desired wells. A PCR reaction can then be performed and analyzed for each well.

Expression monitoring is performed to identify compounds that modify expression of one or more cancer-associated sequences, eg, a polynucleotide sequence set forth in Figure 1. Generally, a test modulator is added to the cells prior to analysis. Furthermore, screenings are also provided to identify agents that modulate cancer, modulate cancer proteins according to the invention, bind to a cancer protein according to the invention or interfere with the binding of cancer protein according to the invention and an antibody or other binding partner.

In one embodiment, high-throughput screening methods comprise providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such "combinatorial chemical libraries" are then screened in one or more assays to identify those library members (particular chemical species or subclasses) that exhibit a desired characteristic activity. The compounds thus identified may serve as conventional "lead compounds," as compounds for screening, or as therapeutic agents.

In certain embodiments, combinatorial libraries of potential modulators are screened for an ability to bind to the cancer polypeptide or to modulate activity. Conventionally, new chemical entities with useful properties are formed by identifying a chemical compound (termed a "lead compound") with some desirable property or activity, e.g., inhibitory activity, producing variants of the "lead compound" and evaluating properties and activity of the variant compounds. High-throughput screening (HTS) methods are often used for such an analysis.

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

If necessary, the target sequence is prepared using known techniques. For example, a sample is processed to lyse the cells, using known lysis buffers, electroporation, etc., with purification and/or amplification such as PCR performed as appropriate. For example, an in vitro transcription with tags covalently bound to the nucleotides is performed. In general, 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, a chemiluminescent, a chemical or a radioactive signal, to provide a method for detecting the specific binding of the target sequence to a probe. The label can also be an enzyme, such as alkaline phosphatase or horseradish peroxidase, which when provided with a suitable substrate yields a product that is detected.

Alternatively, the label is a labeled compound or small molecule, such as an enzyme inhibitor, that binds but is not catalyzed or altered by the enzyme.

The label can also be a group or compound, such as, an epitope tag or biotin that specifically binds to streptavidin. For the biotin example, streptavidin is labeled as described above, thereby providing a detectable signal for the bound target sequence. Unbound labeled streptavidin is typically removed before analysis.

As will be understood by those skilled in the art, these assays may be direct hybridization assays or may comprise "sandwich assays", involving the use of multiple probes, as generally described in U.S. Pat. Patents No. 5, 681,702; 5,597,909; 5,545,730; 5,594,117; 5,591,584; 5,571,670; 5,580,731;

5,571,670; 5,591,584; 5,624,802; 5,635,352; 5,594,118; 5,359,100; 5,124, 246; and 5,681,697. In these embodiments, in general, the target nucleic acid is prepared as described above and then added to the biochip comprising an array of nucleic acid probes, under conditions that allow formation of a hybridization complex.

A number of hybridization conditions are used in the present invention, including high, moderate and low stringency conditions as described above. The assays are generally run under stringency conditions that allow formation of the labeled probe hybridization complex only in the presence of target. Stringency 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 can also be used to control non-specific binding, which generally is described in U.S. Patent No. 5,681,697. Accordingly, it may be desirable to perform certain steps at higher stringency conditions to reduce non-specific binding.

The reactions described here can be carried out in a number of ways.

Components of the reaction may be added simultaneously or sequentially, in different orders, preferred embodiments of which are described below. In addition, the reaction may include a variety of other reagents. These include salts, buffers, neutral proteins, e.g. albumin, detergents, etc. that can be used to facilitate optimal hybridization and detection and/or reduce non-specific or background interactions. Reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may also be used as appropriate, depending on sample preparation methods and purity of the target. The assay data are analyzed to determine the expression levels of individual genes and changes in expression levels between conditions, forming a gene expression profile.

Biological activity-related analyses

The invention provides methods for identifying or screening for a compound that modulates the activity of a cancer-related gene or protein according to the invention. The methods comprise the addition of a test compound, as defined above, to a cell comprising cancer protein according to the invention. The cells contain a recombinant nucleic acid which codes for a cancer protein according to the invention. In another embodiment, a library of candidate agents is tested on a variety of cells.

In one aspect, the experiments are evaluated in the presence or absence or before or after exposure to physiological cues, e.g. hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogens or other cells (ie, cell-cell contacts). In another example, the determinations are performed at different stages of the cell cycle process. In this way, compounds that modulate genes or proteins according to the invention are identified. Compounds with pharmacological activity are capable of enhancing or interfering with the activity of the cancer protein according to the invention. Once identified, similar structures are evaluated to identify critical structural features of the compound.

In one embodiment, a method of modulating (eg, inhibiting) cancer cell division is provided; the method comprises the administration of a cancer modulator. In another embodiment, a method of modulating (eg, inhibiting) cancer is provided; the method comprises the administration of a cancer modulator. In a further embodiment, methods are provided for treating cells with cancer; the method comprises the administration of a cancer modulator. Methods for treating individuals with cancer are also described.

In one embodiment, a method is provided for modulating the status of a cell expressing a gene according to the invention. As used herein, status includes such parameters known in the art as growth, proliferation, survival, function, apoptosis, senescence, localization, enzymatic activity, signal transduction, etc. of a cell. In one embodiment, the cancer inhibitor is an antibody as described above. In another embodiment, the cancer inhibitor is an antisense molecule. A number of cell growth, proliferation and metastasis experiments are known to those skilled in the art, as described herein.

High-throughput screening to identify modulators

The analyzes to identify suitable modulators are amenable to high-throughput screening. Accordingly, preferred assays detect enhancement or inhibition of cancer gene transcription, inhibition or enhancement of polypeptide expression, and inhibition or enhancement of polypeptide activity.

In one embodiment, modulators evaluated in high-throughput screening methods are proteins, often naturally occurring proteins or fragments of naturally occurring proteins. Accordingly, for example, cellular extracts containing proteins or random or controlled cleavages of proteinaceous cellular extracts are used. In this way, libraries of proteins are prepared for screening by the methods according to the invention.

Particularly preferred in this embodiment are libraries of bacterial, fungal, viral and mammalian proteins, the latter being preferred and human proteins being particularly preferred. Particularly useful test compound will be directed against the class of proteins to which the target belongs, eg, substrates for enzymes, or ligands and receptors.

Application of softagar growth and colony formation to identify and characterize modulators

Normal cells require a solid substrate to attach and grow. When cells are transformed, they lose this phenotype and grow isolated from the substrate. For example, transformed cells can be grown in stirred suspension culture or suspended in semi-solid medium, such as semi-solid or soft agar. The transformed cells, when transfected with tumor suppressor genes, can regenerate normal phenotype and again require a solid substrate to attach and grow. Softagar growth or colony formation assays are used to identify modulators of cancer sequences that, when expressed in host cells, inhibit abnormal cellular proliferation and transformation. A modulator reduces or eliminates the ability of host cells to grow suspended in solid or semi-solid medium, such as agar.

Techniques for softagar growth or colony formation in suspension experiments are described in Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd ed., 1994). See also, the method section according to Garkavtsev et al. (1996), supra.

Evaluation of contact inhibition and growth density limitation to identify and characterize modulators

Normal cells typically grow in a planar and organized pattern in cell culture until they encounter other cells. When the cells come into contact with each other, their contact is inhibited and they stop growing. However, transformed cells are not contact inhibited and continue to grow to high density in disorganized foci. Thus, transformed cells grow to a higher saturation density than corresponding normal cells. This is detected morphologically by the formation of a disoriented monolayer of cells or cells in foci. Alternatively, the labeling index with (<3>H)-thymidine at saturation density is used to measure density limitation of growth, similarly an MTT or Alamar blue analysis will reveal the proliferation capacity of cells and the ability of modulators to influence the same. See Freshney (1994), supra. Transformed cells, when transfected with tumor suppressor genes, can regenerate a normal phenotype and become contact inhibited and would grow to a lower density.

In this experiment, labeling index with 3H)-thymidine at saturation density is a preferred method for measuring density limitation of growth. Transformed host cells are transfected with cancer-associated sequence and are cultured for 24 hours at saturating density in non-limiting medium conditions. The percentage of cells labeled with (<3>H)-thymidine is determined by incorporated cpm.

Contact independent growth is used to identify modulators of cancer sequences, which had led to abnormal cellular proliferation and transformation. A modulator reduces or eliminates contact-independent growth and returns the cells to a normal phenotype.

Evaluation of growth factor or serum dependence to identify and characterize modulators

Transformed cells have lower serum dependence than their normal counterparts (see, e.g., Temin, J. Natl. Cancer Inst. 37:167-175 (1966); Eagle et al., J. Exp. Med 131:836-879 (1970)); Freshney, supra. This is partly 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 to that of control. For example, growth factor or serum dependence of a cell is monitored by methods to identify and characterize compounds that modulate cancer-associated sequences according to the invention.

Application of tumor-specific marker levels to identify and characterize modulators

Tumor cells release an increased amount of certain factors (called "tumor-specific markers") more than their normal counterparts. For example, plasminogen activator (PA) is released from human glioma at a higher level than from normal brain cells (see, e.g., Gullino, Angiogenesis, Tumor Vascularization and Potential Interference with Tumor Growth, in Biological Responses in Cancer, pp. 178- 184 (Mihich (ed.) 1985)). Similarly, Tumor Angiogenesis Factor (TAF) is released at a higher level in tumor cells than their normal counterparts. See, e.g., Folkman, Angiogenesis and Cancer, Sem. Cancer Biol. (1992)), while bFGF is released from endothelial tumors (Ensoli, B et al.).

Various techniques 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 & Beers, J. Biol. Chem. 251:5694-5702 (1976); Whur et al., Br. J. Cancer 42:305312 (1980); Gullino, Angiogenesis, Tumor Vascularization and Potential Interference with Tumor Growth, in Biological Responses in Cancer, pp. 178-184 (Mihich (ed.) 1985); Freshney, Anticancer Res. 5:111-130 (1985). For example, tumor-specific marker levels are monitored by methods to identify and characterize compounds that modulate cancer-associated sequences according to the invention.

Invasiveness into matrigel to identify and characterize modulators The degree of invasiveness into Matrigel or an extracellular matrix constituent can be used as a measure to identify and characterize compounds that modulate cancer-associated sequences. Tumor cells show a positive correlation between malignant disease and invasiveness of cells into Matrigel or any other extracellular matrix constituent. In this experiment, tumorigenic cells are typically used as host cells. Expression of a tumor suppressor gene in these host cells would reduce invasiveness of the host cells. Techniques described in Cancer Res.1999; 59:6010; Freshney (1994), supra, can be applied. Briefly, the level of host cell invasion is measured using filters coated with Matrigel or some other extracellular matrix constituent. Penetration into the gel or through to the distal side of the filter is assessed as invasiveness and assessed histologically by the number of cells and distance moved or by pre-labeling the cells with <125>1 and counting the radioactivity on the distal side of the filter or the bottom of the plate . See, e.g., Freshney (1984), supra.

Evaluation of tumor growth in vivo to identify and characterize modulators

Effects of cancer-associated sequences on cell growth are tested in transgenic or immune-suppressed organisms. Transgenic organisms are produced by a number of methods known in the field. For example, knock-out transgenic organisms, eg, mammals such as mice, are produced in which a cancer gene is interrupted or in which a cancer gene is inserted. Knock-out transgenic mice are produced by insertion of a marker gene or other heterologous gene into the endogenous cancer gene site 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 produce transgenic chimeric animals, eg, mice, a DNA construct is introduced into the nuclei of embryonic stem cells. Cells containing the newly engineered genetic lesion are injected into a host mouse embryo, which is reimplanted into a recipient female. Some of these embryos develop into chimeric mice that have germ cells, some of which are derived from the mutant cell line. Therefore, by breeding the chimeric mice it is possible to obtain a new line of mice containing the introduced genetic lesion (see, e.g., Capecchi et al., Science 244:1288 (1989)). Chimeric mice can be derived according to US Patent 6,365,797, granted April 2, 2002; US Patent 6,107,540 granted 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, various immunosuppressed or immunodeficient host animals can be used. For example, a genetically athymic "nude" mouse (see, e.g., Giovanella et al., J. Natl. Cancer Inst.52:921 (1974)), a SCID mouse, a thymectornized mouse, or an irradiated mouse (see (eg, Bradley et al., Br. J. Cancer 38:263 (1978); Selby et al., Br. J. Cancer 41:52 (1980)) is used as a host.

Transplantable tumor cells (typically about 10<6 > cells) injected into isogenic hosts produce invasive tumors in a high proportion of cases, whereas normal cells of similar origin will not. In hosts that developed invasive tumors, cells expressing cancer-associated sequences are injected subcutaneously or orthotopically. Mice are then divided into groups, comprising control groups and treated experimental groups) e.g. treated with a modulator). After an appropriate period of time, preferably 4-8 weeks, tumor growth is measured (eg, by volume or by its two largest dimensions or weight) and compared to the control. Tumors that have statistically significant reduction (using, e.g., Student's T test) are indicated as having inhibited growth.

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

Alternatively, a reporter gene system can be constructed using a cancer protein promoter operably linked to a reporter gene such as luciferase, green fluorescent protein, CAT or β-gal. The reporter construct is typically transfected into a cell. After treatment with a potential modulator, the amount of reporter gene transcription, translation or activity is measured according to standard techniques known to those 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 for individual genes and gene products. That is, once one has identified a particular differentially expressed gene as important in a particular condition, screening for modulators of expression of the gene or gene product itself is performed.

In one embodiment, screening for modulators of expression of specific gene(s) is performed. Typically, expression of only one or a few genes is evaluated. In another embodiment, screens are designed to first find compounds that bind to differentially expressed proteins. These compounds are then evaluated for the ability to modulate differentially expressed activity. Furthermore, once initial candidate compounds are identified, variants can be further screened to better evaluate structure-activity relationships.

Binding experiments to identify and characterize modulators

In binding experiments according to the invention, a purified or isolated gene product according to the invention is generally used. For example, antibodies are formed to a protein according to the invention and immunoassays are run to determine the amount and/or localization of the protein. Alternatively, cells containing the cancer proteins are used in the experiments.

Accordingly, the methods comprise combining a cancer protein according to the invention and a candidate compound such as a ligand, and determining the binding of the compound to the cancer protein according to the invention. Preferred embodiments employ the human cancer protein; animal models of human disease can also be developed and used. Likewise, other analogous mammalian proteins can also be used as understood by those skilled in the art. Furthermore, in some embodiments variant or derived cancer proteins are used.

In general, the cancer protein according to the invention or the ligand is non-volatilely bound to an insoluble carrier. The carrier may, for example, be one having isolated sample-receiving areas (a microtiter plate, an array, etc.). The insoluble carriers can be made of any composition to which the mixtures can be bound, are readily separated from soluble material, and are otherwise compatible with the general screening method. The surface of such carriers may be solid or porous and of any suitable shape.

Examples of suitable insoluble supports include microtiter plates, matrices, membranes and beads. These are typically made of glass, plastic (eg, polystyrene), polysaccharide, nylon, nitrocellulose or Teflon™, etc. Microtiter plates and arrays are particularly convenient because a large number of experiments can be performed simultaneously, using small amounts of reagents and samples. The particular manner of binding the composition to the carrier is not important as long as it is compatible with the reagents and general methods of the invention, maintains the activity of the composition and does not spread easily. Preferred methods of binding include the use of antibodies that do not sterically block either the ligand-binding site or the activation sequence when the protein is attached to the support, direct binding to "sticky" or ionic supports, chemical cross-linking, synthesis of the protein or agent on the surface, etc. After binding of the protein or ligand/binding agent to the carrier, excess unbound material is removed by washing. The areas receiving the samples can then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or group.

When a cancer protein according to the invention is first bound to the carrier and a test compound is added to the analysis. Alternatively, the candidate binder is bound to the carrier and the cancer protein of the invention is then added. Binding agents include specific antibodies, non-natural binding agents identified in chemical library screenings, peptide analogs, etc.

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

A determination of binding of the test compound (ligand, binding agent, modulator, etc.) to the cancer protein according to the invention can be carried out in several ways. The test compound may be labeled and binding determined directly, e.g., by attaching all or part of the cancer protein of the invention to a solid support, adding labeled candidate compound (e.g., a fluorescent label), washing off excess reagent and determining whether the label is present on the solid support. Different blocking and washing steps can be used as appropriate.

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

Competitive binding to identify and characterize modulators

In one embodiment, the binding of the "test compound" is determined by competitive binding assays with a "competitor." The competitor is a binding group that binds to the target molecule (eg, cancer protein according to 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 will displace 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 carried out at a temperature that facilitates optimal activity, typically between four and 40ºC. Incubation periods are typically optimized, e.g., to facilitate rapid high-throughput screening; typically between zero and one hour will be sufficient.

Excess reagent is generally removed or washed away. The second component is then added and the presence or absence of the labeled component is followed to indicate binding.

In one embodiment, the competitor is added first, followed by the test compound. Displacement of the competitor is an indication that the test compound binds to the cancer protein and is thus able to bind to and potentially modulate the activity of the cancer protein. In this embodiment, one or the other component may be labeled. Accordingly, for example, if the competitor is labeled, the presence of label in the post-test compound washing solution indicates displacement by the test compound. Alternatively, if the test compound is labeled, the presence of the label on the support will indicate displacement.

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

Accordingly, the competitive binding methods include differential screening to identify agents that can modulate the activity of the cancer proteins according to the invention. In this embodiment, the methods comprise combining the cancer protein and a competitor in a first sample. A second sample comprises a test compound, the cancer protein and a competitor. The binding of the competitor is determined for both samples and a change or difference in binding between the two samples indicates the presence of an agent capable of binding to the cancer protein and potentially modulating its activity. Thus, if the binding of the competitor is different in the second sample relative to the first sample, the agent is able to bind to the cancer protein.

Alternatively, differential screening is used to identify drug candidates that bind to the native cancer protein but cannot bind to modified cancer proteins. For example, the structure of the cancer protein is modeled and used in rational drug design to synthesize agents that interact with this site, agents that generally do not bind to site-modified proteins. Furthermore, such drug candidates that affect the activity of a native cancer protein are also identified by screening drugs for the ability to either promote or reduce the activity of such proteins.

Positive controls and negative controls can be used in the experiments.

Preferably, control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples takes place for a period of time sufficient to allow binding of the agent to the protein. After incubation, samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, when a radioactive label is used, the samples can be counted in a scintillation counter to determine the amount of bound compound.

A number of other reagents can be included in the screening analysis. These include reagents such as salts, neutral proteins, e.g. albumin, detergents, etc. which are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also, reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., can be used. The mixture of components is added in an order that allows the desired bond.

Use of polynucleotides to down-regulate or inhibit a protein according to the invention.

Polynucleotide modulators of cancer 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, conjugation of the ligand binding molecule does not substantially prevent the ability of the ligand binding molecule to bind to its corresponding molecule or receptor or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell. Alternatively, a polynucleotide modulator of cancer can be introduced into a cell containing the target nucleic acid sequence, for example, by forming a polynucleotide-lipid complex, as described in WO 90/10448. It will be understood that the use of antisense molecules or knock out and knock in models can also be used in screening analyzes as described above, in addition to methods of treatment.

Inhibitory and antisense nucleotides

In certain embodiments, the activity of cancer-associated protein is down-regulated or completely inhibited, using antisense polynucleotide or inhibitory small nuclear RNA (snRNA), i.e., a nucleic acid complementary to, and which can preferentially hybridize specifically to, a coding mRNA nucleic acid sequence, eg, cancer protein according to the invention, mRNA or a subsequence thereof. Binding of the antisense polynucleotide to the mRNA reduces the translation and/or stability of the mRNA.

In the context of the present invention, antisense polynucleotides may comprise naturally occurring nucleotides or synthetic species formed from naturally occurring subunits or close homologues thereof. Antisense polynucleotides may also have altered sugar groups or inter-sugar linkages. Examples of these are phosphorothioate and other sulfur-containing species known for use in the area. Analogues are covered by the present invention as long as they function effectively to hybridize with nucleotides according to the invention. See, e.g., Isis Pharmaceuticals, Carlsbad, CA; Sequitor, Inc., Natick, MA.

Such antisense polynucleotides can be readily synthesized using recombinant methods or can be synthesized in vitro. Equipment for such synthesis is sold by many suppliers, including Applied Biosystems. 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. Sense oligonucleotides can, for example, be used to block transcription by binding to the anti-sense strand. The antisense and sense oligonucleotide comprise a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences for cancer molecules. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment of generally at least about 12 nucleotides, preferably from about 12 to 30 nucleotides. The ability to obtain an antisense or a sense oligonucleotide, based on a cDNA sequence encoding a given protein is described in, e.g., Stein & Cohen (Cancer Res. 48:2659 (1988) and van der Krol et al. (BioTechniques 6:958 (1988)).

Ribozymes

In addition to antisense polynucleotides, ribozymes can be used to target and inhibit transcription of cancer-associated nucleotide sequences. A ribozyme is an RNA molecule that catalytically cleaves other RNA molecules. Various types of ribozymes have been described, including group I ribozymes, "hammerhead" ribozymes, hairpin ribozymes, RNase P and axhead ribozymes (see, e.g., Castanotto et al., Adv. in Pharmacology 25: 289-317 (1994) for a general overview of the properties of different ribozymes).

The general features 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, e.g., 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)).

Application of modulators in phenotypic screening

In one embodiment, a test compound is administered to a population of cancer cells, which have an associated cancer expression profile. By "administering" or "contacting" here is meant that the modulator is added to the cells in such a way that it allows the modulator to act on the cell, whether by uptake and intracellular action or by action on the cell surface. In some embodiments, a nucleic acid encoding a proteinaceous agent (ie, a peptide) is placed into a viral construct such as an adenoviral or retroviral construct and added to the cell, such that expression of the peptide agent is completed, e.g. ., 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 are incubated under preferably physiological conditions for a period of time. The cells are then harvested and a new gene expression profile is formed. Thus, e.g., cancer tissue is screened for agents that modulate, e.g., induce or suppress, the cancer phenotype. A change in at least one gene, preferably many, of the expression profile indicates that the agent has an effect on cancer activity. Similarly, alteration of biological function or a signaling pathway is indicative of modulator activity. By defining such a signature for the cancer phenotype, screens for new drugs that change the phenotype are constructed. With this method, the drug target does not need to be known and does not need to be represented in the original gene/protein expression screening platform, nor does the level of transcript for the target protein need to change. The modulator-inhibitory function will serve as a surrogate marker As described above, screens are performed to assess genes or gene products. That is, when a particular differentially expressed gene is identified as important in a particular condition, screening for modulators of either expression of the gene or the gene product itself is performed.

Use of modulators to influence peptides according to the invention Measurements of cancer polypeptide activity or of the cancer phenotype are carried out using a series of tests. For example, the effects of modulators on the function of cancer polypeptide(s) are measured by examining parameters described above. A physiological change affecting activity is used to assess the effect of a test compound on the polypeptides of the present invention. When the functional results are determined using intact cells or animals, a number of effects can be assessed such as, in the case of cancer associated with solid tumors, tumor growth, tumor metastasis, neovascularization, hormone release, transcriptional changes of both known and uncharacterized genetic markers ( eg, by Northern blot), changes in cell metabolism such as cell growth or pH changes and changes in intracellular second messengers such as cGNIP.

Methods for the identification of characterizing cancer-associated sequences Expression of different gene sequences is correlated with cancer. Accordingly, disorders based on mutant or variant cancer genes are determined. In one embodiment, the invention provides methods for identifying cells containing variant cancer genes, e.g., determining the presence of, all or part of, the sequence of at least one endogenous cancer gene in a cell. This is accomplished using any number of sequencing techniques. The invention includes methods for identifying the cancer genotype of an individual, e.g. determining all or part of the sequence of at least one gene according to the invention in the individual. This is generally performed in at least one tissue of the subject, eg, a tissue listed in Table I and may include evaluation of multiple tissues or different samples of the same tissue. The method may comprise comparing the sequence of the sequenced gene with a known cancer gene, i.e., a wild-type gene to determine the presence of family members, homologies, mutations or variants. The sequence of all or part of the gene can then be compared to the sequence of a known cancer gene to determine if any differences exist. This is performed using any number of known homology programs, such as BLAST, Bestfit, etc. The presence of a difference in the sequence between the cancer gene from the patient and the known cancer gene correlates with a disease state or a predisposition to a disease state , as described here.

In a preferred embodiment, the cancer genes are used as probes to determine the number of copies of the cancer gene in the genome. The cancer genes are used as probes to determine the chromosomal location of the cancer genes.

Information such as chromosomal localization can be used in providing a diagnosis or prognosis especially when chromosomal abnormalities such as translocations and the like are identified in the cancer gene locus.

XIV.) RNAi and therapeutic use of small interfering RNAs (siRNAs) The present invention also relates to siRNA oligonucleotides, in particular double-stranded RNAs comprising at least a fragment of the PSCA coding region or 5” UTR regions or complement or any antisense oligonucleotide specific to the PSCA sequence. In one embodiment, such oligonucleotides are used to elucidate a function of PSCA or are used to screen for or evaluate modulators of PSCA function or expression. In another embodiment, gene expression of PSCA is reduced using siRNA transfection and results in significantly reduced proliferative capacity of transformed cancer cells endogenously expressing the antigen; cells treated with specific PSCA siRNAs show reduced survival as measured, e.g., by a metabolic readout of cell viability, consistent with the reduced proliferative capacity. Accordingly, PSCA siRNA mixtures comprise siRNA (double-stranded RNA) corresponding to the nucleic acid ORF sequence 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 consecutive RNA nucleotides in length and contain sequences that are complementary and non-complementary to at least part of the mRNA coding sequence. In a preferred embodiment, the subsequences are 19-25 nucleotides in length, most preferably 21-23 nucleotides in length.

RNA interference is a new method for inactivating ("silencing") genes in vitro and in vivo, therefore small double-stranded RNAs (siRNAs) are valuable therapeutic agents. The power of siRNAs to inactivate specific gene activities has now been brought to animal models of disease and is also being used in humans. For example, hydrodynamic infusion of a solution of siRNA into a mouse with an siRNA against a specific target has been proven to be therapeutically effective.

The pioneering work of Song et al. indicate that one type of fully natural nucleic acid, small interfering RNAs (siRNAs), served as therapeutic agents even without further chemical modification (Song, E., et al. “RNA interference targeting Fas protects mice from fulminant hepatitis” Nat. Med .9(3): 347-51(2003)). This work provided the first in vivo evidence that infusion of siRNAs into an animal could alleviate disease. In this case, the authors gave mice injections of siRNA designed to silence the FAS protein (a cell death receptor that, when over-activated during an inflammatory response, induces hepatocytes and other cells to die). The following day, the animals were given an antibody specific to Fas. Control mice died of acute liver failure within a few days, while over 80% of the siRNA-treated mice remained free of severe disease and survived. Approximately 80% to 90% of their liver cells incorporated the naked siRNA oligonucleotides. Furthermore, the RNA molecules worked for 10 days before losing their effect after 3 weeks.

For use in human therapy, siRNA is delivered by efficient systems that induce long-lasting RNAi activity. An important caveat for clinical application is the delivery of siRNAs to appropriate cells. Hepatocytes appear to be particularly susceptible to exogenous RNA. Today, targets located in the liver are attractive because the liver is an organ that can be easily targeted by nucleic acid molecules and viral vectors. However, other tissue and organ targets are also preferred.

Formulations of siRNAs with compounds that promote passage through cell membranes are used to improve administration of siRNAs in therapy.

Chemically modified synthetic siRNA, which is resistant to nucleases and has serum stability with accompanying increased duration of RNAi effects, is a further embodiment.

Accordingly, siRNA technology is a therapeutic agent for human malignancy by delivering siRNA molecules targeting PSCA to individuals with the cancers listed in Table 1. Such administration of siRNAs leads to reduced growth of cancer cells expressing PSCA and provides a anti-tumor therapy, reduces the morbidity and/or mortality associated with malignant disease.

The effectiveness of this form of gene product "knockdown" is significant when measured in vitro or in vivo. Efficacy in vitro is readily demonstrable through the application of siRNAs to cells in culture (as described above) or to aliquots of cancer patient biopsies when in vitro methods are used to detect the reduced expression of PSCA protein.

XV.) Kit/Articles for manufacture

For use in the laboratory, prognostic, prophylactic, diagnostic and therapeutic uses described herein, the kit is within the scope of the invention. Such kits may comprise a carrier, package or container which is divided into compartments to receive one or more containers such as vials, tubes and the like, where each of the container(s) comprises one of the separate elements to be used in the method, together with a mark or attachment including instructions for use, such as an application described here. For example, the container(s) may comprise a probe that is or may be detectably labeled. Such a probe can be an antibody or polynucleotide specific for a protein or a gene or message according to the invention, respectively. When the method uses nucleic acid hybridization to detect the target nucleic acid, the kit can also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence. Kits may comprise a container comprising a reporter, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, fluorescent or radioisotope label; such a reporter can be used with, for example, a nucleic acid or antibody. The kit may comprise all or part of the amino acid sequences in Figure 1, Figure 2 or Figure 3 or analogues thereof or a nucleic acid molecule that codes for such amino acid sequences.

The kit according to the invention will typically comprise the container described above and one or more other containers connected thereto comprising materials desirable from a commercial and user point of view, including buffers, diluents, filters, needles, syringes; carrier, package, container, vial and/or tube labels listing contents and/or instructions for use and package inserts with instructions for use.

A label may be present on or with the container to indicate that the composition is being used for a specific treatment or non-therapeutic use, such as a prognostic, prophylactic, diagnostic or laboratory use and may also indicate guidance for either in vivo or in vitro use , such as those described here. Instructions for use and or other information may also be included in an appendix or brand(s) that are included with or in the kit. The mark may be on or associated with the container. A mark may be on a container when the letters, numbers or other characters forming the mark are cast or etched on the container itself; a mark may be associated with a container when it is present in a container or carrier that also houses the container, eg, as a package insert. The mark may indicate that the composition is used for the diagnosis, treatment, prophylaxis or prognosis of a condition, such as a neoplasia in a tissue listed in Table I.

The terms "kit" and "article for manufacture" can be used as synonyms.

In another embodiment according to the invention, an article(s) is provided for production containing mixtures, such as amino acid sequence(s), small molecule(s), nucleic acid sequence(s) and/or antibody(s), e.g., materials applicable for diagnosis, prognosis, prevention and/or treatment of tissue neoplasias such as those listed in Table I. The articles for manufacture typically comprise at least one container and at least one brand. Suitable containers include, for example, bottles, vials, syringes and test tubes. The containers can be formed from a variety of materials such as glass, metal or plastic. The container 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 examining the mRNA expression profile of a cell, together with reagents used for this purpose. In another embodiment, a container comprises an antibody, binding fragment thereof or specific binding protein for use in evaluating protein expression of PSCA in cells and tissues or for relevant laboratory, prognostic, diagnostic, prophylactic and therapeutic purposes; indications and/or instructions for use for such applications may be included in or with such a container, as well as reagents and other mixtures or tools used for these purposes. In another embodiment, a container comprises materials for eliciting a cellular or humoral immune response, along with associated indications and/or directions for use. In another embodiment, a container comprises materials for adoptive immunotherapy, such as cytotoxic T cells (CTL) or helper T cells (HTL), along with associated indications and/or directions for use; reagents and other mixtures or tools used for such purposes may also be included.

Alternatively, the container may contain a composition effective for the treatment, diagnosis, prognostication, or prophylaxis of a condition and may have a sterile access opening (for example, the container may be an intravenous solution bag or a vial having a cap penetrable by a hypodermic injection needle). The active agents in the mixture may be an antibody capable of specifically binding PSCA and modulating the function of PSCA.

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 further include other materials desirable from a commercial and user point of view, including other buffers, diluents, filters, whisks, needles, syringes and/or package inserts with indications and/or instructions for use.

EXAMPLES:

Various aspects of the invention are further described and illustrated through the many examples that follow, none of which are intended to limit the scope of the invention.

Example 1

Expression analysis of PSCA variants in normal tissue and patient samples

Previously, PSCA, here referred to as PSCA v.1, was identified as an antigen expressed in prostate cancer. Its expression was detected in more than 80% of primary prostate cancer and in the majority of 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. By immunohistochemical analysis, PSCA has been shown to be overexpressed on the cell surface of most urothelial cancers ("urothelial transitional carcinoma") and in 60% of primary pancreatic adenocarcinomas. PSCA expression data are reported in the patent publications (PCT/US98/04664, PCT/US/28883, PCT/US00/19967) and in the articles (Saffran et al., Proc Natl Acad Sci US A.2001 Feb 27; 98(5): 2658-2663; Amara et al., Cancer Res.2001 Jun 15; 61(12): 4660-65; Reiter et al., Proc Natl Acad Sci USA.1998 Feb 17; 95(4): 1735-40; Argani et al., Cancer Res.2001 Jun 1;61(11):4320-24).

Specific expression of different PSCA variants has been studied in normal and cancer patient samples. Primers were designed to differentiate between PSCA v.1/v.2/v.4, PSCA v.3 and PSCA v.5. PSCA v.1/v.2/v.4 leads to a PCR product of 425 bp, PSCA v.3 leads to a PCR product of 300 bp, while PSCA v.5 leads to a PCR product of 910 bp in size (Figure 1I(a).

First strand cDNA was prepared from normal bladder, brain, heart, kidney, liver, lung, prostate, spleen, skeletal muscle, testis, pancreas, colon, stomach, collections of prostate cancer, bladder cancer, kidney cancer, colon cancer, lung cancer, ovarian cancer, breast cancer, cancer metastasis and pancreatic cancer (Figure 1I(b).

Normalization was performed by PCR using primers for actin. Semiquantitative PCR, using the variant-specific primers, was performed at 30 cycles of amplification.

Results show expression of PSCA v.5 mainly in breast cancer, cancer metastasis and pancreatic cancer and at a lower level in colon cancer and lung cancer.

PSCA v.1/v.2/v.4 PCR product was detected in prostate cancer, bladder cancer, kidney cancer, colon cancer, lung cancer, ovarian cancer, breast cancer, cancer metastasis and pancreatic cancer. In normal tissue, PSCA v.1/v.2/v.4 PCR product was detected only in prostate, stomach and at a lower level in kidney and lung, while PSCA v.5 was not detected in any normal tissue. PSCA v.3 PCR-detected product was not detected in any of the samples tested.

Primers were designed to distinguish between PSCA v.4 and PSCA v.5 (Figure 1J(a). PSCA v.4 leads to a PCR product of 460 bp, while PSCA v.5 leads to a PCR product of a size of 945 bp.

First strand cDNA was prepared from normal bladder, brain, heart, kidney, liver, lung, prostate, spleen, skeletal muscle, testis, pancreas, colon, stomach, prostate cancer collections, bladder cancer and multi-xenograft collection (prostate cancer, kidney cancer and bladder cancer xenografts) (Figure 1J(b). Normalization was performed by PCR using primers for actin. Semi-quantitative PCR using the variant-specific primers was performed at 30 cycles of amplification.

Results show expression of PSCA v.4 in prostate cancer, bladder cancer and multixenograft collections, normal kidney and prostate. PSCA v.5 was detected only in normal prostate and bladder cancer.

The limited expression of PSCA variants in normal tissues and the expression detected in cancer patient samples indicate that PSCA variants are therapeutic, prognostic, laboratory, prophylactic and diagnostic targets for human cancer.

Example 2

Splicing variants of PSCA

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 transcripts from the same gene but start transcription at different points. Splice variants are mRNA variants spliced differently from the same transcript. In eukaryotes, when a multi-exon gene is transcribed from genomic DNA, the initial RNA is spliced to produce functional mRNA, which has only exons and is used for translation into an amino acid sequence.

Consequently, a given gene can have zero to many alternative transcripts and each transcript can have zero to many splice variants. Each transcript variant has a unique exon composition and may have different coding and/or non-coding (5' or 3' end) parts from the original transcript. Transcript variants can code for the same, similar or different proteins, where such proteins have the same or a similar function or a different function. The variant proteins can be expressed in the same tissue at the same time, in different tissues at the same time, or in the same tissue at different times or in different tissues at different times. Proteins encoded by a transcript variant may have similar or different subcellular or extracellular localization (eg, secreted versus intracellular).

Transcript variants are identified by a number of methods known in the field. For example, alternative transcripts and splice variants are identified by full-length cloning experiments or by using full-length transcript and EST sequences. First, all human ESTs were grouped into clusters that show direct or indirect identity with each other. Then, ESTs in the same cluster were further grouped into sub-clusters and assembled into a consensus sequence. The original gene sequence is compared to the consensus sequence(s) or other full-length sequences. Each consensus sequence is a potential splice variant for this gene. Many methods of confirmation are known in the art, such as identification of the variant by Northern analysis, full-length cloning or the use of probe libraries, etc.. Even when a variant is identified that is not yet a full-length clone, this part of the variant is very useful as a research tool, for example, for antigen formation or for further cloning of the full-length splice variant, using techniques known in the field.

Furthermore, computer programs are available in the field that identify transcript variants based on genomic sequences. Genomic-based transcript variant identification programs include FgenesH (A. Salamov and V. Solovyev, "Ab initio gene finding in Drosophila genomic DNA," Genome Research.

2000 April; 10(4):516-22); Grail (URL compbio.ornl.gov/Grail-bin/EmptyGrailForm) and GenScan (URL genes.mit.edu/GENSCAN.html). For a general discussion of identification protocols for splice variants see, e.g. Southan, C., A genomic perspective on human proteases, FEBS Lett.2001 Jun 8; 498(2-3):214-8; de Souza, S.J., et al., Identification of human chromosome 22 transcribed sequences with ORF expressed sequence tags, Proc. Natl Acad Sci U S A. 2000 Nov 7;

97(23):12690-3.

To further confirm the parameters of a transcript variant, a number of techniques are available in the field, such as full-length cloning, proteomic validation, PCR-based validation and 5' RACE validation, etc. (see e.g. Proteomic Validation: Brennan, S.O., et al., Albumin banks peninsula: a new termination variant characterized by electrospray mass spectrometry, Biochem Biophys Acta.1999 Aug 17;1433(1-2):321-6; Ferranti P, et al., Differential splicing of pre -messenger RNA produces multiple forms of mature caprine alpha(s1)-casein, Eur J Biochem.

1997 Oct 1;249(1):1-7. For PCR-based Validation: Wellmann S, et al., Specific reverse transcription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCycler technology, Clin Chem.2001 Apr;

47(4):654-60; Jia, H.P., et al., Discovery of new human beta-defensins using a genomics-based approach, Gene.2001 Jan 24; 263(1-2):211-8. For PCR-based and 5' RACE validation: Brigle, K.E., et al., Organization of the murine reduced folate carrier gene and identification of variant splice forms, Biochem Biophys Acta. 1997 Aug 7; 1353(2): 191-8).

It is known in the art that genomic regions are modulated in cancer. When the genomic region to which a gene maps is modulated in a particular cancer, the alternative transcripts or splice variants of the gene are also modulated. It is described here that PSCA has a specific expression profile related to cancer (see, for example, Table I). Alternative transcripts and splice variants of PSCA are also implicated in cancer, for example in one or more of these tissues and also in certain additional tissues. The variants therefore serve as tumor-associated markers/antigens.

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

Example 3

Single nucleotide polymorphisms of PSCA

A single nucleotide polymorphism (SNP) is a single base pair variation in 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 alternative forms of a given gene, which differ in DNA sequence and affect a product (RNA and/or protein).

A SNP that occurs in a cDNA is called a cSNP. This cSNP can change amino acids of the protein coded for by the gene and thus change the function of the protein. Some SNPs cause inherited diseases; others contribute to quantitative variations in phenotype and responses to environmental factors including diet and drugs among individuals. Therefore, the existence of a SNP and/or combinations of alleles (termed haplotypes) has many useful applications, such as diagnosis of inherited diseases, determination of drug responses and dose, identification of genes responsible for diseases and analysis of the genetic relatedness between individuals (P. Nowotny, J. M. Kwon and A. M. Goate, "SNP analysis to dissect human traits," Curr. Opin. Neurobiol. 2001 Oct;11(5):637-641; M. Pirmohamed and B. K. Park, "Genetic susceptibility to adverse drug reactions, " Trends Pharmacol. Sci.2001 Jun; 22(6):298-305; J. H. Riley, C. J. Allan, E. Lai, and A. Roses, " The use of single nucleotide polymorphisms in the isolation of common disease genes," Pharmacogenomics.

2000 Feb; 1(1):39-47; R. Judson, J. C. Stefens, and A. Windemuth, “The predictive power of haplotypes in clinical response,” Pharmacogenomics.2000 Feb; 1(1):15-26).

SNPs are identified by a number of methods known in the field (P. Bean, “The promising voyage of SNP target discovery,” Am. Clin. Lab. 2001 Oct-Nov; 20(9):18-20; K. M. Weiss, "In search of human variation," Genome Res.1998 Jul; 8(7):691-697; M. M. She, "Enabling large-scale pharmacogenetic studies by highthroughput mutation detection and genotyping technologies," Clin. Chem. 2001 Feb; 47 (2):164-172). For example, SNPs are identified by sequencing DNA fragments that show polymorphism by gel-based methods such as restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). They can also be found by direct sequencing of DNA samples collected from different individuals or by comparing sequences from different DNA samples. With the rapid accumulation of sequence data in public and private databases, one also finds SNPs by comparing sequences using computer programs (Z. Gu, L. Hillier, and P. Y. Kwok, “Single nucleotide polymorphism hunting in cyberspace,” Hum. Mutat .1998;12(4):221-225). SNPs can be verified and the genotype or haplotype of an individual can be determined by a variety of methods including direct sequencing and high-throughput microarrays (P. Y. Kwok, “Methods for genotyping single nucleotide polymorphisms,” Annu. Rev. Genomics Hum. Genet. 2001; 2 :235-258; M. Kokoris, K. Dix, K. Moynihan, J. Mathis, B. Erwin, P. Grass, B. Hines, and A. Duesterhoeft, “High-throughput SNP genotyping with the Masscode system,” Mol Diagn 2000 Dec;5(4):329-340).

Using the methods described above, thirteen SNPs were identified in the PSCA v.2 transcript. Variant 2 was used, rather than, for example, variant 1, as it had fewer ambiguous bases than variant 1. Accordingly, SNPs were identified in PSCA v.2, at positions 57 (t/c), 367 (c/t), 424 (a/c), 495 (c/g), 499 (c/t), 563 (c/t), 567 (g/a), 627 (g/a), 634 (t/g), 835 (g/a), 847 (g/a), 878 (g/a) and 978 (c/g). The transcripts or proteins with alternative alleles were designated variant PSCA v.6 through v.18, as shown in Figure 1B and Figure 1G.

The nucleotide change in v.6 changed the start codon of v.1 and consequently translation would not start until the next ATG (AUG in mRNA), resulting in a protein 9 AA shorter than v.1 protein. The nucleotide changes for v.7 and v.8 were silent at the protein level.

Twelve of these 13 SNPs were also present in variant 4. The 12 SNP variants relative to PSCA v.4 are designated PSCA v.19 through v.30.

Variants 19 through 27 code for alternative amino acids as shown in Figure 1H.

Example 4

Production of recombinant PSCA in prokaryotic systems

To express recombinant PSCA and PSCA variants in prokaryotic cells, full-length or partial-length PSCA and PSCA variant cDNA sequences are cloned into any of a variety of expression vectors known in the art. One or more of the following regions of PSCA variants are expressed: the full-length sequence presented in Figure 1 or any 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive amino acids from PSCA, variants or analogues thereof.

A. In vitro transcription and translation constructs:

pCRII: To generate PSCA sense and antisense RNA probes for RNA in situ studies, pCRII constructs (Invitrogen, Carlsbad CA) are prepared encoding either whole or fragments of PSCA cDNA. The pCRII vector has the Sp6 and T7 promoters flanking the insert to drive the transcription of 1 PSCA RNA for use as probes in RNA in situ hybridization experiments. These probes are used to analyze cell and tissue expression of PSCA 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 constructs:

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

pMAL constructs: To produce, in bacteria, recombinant PSCA proteins fused to maltose binding protein (MBP), all or part of the PSCA cDNA protein coding sequence is fused to the MBP gene by cloning into pMAL-c2X and pMAL -p2X vectors (New England Biolabs, Beverly, MA).

These constructs allow controlled expression of recombinant PSCA protein sequences with MBP fused at the amino-terminal end and a 6X His epitope tag at the carboxy-terminal end. MBP and 6X His-tags allow purification of the recombinant protein from induced bacteria with the appropriate affinity matrix and allow recognition of the fusion protein by anti-MBP and anti-His antibodies. The 6X His epitope tag is formed by adding 6 histidine codons to the 3' cloning primer. A Factor Xa recognition site allows cleavage of the pMAL tag from PSCA. pMAL-c2X and pMAL-p2X vectors are optimized to express the recombinant protein in the cytoplasm or periplasm, respectively. Periplasmic expression improves folding of proteins with disulfide bonds. pET constructs: To express PSCA in bacterial cells, all or part of the PSCA cDNA protein coding sequence is cloned into the pET family of vectors (Novagen, Madison, WI). These vectors allow tightly controlled expression of recombinant PSCA protein in bacteria with and without fusion to proteins that promote solubility, such as NusA and thioredoxin (Trx) and epitope tags, such as 6X His and S-Tag™ that aid in purification and detection of the recombinant protein. For example, constructs are made using the pET NusA fusion system 43.1 so that regions of the PSCA protein are expressed as amino-terminal fusions to NusA.

C. Yeast constructs:

pESC constructs: To express PSCA in the yeast species Saccharomyces cerevisiae for recombinant protein production and functional studies, all or part of the PSCA cDNA protein coding sequence is cloned into the pESC family of vectors each containing 1 of 4 selectable markers, HIS3 , TRP1, LEU2, and URA3 (Stratagene, La Jolla, CA). These vectors allow controlled expression from the same plasmid of up to 2 different genes or cloned sequences containing either Flag™ or Myc epitope tags in the same yeast cell. This system is applicable to confirm protein-protein interactions of PSCA. In addition, expression in yeast produces similar post-translational modifications, such as glycosylations and phosphorylations, as those found when expressed in eukaryotic cells.

pESP constructs: To express PSCA in the yeast species Saccharomyces pombe, all or part of the PSCA cDNA protein coding sequence was cloned into the pESP family of vectors. These vectors allow controlled high level expression of a PSCA protein sequence fused at either the amino-terminal or at the carboxy-terminal end of GST which aids in purification of the recombinant protein. A Flag™ epitope tag allows detection of the recombinant protein with anti-Flag™ antibody.

Example 5

Production of recombinant PSCA in higher eukaryotic systems

A. Mammalian constructs:

To express recombinant PSCA in eukaryotic cells, full-length or partial-length PSCA cDNA sequences or variants thereof can 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 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 consecutive amino acids from PSCA v.1, PSCA variants or analogs thereof.

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

pcDNA4/HisMax Constructs: To express PSCA in mammalian cells, a PSCA ORF, or portions thereof, of PSCA is cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter and the SP16 translational enhancer. The recombinant protein has XpressTM and six histidine (6X His) epitopes fused to the amino-terminal end. The pcDNA4/HisMax vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to improve mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The zeocin resistance gene allows selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin allows selection and maintenance of the plasmid in E. coli.

pcDNA3,1/MycHis constructs: To express PSCA in mammalian cells, a PSCA ORF or parts thereof, of PSCA with a consensus Kozak translation initiation site was cloned into pcDNA3,1/MycHis Version A (Invitrogen, Carlsbad, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the myc epitope and the 6X His epitope fused to the carboxy-terminal end. The pcDNA3,1/MycHis vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to improve mRNA stability, along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The neomycin resistance gene can be used, as it allows selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin allows selection and maintenance of the plasmid in E. coli.

pcDNA3,1/CT-GFP-TOPO construct: To express PSCA in mammalian cells and to allow detection of the recombinant proteins using fluorescence, a PSCA ORF or parts thereof, with a consensus Kozak translation initiation site is cloned into pcDNA3 ,1/CT-GFP-TOPO (Invitrogen, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have Green Fluorescent Protein (GFP) fused to the carboxy terminus which facilitates non-invasive, in vivo detection and cell biology studies. The pcDNA3,1CT-GFP-TOPO vector also contains the bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to enhance mRNA stability along with the SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen. The neomycin resistance gene allows selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin allows selection and maintenance of the plasmid in E. coli. Additional constructs with an amino-terminal GFP fusion are made in pcDNA3.1/NT-GFP-TOPO spanning the full length of a PSCA protein.

PAPtag: A PSCA ORF or part thereof is cloned into pAPtag-5 (GenHunter Corp. Nashville, TN). This construct generates an alkaline phosphatase fusion at the carboxy-terminal end of a PSCA protein while fusing the IgGκ signal sequence to the amino-terminal end. Constructs are also made in which alkaline phosphatase with an amino-terminal IgGκ signal sequence is fused to the amino-terminal end 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 driven from the CMV promoter and the recombinant proteins also contain myc and 6X His epitopes fused at the carboxy-terminal end which facilitates detection and purification. The zeocin resistance gene present in the vector allows selection of mammalian cells expressing the recombinant protein and the ampicillin resistance gene allows selection of the plasmid in E. coli.

ptag5: A PSCA ORF or part thereof was cloned into pTag-5. This vector is similar to pAPtag but without the alkaline phosphatase fusion. This construct generates PSCA protein with an amino-terminal IgGκ signal sequence and myc and 6X His epitope tags at the carboxy terminus that facilitate 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 or receptors that interact with the PSCA proteins.

Protein expression is driven from the CMV promoter. The zeocin resistance gene present in the vector allows 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 parts thereof, was cloned into psecFc. The psecFc vector was assembled by cloning the human immunoglobulin G1 (IgG) Fc (hinge, CH2, CH3 regions) into pSecTag2 (Invitrogen, California). This construct generates an IgG1 Fc fusion at the carboxy-terminal end of the PSCA proteins, while fusing the IgGK signal sequence to the N-terminus. PSCA fusions using the murine IgG1 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 or receptors that interact with PSCA protein. Protein expression is driven from the CMV promoter. The hygromycin resistance gene present in the vector allows selection of mammalian cells expressing the recombinant protein and the ampicillin resistance gene allows selection of the plasmid in E. coli.

Figure 8 shows expression and purification of PSCA.psecFc protein from 293T cells.

pSRα constructs: To prepare mammalian cell lines constitutively expressing PSCA, the PSCA ORF or parts thereof, of PSCA were cloned into pSRα constructs. Amphotropic and ecotropic retroviruses were generated by transfection of pSRα constructs into the 293T-10A1 packaging line or cotransfection of pSRα and a helper plasmid (containing deleted packaging sequences) into the 293 cells, respectively. The retrovirus is used to infect a variety of mammalian cell lines, resulting in the integration of the cloned gene, PSCA, into the host cell lines. Protein expression is driven from a long terminal repeat (LTR). The neomycin resistance gene present in the vector allows selection of mammalian cells expressing the protein and the ampicillin resistance gene and ColE1 origin allows selection and maintenance of the plasmid in E. coli. The retroviral vectors can then be used for infection and formation of different cell lines using, for example, PC3, NIH 3T3, TsuPr1, 293 or rat-1 cells.

Figure 6 shows expression of PSCA in recombinant murine, rat and human cell lines using the PSCA.pSRα 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 generated by G418 drug selection.

PSCA expression was determined by FACS labeling with 1G8 anti-PSCA MAb (5 µg/ml). Shown is the FACS profile of each cell line showing a fluorescent shift only in the PSCA-infected line indicating cell surface PSCA expression. These lines are applicable in MAb development as immunogens, MAb screening reagents and in functional experiments.

Additional pSRα constructs are prepared that fuse an epitope tag such as the FLAG™ tag to the carboxy-terminal end of PSCA sequences to allow detection using anti-Flag antibodies. For example, the FLAG™ sequence 5' gat tac aag gat gac gac gat aag 3' (SEQ ID NO: 19) is added to the cloning primer at the 3' end of the ORF. Additional pSRα constructs are prepared to produce both amino-terminal and carboxyl-terminal GFP and myc/6X His fusion proteins of the full-length PSCA proteins.

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

Regulated expression systems: To control expression of PSCA in mammalian cells, the coding sequences of PSCA or parts thereof are cloned into regulated mammalian expression systems such as the T-Rex System (Invitrogen), the GeneSwitch System (Invitrogen) and the tightly regulated Ecdyson System ( Stratagen). These systems allow the study of the temporal and concentration-dependent effects of recombinant PSCA. These vectors are then used to control expression of PSCA in different cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

B. Baculovirus expression systems

To produce recombinant PSCA proteins in a baculovirus expression system, the PSCA ORF or parts thereof are cloned into the baculovirus transfer vector pBlueBac 4.5 (Invitrogen), which provides a His-tag at the amino-terminal end. Specifically, pBlueBac-PSCA is cotransfected with helper plasmid pBac-N-Blue (Invitrogen) into SF9 (Spodoptera frugiperda) insect cells to generate recombinant baculovirus (see Invitrogen instruction manual for details). Baculovirus is then collected from cell supernatant and purified by plaque assay.

Recombinant PSCA protein is then produced by infection of HighFive insect cells (Invitrogen) with purified baculovirus. Recombinant PSCA protein can be detected using anti-PSCA or anti-His tag antibody. PSCA protein can be purified and used in various cell-based experiments or as an immunogen to generate polyclonal and monoclonal antibodies specific for PSCA.

C. Expression vectors for PSCA orthologs

Mouse and monkey orthologs of PSCA were cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, CA). Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the myc epitope and the 6X His epitope fused to the carboxy-terminal end. These vectors allow expression of PSCA orthologs to analyze cross-reactivity of monoclonal anti-human PSCA antibodies.

Mouse and monkey orthologs of PSCA were also cloned into pSRα constructs. The pSRα constructs allowed generation of mammalian cell lines constitutively expressing PSCA orthologs. Protein expression is driven from the cytomegalovirus (CMV) promoter. The recombinant proteins have the myc epitope and the 6X His epitope fused to the carboxy-terminal end. These vectors allow the expression of PSCA orthologs to analyze the cross-reactivity of monoclonal anti-human PSCA antibodies and to study the functional activity of PSCA orthologs.

Amphotropic and ecotropic retroviruses were generated by transfection of pSRα constructs into the 293T-10A1 packaging line or co-transfection of pSRα and a helper plasmid (containing deleted packaging sequences) into the 293 cells, respectively. The retrovirus is used to infect a variety of mammalian cell lines, resulting in the integration of the cloned gene, the PSCA ortholog, into the host cell lines.

Figure 7 shows expression of mouse and simian PSCA.pcDNA3,1/MycHis after transfection into 293T cells. 293T cells were transfected with either mouse PSCA.pcDNA3,1/MycHis or simian PSCA.pcDNA3,1/MycHis or pcDNA3, 1/MycHis vector control. Forty hours later, cells were collected and analyzed by flow cytometry using anti-PCSA monoclonal antibodies.

Example 6

Antigenicity profiles and secondary structure

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

These profiles: Hydrophilicity, (Hopp T.P., Woods K.R., 1981. Proc. Natl. Acad. Sci. U.S.A.78:3824-3828); Hydropathicity, (Kyte J., Doolittle R.F., 1982. J. Mol. Biol. 157:105-132); Percent available residues (Janin J., 1979 Nature 277:491-492); Average flexibility, (Bhaskaran R. and Ponnuswamy P.K., 1988. Int. J. Pept. Protein Res.32:242-255); Beta-turn (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294); and possibly others available in the area, such as on the ProtScale web page, were used to identify antigenic regions of each of the PSCA variant proteins. Each of the above amino acid profiles for PSCA variants was generated using the following ProtScale parameters for analysis: 1) A window size of 9; 2) 100% weight of window edges compared to window center; and, 3) amino acid profile values normalized to lie between 0 and 1.

Hydrophilicity, Hydropathicity and Percent Available Residues profiles were used to determine stretches of hydrophilic amino acids (ie, values greater than 0.5 on the Hydrophilicity and Percent Available Residues profile and values less than 0.5 in the Hydropathicity profile). Such regions are probably exposed to the aqueous environment, are present on the surface of the protein and thus available for immune recognition, such as with antibodies.

Average flexibility and Beta-turn profiles determine stretches of amino acids (ie, values greater than 0.5 on the Beta-turn profile and Average flexibility profile) that are not constrained into secondary structures such as beta-sheets and alpha-helices. Such regions are also more likely to be exposed in the protein and thus available for immune recognition, such as with antibodies.

Antigenic sequences of the PSCA variant proteins indicated, e.g., by the profiles described above, are used to prepare immunogens, either peptides or nucleic acids encoding them, to generate therapeutic and diagnostic anti-PSCA antibodies. The immunogen can be any 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more than 50 consecutive amino acids or the corresponding nucleic acids encoding them, from the PSCA protein variants listed in Figure 1 from which the amino acid profiles can be deduced because the variant contains sequence similar to a variant shown.

In particular, peptide immunogens according to the invention can include, a peptide region of at least 5 amino acids according to Figure 1 in any integer increment that includes an amino acid position that has 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 comprising an amino acid position having a value less than 0.5 in the Hydropathicity profile; a peptide region of at least 5 amino acids of Figure 1 in any integer increment that includes an amino acid position that has a value greater than 0.5 in the Percent Available Residues profiles; a peptide region of at least 5 amino acids of Figure 1 in any integer increment that includes an amino acid position that has a value greater than 0.5 in the Average Flexibility profiles; and, 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 Beta-turn profile. Peptide immunogens according to the invention may also comprise nucleic acids which code for any of the foregoing.

All immunogens according to the invention, peptide or nucleic acid, can be included in human unit dose form or included in a mixture that includes a pharmaceutical additive compatible with human physiology.

The secondary structure of PSCA variants 1, 3, 4 and 6, i.e. the putative presence and localization of alpha-helices, extended strands and random coils, is predicted from the primary amino acid sequence using the HNN -Hierarchical Neural Network method (NPS@ : Network Protein Sequence Analysis TIBS 2000 March Vol.25, No 3 [291]:147-150 Combet C., Blanchet C., Geourjon C. and Deléage G., http://pbil.ibcp.fr/cgibin/npsa_automat .pl?page=npsa_nn.html), available from the ExPasy molecular biology server located in the World Wide Web at (.expasy.ch/tools/). The analysis indicates that PSCA variant 1 is composed of 30.89% alpha helix, 21.95% extended strand and 47.15% random coil. PSCA protein variant 3 is composed of 14.89% alpha helix, 8.51% extended strand and 76.60% random coil. PSCA protein variant 4 is composed of 9.52% alpha helix, 8.99% extended strand and 81.48% random coil. PSCA protein variant 6 is composed of 24.56% alpha helix, 21.93% extended strand and 53.51% random coil.

Analysis for the potential presence of transmembrane domains in the PSCA variant proteins was performed using a number of transmembrane prediction algorithms available from the ExPasy molecular biology server located in the World Wide Web at (.expasy.ch/tools/).

Example 7

Preparation of PSCA polyclonal antibodies

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

Typically, the immunizing agent and/or adjuvant will be injected into the mammal by multiple subcutaneous or intraperitoneal injections. In addition to immunizing with a full-length PSCA protein variant, computer algorithms are used in the design of immunogens that, based on amino acid sequence analysis, contain features to be antigenic and available for recognition by the immune system of the immunized host (see the example labeled “Antigenicity Profiles and Secondary Structure”). Such regions would be predicted to be hydrophilic, flexible, in beta-turn conformations and to be exposed on the surface of the protein.

For example, recombinant bacterial fusion proteins or peptides containing hydrophilic, flexible, beta-turn 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 "Generation of PSCA Monoclonal Antibodies ( MAbs)”. 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 being immunized. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. In one embodiment, a peptide encoding amino acids 103-133 of PSCA variant 4 is conjugated to KLH and used to immunize a rabbit. Alternatively, the immunizing agent may comprise all or parts of the PSCA variant proteins, analogues or fusion proteins thereof. For example, the PSCA variant amino acid sequences can be fused using recombinant DNA techniques to any of a number of fusion protein partners well known in the art, such as glutathione S-transferase (GST) and HIS-tagged fusion proteins. In one embodiment, the PSCA variant 1 sequence, amino acids 18-98 was fused to GST using recombinant techniques in the pGEX expression vector, expressed, purified and used to immunize both rabbits and mice to generate polyclonal and monoclonal antibodies, respectively.

Such fusion proteins are purified from induced bacteria using appropriate affinity matrices.

Other recombinant bacterial fusion proteins that can be used include maltose binding protein, LacZ, thioredoxin, NusA or an immunoglobulin constant region (see the section entitled “Production of PSCA in prokaryotic systems” and Current Protocols in Molecular Biology, Volume 2, Unit 16, Frederick M. Ausubul et al. eds., 1995; Linsley, P.S., Brady, W., Urnes, M., Grosmaire, L., Damle, N. and Ledbetter, L. (1991) J. Exp. Med. 174, 561-566 ).

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

During the immunization protocol, it is useful to mix or emulsify the antigen in adjuvants that promote the immune response in the host animal.

Examples of adjuvants include, but are not limited to, complete Freund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).

In a typical procedure, rabbits are initially immunized subcutaneously with up to 200 µg, typically 100-200 µg, of fusion protein or peptide conjugated to KLH mixed in complete Freund's adjuvant (CFA). Rabbits are then injected subcutaneously every other week with up to 200 µg, typically 100-200 µg, of the immunogen in incomplete Freund's adjuvant (IFA). Test swabs are taken approximately 7-10 days after each immunization and 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 GST fusion of PSCA variant 3 or 4 protein, the respective full-length PSCA variant cDNA is cloned into the pCDNA 3.1 myc-his expression vector ( Invitrogen, see the example entitled “Production of Recombinant PSCA in Eukaryotic Systems”). After transfection of the constructs into 293T cells, cell lysates are probed with anti-variant serum and with anti-His antibody (Santa Cruz Biotechnologies) to determine specific reactivity of denatured variant protein using the Western blot technique. In addition, immune sera are tested by fluorescence microscopy, flow cytometry and immunoprecipitation against 293T and other recombinant PSCA variant-expressing cells to determine specific recognition of native protein. Western blot, immunoprecipitation, fluorescence microscopy and flow cytometry techniques using cells endogenously expressing PSCA are also performed to test reactivity and specificity.

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

Example 8

Preparation of PCSA monoclonal antibodies (MAbs)

In one embodiment, therapeutic monoclonal antibodies (“MAbs”) against PSCA and PSCA variants include those that react with epitopes specific to each protein or specific to sequences common to the variants that would bind, internalize, disrupt or modulate the biological function of PSCA or PSCA -variants, for example those that would disrupt the interaction with ligands, substrates and binding partners. Immunogens for generation of such MAbs include those designed to encode or contain the extracellular domain or the entire PSCA protein sequence, regions predicted to contain functional motifs and regions of the PSCA protein variants predicted to be antigenic from data analysis of the amino acid sequence. Immunogens include peptides, recombinant bacterial proteins such as GST-PSCA fusion proteins (Figure 8) and His-tagged PSCA pET vector protein (Figure 6) and mammalian expressed purified His-tagged proteins (Figure 7) and human and murine IgG FC fusion proteins. In addition, cells are engineered through retroviral transduction to express high levels of PSCA variant 1, such as RAT1-PSCA. 293T-PSCA, 3T3-PSCA or 300.19-PSCA used to immunize mice (Figure 5).

To generate MAbs against PSCA, mice are first immunized in the footpad (FP) with, typically, 5-50 µg of protein immunogen or between 10<6> and 10<7>1 PSCA-expressing cells mixed in a suitable adjuvant. Examples of suitable adjuvants for FP immunizations are TiterMax (Sigma) for the initial FP injection and alum gel with Immuneasy (Qiagen). After an initial injection, mice were then immunized twice per week until the time they were killed and B cells obtained from the lymph node used for fusion.

During the immunization protocol, test swabs are taken to monitor the titer and specificity of the immune response. In most cases, once appropriate reactivity and specificity are achieved as determined by ELISA, Western blotting, immunoprecipitation, fluorescence microscopy or flow cytometry analyses, fusion and hybridoma formation are then performed using electrocell fusion (BTX, ECM2000).

In one embodiment, the invention provides monoclonal antibodies designated H1-1.10. The antibodies were identified and shown to react and bind to cell surface or immobilized PSCA.

MAbs against PSCA were generated using XenoMouse technology<® >in which the murine heavy and kappa light chain loci have been inactivated and most of the human heavy and kappa light chain immunoglobulin loci have been inserted. H1-1.10 was generated after immunization of human gamma 1-producing Xenomus with a cell-based immunization using B300.19/PSCA cells. Anti-PSCA MAbs, H1-1.10 bind endogenous cell surface PSCA expressed in prostate cancer xenograft cells.

The antibodies designated H1-1.10 were sent (via Federal Express) to the American Type Culture Collection (ATCC), P.O. Box 1549, Manassas, VA 20108 on May 4, 2005 and assigned Accession No. PTA-6697.

DNA coding sequences for anti PSCA MAb H1-1.10 were determined after isolation of mRNA from the respective hybridoma cells with Trizol reagent (Life Technologies, Gibco BRL). Total RNA was purified and quantified. First strand cDNAs were generated from total RNA with oligo (dT)1218 priming using the Gibco BRL Superscript Preamplification system. First strand cDNA was amplified using human immunoglobulin variable heavy chain primers and human immunoglobulin variable light chain primers. PCR products were cloned into pCRScript vector (Stratagene, La Jolla). Many clones were sequenced and the variable heavy and light chain regions determined. The nucleic acid and amino acid sequences of the variable heavy and light chain regions are listed in Figures 2A and 2B and Figures 3A and 3B. Compilation of PSCA H1-1.10 antibodies with germ cell V-D-J sequences is shown in Figures 4A and 4B.

Example 9

Screening and identification of PSCA antibodies

Antibodies generated using the methods set forth in the example designated “Generation of PSCA Monoclonal Antibodies (MAbs)” were initially screened using F-MAT and then screened and identified using a combination of assays including ELISA, FACS, epitope clustering and affinity to PSCA expressed on the cell surface.

A. PSCA human MAb screening by FACS.

Primary hybridoma screening for MAbs for FACS was performed by FACS.

The procedure is as follows: 50 µl/well of hybridoma supernatant (neat) or purified antibodies (in serial dilutions) are added to 96-well FACS plates and mixed with FACS-expressing cells (endogenous or recombinant, 50,000 cells/well) . The mixture is incubated at 4ºC for two hours. At the end of incubation, the cells are washed with FACS buffer and incubated with 100 µl detection antibody (anti-hIgG-PE) for 45 minutes at 4ºC. At the end of the incubation, the cells are washed with FACS buffer, fixed with Formaldehyde and analyzed using FACScan. Data are analyzed using CellQuest Pro software.

Filled histograms indicate data with a negative control IgG1 and open histograms represent data from PSCA-positive cells (Figure 9).

Positive hybridomas identified from primary screens are transferred to 24-well plates and supernatants collected for confirmatory screens.

Confirmatory screenings included FACS analysis for B300,19-PSCA/300,19-neo, Rat1-PSCA/Rat1-neo, PC3-PSCA/neo, SW780 (bladder cancer cell line), LAPC9AI (prostate cancer cell line), HPAC (pancreatic cancer cell line) and ELISA assays using Tag5-PSCA, GST-PSCA, GST-PSCA N-term, Med. C-Term and pET-PSCA.

B. PSCA H1-1.10 human MAb relative affinity assay

The hybridoma supernatants were tested to determine their relative binding affinity to cell surface PSCA. Hybridoma supernatants were serially diluted in FACS buffer (FB), from µg/ml to sub-ng/ml; and evaluated in a FACS binding assay using LAPC9AI cells. High affinity antibodies gave high MFI values. MFI values for each point were obtained using CellQuest Pro software and used for affinity calculation using Graphpad Prism software (Table VII and Table VIII): Sigmoidal Dose-Response (variable slope) equation. The results of the relative affinity assay are set forth in Table IX.

B. Epitope grouping

C. PSCA antibodies were grouped according to epitope by judging their binding pattern on LAPC9AI cells. Briefly, a small amount of each of the antibodies was biotinylated; then each of the biotinylated antibodies was incubated with LAPC9AI in the presence of excess (100x) amount of non-biotinylated antibodies at 4°C for 1 hour during the incubation. In general, an excess amount of antibodies will compete with biotinylated antibodies if they bind to the same epitope. At the end of incubation, 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 is background control for each biotinylated antibody. Cells without color indicate that the two antibodies compete with each other (low MFI), high MFI (highlighted in blue) indicates that the two antibodies bind to two distinct epitopes. The antibodies that have the same binding pattern bind to the same epitope among the antibodies. There are 6 epitope groups within the antibodies tested. Table XI shows that PSCA H1-1.10 binds to its unique epitope.

Example 10

Characterization and expression of PSCA antibodies

D. Cross-reactivity with monkey PSCA and mouse PSCA

MAbs were screened and characterized for their ability to react with PSCA of mouse and monkey origin. This property is useful to understand the consequences of MAb engagement of PSCA for cells and tissues using mouse and monkey animal models. The cynomolgus monkey and mouse PSCA genes were cloned, expressed in a retrovirus, and transiently infected into 293-T cells. Test antibodies were incubated with either monkey or mouse PSCA-expressing 293-T cells. 293T-neo were used as negative controls. Bound antibodies were detected with anti-hIgG-PE detection antibody. The results presented in Table X show that PSCA MAb H1-1.10 does not cross-react with monkey-PSCA or mouse-PSCA.

Example 11

PSCA antibody internalization

Internalization of H1-1.10 was studied using PC3-PSCA cells. Briefly, H1-1.10 was incubated with cells at 4°C 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 either 37°C to allow antibody internalization or at 4°C as a control (no internalization). An acid wash after 37°C/4°C incubation was used to remove PSCA 4,121 bound on the cell surface. Subsequent permeablization allowed detection of antibodies bound to internalized PSCA. After incubation with secondary detection antibodies, cells were analyzed using FACS or observed under a fluorescence microscope. Approximately 30% of H1-1.10 was internalized after incubation at 37°C for two hours (Figure 10).

Example 12

Antibody-mediated killing

PSCA H1-1.10 MAb mediates killing in PC-3/PSCA cells when co-incubated with a conjugated secondary antibody. PC-3 cells engineered to express either the neomycin resistance gene (neo) or PSCA were plated in triplicate into 96-well tissue culture plates. After allowing the cells to adhere overnight, medium was removed and replaced with fresh medium containing the indicated concentrations of anti-PSCA MAb H1-1.10 or H1-1.10 together with a 3-fold excess of the indicated saporin-conjugated secondary antibody ( Advanced Targeting systems, San Diego, CA). The cells were incubated for 4 days and percent viability determined using an MTT assay (Promega Corp). The PSCA antibody H1-1.10 was specific as it did not mediate killing of cells not expressing PSCA (Panel A). H1-1.10 mediated cell killing when incubated with a secondary antibody recognizing the human Fc region but not with a secondary antibody recognizing the Fc region of a goat antibody (Figure 11 - Panel B). These results indicate that drugs or cytotoxic proteins can be selectively delivered to PSCA-expressing cells using an appropriate anti-PSCA MAb.

In addition, PSCA H1-1.10 MAb mediates killing in LNCaP/PSCA cells coincubated with a conjugated secondary antibody. LNCap cells engineered to express either the neomycin resistance gene (neo) or PSCA were plated in triplicate in 96-well tissue culture plates. After allowing cells to adhere overnight, medium was removed and replaced with fresh medium containing the indicated concentrations of anti-PSCA MAb H1-1.10 or H1-1.10 together with a 3-fold excess of anti-human or anti-human saporin -conjugated secondary antibody (Advanced Targeting systems, San Diego, CA). The cells were incubated for 4 days and percent viability determined using an MTT assay (Promega Corp). The PSCA antibody H1-1.10 was specific as it did not mediate killing of LNCap cells that do not express PSCA (Figure 12).

Example 13

Antibody-mediated immune cytotoxicity

PSCA antibodies were evaluated to determine their ability to mediate immune-dependent cytotoxicity. . H1-1.10 (0–50 µg/ml) was diluted with RHB buffer (RPMI 1640, Gibco Life Technologies, 20 mM HEPES). B300.19-PSCA-expressing cells were washed in RHB buffer and resuspended at a density of 10<6 >cells/ml. In a typical assay, 50 µl of PSCA antibody, 50 µl of diluted rabbit complement serum (Cedarlan, Ontario, Can) and 50 µl of a cell suspension were added together into a flat-bottomed tissue culture 96-well plate. The mixture was incubated for 2 hours. at 37°C in a 5% CO2 incubator to facilitate complement-mediated cell lysis.

50 µl of Alamar Blue (Biosource Intl. Camarillo, CA) was added to each well and incubation continued for an additional 4-5 hours at 37°C. The 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 H1-1.10, a human Ig1, was able to mediate complement-dependent lysis of target cells (Figure 13).

ADCC (Antibody-Dependent Cellular Cytotoxicity) is an immune-mediated lytic attack on cells bound by an antibody targeting a specific cell surface antigen. Immune cells recognize the Fc part of the antibody through binding to Fcγ receptors on the surface of leukocytes, monocytes and NK cells, which triggers a lytic attack resulting in cell death. In general, Panc0203 cells are incubated in vitro with <51>chromium for 1 hour. After washing with fresh medium, the labeled cells are incubated with 2.5 μg/ml human PSCA MAbs and freshly isolated mononuclear cells from peripheral blood at different effector to target cell ratios (E:T ratios). After 4 hours at 37C, the cells are carefully centrifuged and the supernatant containing <51>Cr released from the cells is counted in a Beta counter.

The results show that mediated antibody-dependent cell killing increases when the effector to target cell ratio increases. The specificity of the assay is determined by showing that an IgG1 control MAb and incubation of target and effector cells in the absence of antibody did not cause cell killing.

Example 14

Preparation of F(Ab')2 fragments

Preparation of F(Ab')2 fragments of MAbs is useful for investigating the effects of MAb molecules that retain their bivalent antigen binding site but lack the immune effector Fc domain in in vitro and in vivo therapeutic models. The procedure follows, 20 mg of MAb H1-1.10 in 20 mM sodium acetate buffer pH 4.5 is incubated with and without immobilized pepsin (Pierce. Rockford IL) for the indicated number of times. Intact MAb and cleaved Fc fragments are removed by protein A chromatography. An SDS-PAGE Coomasie-labeled gel of intact uncleaved non-reduced MAb, non-reduced aliquots of cleaved material taken at the indicated time points and a reduced sample of the final cleaved F(ab')2 product is observed. This reagent can be used to treat animals that have PSCA-expressing tumors. The antitumor activity observed with this antibody fragment can distinguish intrinsic biological activity from activity mediated by immune-dependent 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 human heavy chain IgG1 and light chain Igκ constant regions, respectively. The complete anti-PSCA human heavy chain and light chain cassettes were cloned downstream of the CMV promoter/enhancer in a cloning vector. A polyadenylation site was included downstream of the MAb coding sequence. The recombinant anti-PSCA MA expressing constructs were transfected into 293T, Cos and CHO cells. The H1-1.10 antibody secreted from recombinant 293-T cells is evaluated for binding to the cell surface of PSCA and can then be compared to the same antibody produced from the original hybridoma.

Example 16

HLA class I and class II binding assays

HLA class I and class II binding assays using purified HLA molecules are performed according to described protocols (eg, PCT publications WO 94/20127 and WO 94/03205; Sidney et al., Current Protocols in Immunology 18 ,3,1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); Sette, et al., Mol.

Immunol. 31:813 (1994)). Briefly, purified MHC molecules (5 to 500 nM) are incubated with various unlabeled peptide inhibitors and 1-10 nM <125>I-radiolabeled probe peptides as described. After incubation, MHC-peptide complexes are separated from free peptide by gel filtration and the fraction of peptide bound is determined. Typically, in preliminary experiments, each MHC preparation is 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 experiments are performed using these HLA concentrations.

Since under these conditions [label]<[HLA] and IC50≥[HLA], the measured IC50 values are reasonable estimates of the correct KD values. Peptide inhibitors are typically tested at concentrations ranging from 120 µg/ml to 1.2 ng/ml and are tested in two to four completely independent experiments. To allow comparison of the data obtained in different experiments, a relative binding number is calculated for each peptide by dividing the IC50 of a positive control for inhibition by the IC50 of each tested peptide (typically unlabelled versions of the radiolabelled probe peptide). For database purposes and inter-experiment comparisons, relative binding values are compiled. These values can then be converted back to IC50 nM values by dividing the IC50 nM of the positive controls for inhibition by the relative binding of the peptide of interest. This method of data compilation is accurate and consistent for comparison of peptides tested on different days or with different lots of purified MHC.

Binding experiments as described above can be used to analyze HLA supermotif and/or HLA motif-bearing peptides (see Table IV).

Example 17

Construction of “Minigene” multi-epitope DNA plasmids

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

A minigene expression plasmid typically comprises multiple CTL and HTL peptide epitopes. In the present example, HLA-A2, -A3, -B7 supermotif-bearing peptide epitopes and HLA-A1 and -A24 motif-bearing peptide epitopes are used together with DR supermotif-bearing epitopes and/or DR3 epitopes. HLA class I supermotif or motif-bearing peptide epitope derived PSCA, is selected so that multiple supermotifs/motifs are represented to ensure broad population coverage. Similarly, HLA class II epitopes are selected from PSCA to provide broad population coverage, i.e. both HLA DR-1-4-7 supermotif-bearing epitopes and HLA DR-3 motif-bearing epitopes are selected for inclusion in the minigene construct. The selected CTL and HTL epitopes are then inserted into a minigene for expression in an expression vector.

Such a construction can additionally include sequences that direct the HTL epitopes to the endoplasmic reticulum. For example, the Ii protein can be fused to one or more HTL epitopes as described in the site, where the CLIP sequence of the Ii protein is removed and replaced with an HLA class II epitope sequence 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-carrying expression plasmid. Other expression vectors that can be used for minigene mixtures are available and known to those skilled in the art.

The minigene DNA plasmid of this example contains a consensus Kozak sequence and a consensus murine kappa Ig light chain signal sequence followed by CTL and/or HTL epitopes selected according to principles described herein.

The sequence encodes an open reading frame fused to the Myc and His antibody epitope tag encoded by the pcDNA 3.1 Myc-His vector.

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

For example, a minigene is produced as follows. For a first PCR reaction, 5 µg of each of two oligonucleotides are hybridized and extended: In an example using eight oligonucleotides, i.e., four pairs of primers, the oligonucleotides 1+2, 3+4, 5+6 and 7 +8 collected in 100 µl reactions containing Pfu polymerase buffer (1x= 10 mM KCL, 10 mM (NH4)2SO4, 20 mM Tris chloride, pH 8.75, 2 mM MgSO4, 0.1% Triton X-100, 100 µg/ml BSA), 0.25 mM each dNTP and 2.5 U Pfu polymerase. The full-length dimer products are gel-purified and two reactions containing the product of 1+2 and 3+4 and the product of 5+6 and 7+8 are mixed, hybridized and extended for 10 cycles. Half of the two reactions are then mixed and 5 cycles of hybridization and extension performed before flanking primers are added to amplify the full-length product. The full-length product is gel-purified and cloned into pCR-blunt (Invitrogen) and individual clones are screened by sequencing.

Example 18

Plasmid construction and the extent to which it 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 epitope presentation by APC after transduction or transfection of APC with an epitope-expressing nucleic acid construct. Such a study determines "antigenicity" and allows the use of human APC. The assay determines the ability of the epitope to be presented by APC in a context recognized by a T cell by quantifying the density of epitope-HLA class I complexes on the cell surface.

Quantification can be performed by direct measurement of the amount of peptide eluted from the APC (see, e.g., Sijts et al., J. Immunol. 156:683-692, 1996; Demotz et al., Nature 342:682-684, 1989 ); or the number of peptide-HLA class I complexes can be calculated by measuring the amount of lysis or lymphokine release induced by diseased or transfected target cells and then determining the concentration of peptide necessary to achieve equivalent levels of lysis or lymphokine release (see, e.g., Kageyama et al., J. Immunol. 154:567-576, 1995).

Alternatively, immunogenicity is confirmed through in vivo injections into mice and subsequent in vitro assessment of CTL and HTL activity, which is assayed using cytotoxicity and proliferation assays, respectively, as detailed, e.g., in Alexander et al., Immunity 1:751-761, 1994.

For example, to confirm the capacity of a DNA minigene construct containing at least one HLA-A2 supermotif peptide to induce CTLs in vivo, HLA-A2,1/ K<b >transgenic mice, for example, are immunized intramuscularly with 100 µg naked cDNA. As a method for comparing the level of CTLs induced by cDNA immunization, a control group of animals is also immunized with a real peptide mixture comprising multiple epitopes synthesized as a single polypeptide as they would be coded for by the minigene.

Splenocytes from immunized animals are stimulated twice with each of the respective mixtures (peptide epitopes encoded for in the minigene or the polyepitope peptide), then examined 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, thus indicating the in vivo immunogenicity of the minigene vaccine and the polyepitope vaccine.

It has therefore been found that the minigene elicits immune responses directed against the HLA-A2 supermotive peptide epitopes in the same way as the polyepitope peptide vaccine. A similar analysis has also been performed using other HLA-A3 and HLA-B7 transgenic mouse models to assess CTL induction at HLA-A3 and HLA-B7 motif or supermotif epitopes, whereby the minigene has also been found to elicit appropriate immune responses directed against the given epitopes.

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

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

For example, the effectiveness of the DNA minigene used in a prime boost protocol is initially evaluated in transgenic mice. In this example, A2.1/K<b>transgenic mice are immunized IM with 100 µg of a DNA minigene encoding the immunogenic peptides comprising at least one HLA-A2 supermotif-bearing peptide. After an incubation period (ranging from 3-9 weeks), the mouse is boosted IP with 10<7 >pfu/mouse of a recombinant vaccinia virus expressing the same sequence as encoded by the DNA minigene. Control mice are immunized with 100 µg of DNA or recombinant vaccinia without the minigene sequence, or with DNA encoding the minigene but without the vaccinia boost. After a further incubation period of two weeks, splenocytes from the mouse are immediately examined for peptide-specific activity in an ELISPOT assay. In addition, splenocytes are stimulated in vitro with the A2-restricted peptide epitopes encoded in the minigene and recombinant vaccinia, then analyzed for peptide-specific activity in an alpha, beta and/or gamma IFN ELISA.

It has been found that the minigene used in a prime-boost protocol elicits greater immune responses against the HLA-A2 supermotif peptides than with DNA alone. Such analysis can also be performed using HLA-A11 or HLA-B7 transgenic mouse models to assess CTL induction at HLA-A3 or HLA-B7 motif or supermotif epitopes. Application of prime boost protocols to humans is described below in the example entitled “Induction of CTL Responses Using a Prime Boost Protocol.”

Example 19

Polyepitope vaccine mixtures from multiple antigens

The PCSA peptide epitopes of the present invention are used together with epitopes from other target tumor-associated antigens, to prepare a vaccine composition useful for the prevention or treatment of cancers expressing PSCA and such other antigens. For example, a vaccine composition may be administered as a single polypeptide comprising multiple epitopes from PSCA as well as tumor-associated antigens commonly expressed by a target cancer associated with PSCA expression or may be administered as a mixture comprising a cocktail of one or more separate epitopes. Alternatively, the vaccine can be administered as a minigene construct or as dendritic cells that have been loaded with the peptide epitopes in vitro.

Example 20

Application of peptides to assess an immune response

Peptides according to the invention can be used to analyze an immune response for the presence of specific antibodies, CTL or HTL directed against PSCA. Such an analysis can be performed 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, highly sensitive human leukocyte antigen tetrameric complexes (“tetramers”) are used as a cross-sectional analysis of, for example, PSCA HLA-A*0201-specific CTL frequencies from HLA A*0201-positive individuals at different stages of disease or after immunization comprising a PCSA peptide containing an A*0201 motif. Tetrameric complexes are synthesized as described (Musey et al., N. Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201 in this example) and β2-microglobulin are synthesized using a prokaryotic expression system. The heavy chain is modified by deletion of the transmembrane-cytosolic tail and COOH-terminal addition of a sequence containing a BirA enzymatic biotinylation site. The heavy chain, β2-microglobulin and peptide are refolded upon dilution. The 45-kD refolded product is isolated by solid protein liquid chromatography and then biotinylated by BirA in the presence of biotin (Sigma, St. Louis, Missouri), adenosine 5' triphosphate, and magnesium. Streptavidin-phycoerythrin conjugate is added in a 1:4 molar ratio and the tetrameric product is concentrated to 1 mg/ml. The resulting product is referred to as tetrameric phycoerythrin.

For analysis of patient blood samples, approximately one million PBMCs are centrifuged at 300 g for 5 min and resuspended in 50 µl of cold phosphate-buffered saline. Three-color analysis is performed with tetramer-phycoerythrin, together with anti-CD8 three-color and anti-CD38. The PBMCs are incubated with tetramers and antibodies on ice for 30 to 60 min and then washed twice before formaldehyde fixation. Gates are sought to contain >99.98% of control samples. Controls for the tetramers include both A*0201-negative individuals and A*0201-positive non-diseased donors. The percentage of cells labeled with the tetramer is then determined by flow cytometry. The results indicate the number of cells in the PBMC sample that contain epitope-restricted CTLs, thereby easily indicating the magnitude of immune response to the PSCA epitope and consequently the status of exposure to PSCA or exposure to a vaccine that elicits a protective or therapeutic response.

Example 21

Induction of immune responses using a prime boost protocol

A prime boost protocol, in underlying principles similar to that used to confirm the efficacy of a DNA vaccine in transgenic mice, as described above in the example labeled "The plasmid construct and the extent to which it induces immunogenicity," can also be used for the administration of the vaccine for humans. Such a vaccine regimen may comprise an initial administration of, for example, naked DNA followed by a booster using recombinant virus encoding the vaccine or recombinant protein/polypeptide or a peptide mixture administered in an adjuvant.

For example, the initial immunization can be carried out using an expression vector, such as that constructed in the example entitled "Construction of "Minigene" multi-epitope DNA plasmids" in the form of naked nucleic acid administered IM (or SC or ID) in an amount of 0.5-5 mg at multiple sites. The nucleic acid (0.1 to 1000 µg) can also be administered using a gene gun. After an incubation period of 3-4 weeks, a booster dose is then administered. The booster can be recombinant fowlpox virus administered at a dose of 5-10<7 > to 5x10<9 >pfu. An alternative recombinant virus, such as an MVA, canarypox, adenovirus or adeno-associated virus, can also be used as a booster or the polyepitope protein or a mixture of the peptides can be administered. For evaluation of vaccine efficacy, patient blood samples are obtained before immunization as well as at intervals after administration of the initial vaccine and booster doses of the vaccine. Peripheral blood mononuclear cells are isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation, aliquoted into freezing media and stored frozen. Samples are examined for CTL and HTL activity.

Analysis of the results indicates that a magnitude of response sufficient to achieve a therapeutic or protective immunity against PSCA is produced.

Example 22

Complementary polynucleotides

Sequences complementary to the PSCA coding sequences (Figure 1 or Figure 3) or any part thereof, are used to detect, reduce or inhibit expression of naturally occurring PSCA. Although the use of oligonucleotides comprising from approximately 15 to 30 base pairs has been described, essentially the same method is used with smaller (9-mer or 10-mer) or with larger (40-90) sequence fragments. Appropriate oligonucleotides are designed using, e.g., OLIGO 4.06 software (National Biosciences) and the coding sequence of PSCA. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosome binding to a PSCA-encoding transcript.

Example 23

Purification of naturally occurring or recombinant PSCA using PSCA-specific antibodies

Naturally occurring or recombinant PSCA is mainly purified by immunoaffinity chromatography using antibodies specific for PSCA. An immunoaffinity column is constructed by covalently coupling anti-PSCA antibody to 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.

Media containing PSCA is passed over the immunoaffinity column and the column is washed under conditions that allow preferential absorbance of PSCA (eg, high ionic strength buffers in the presence of detergent). The column is eluted under conditions that break antibody/PSCA binding (eg, a buffer of pH 2 to pH 3 or a high concentration of a chaotrope, such as urea or thiocyanation) and the GCR.P is collected.

Example 24

Identification of molecules that interact with PSCA

PSCA or biologically active fragments thereof are labeled with 1211 Bolton-Hunter reagent. (see, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously distributed in the wells of a multi-well plate are incubated with the labeled PSCA, washed and any well with the labeled PSCA complex is analyzed. Data obtained using different concentrations of PSCA are used to calculate values for amount, affinity and association of PSCA with the candidate molecules.

Example 25

In vivo assay for PSCA tumor growth promotion

The effect of the 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 in each flank with 1 x 10<6> of either 3T3 or prostate cancer cell lines (eg PC3 cells) containing tkNeo empty vector or PSCA. At least two strategies can be used: (1) Constitutive PSCA expression under the control of a promoter such as a constitutive promoter obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2) , bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis B virus and Simian Virus 40 (SV40) or from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, provided that such promoters are compatible with the host cell systems and (2) Regulated expression under the control of an inducible vector system, such as ecdysone, tetracycline, etc., provided such promoters are compatible with the host cell systems. Tumor volume is then monitored by krumpasser measurement at the appearance of distinct tumors and followed over time to determine whether PSCA-expressing cells grow at a faster rate and whether tumors produced by PSCA-expressing cells show characteristics of altered aggressiveness (e.g., advanced metastasis, vascularization, reduced susceptibility to chemotherapeutic drugs).

In addition, mice can be implanted with 1 x 10<5> of the same cells orthotopically to determine whether PSCA has an effect on local growth in the prostate and whether PSCA affects the ability of the cells to metastasize (Miki T et al, Oncol Res.2001 ;12:209; Fu X et al, Int J Cancer.1991, 49:938). The effect of PSCA on tumor formation and growth can be assessed by injecting prostate tumor cells intratibially.

The experiment is also applicable to determine the PSCA-inhibiting effect of candidate therapeutic mixtures, such as, for example, PSCA intrabodies, PSCA antisense molecules and ribozymes.

Example 26

PCSA monoclonal antibody-mediated inhibition of tumors in vivo

The significant expression of PSCA on the cell surface of tumor tissue, together with its restrictive expression in normal tissue makes PSCA a good target for antibody therapy. Similarly, PSCA is a target for T-cell-based immunotherapy. Accordingly, the therapeutic efficacy of anti-PSCA MAbs in human prostate cancer xenograft mouse models and human pancreatic cancer xenograft mouse models is being evaluated using recombinant cell lines such as PC3PSCA and 3T3-PSCA (see, e.g., Kaighn, M.E., et al., Invest Urol , 1979.17(1): 16-23), as well as human prostate xenograft models such as LAPC 9AD (Saffran et al PNAS 1999, 10:1073-1078).

Antibody efficacy on tumor growth and metastasis formation is studied, eg, in a mouse orthotopic prostate or pancreatic cancer xenograft model. The antibodies may be unconjugated, as described in this example, or may be conjugated to a therapeutic modality, as understood in the art. Anti-PSCA MAbs inhibit formation of pancreatic, prostate and bladder tumor xenografts. Anti-PSCA Mabs also slow the growth of established orthotopic tumors and prolonged 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 the cancers listed in Table I. (see, e.g., Saffran, D., et al., PNAS 10:1073-1078 or world wide web URL pnas.org/cgi/doi/10,1073/pnas,051624698).

Administration of anti-PSCA Mabs led to retardation of established orthotopic tumor growth and inhibition of metastasis to distant sites, resulting in a significant prolongation of the survival of tumor-bearing mice. These studies indicate that PSCA is an attractive target for immunotherapy and demonstrate the therapeutic potential of anti-PSCA Mabs for the treatment of local and metastatic prostate and pancreatic cancer. This example demonstrates that unconjugated PCSA monoclonal antibodies are effective in inhibiting 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 multiple PSCA MAbs

Materials and methods

PCSA monoclonal antibodies:

Monoclonal antibodies were raised against PSCA as described in the example entitled “Generation of PSCA Monoclonal Antibodies (MAbs).” The antibodies are characterized by ELISA, Western blot, FACS and immunoprecipitation for their capacity 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 tissue and cells with these antibodies is performed.

The monoclonal antibodies are purified from ascites or hybridoma tissue culture supernatants by Protein-G or Protein-A Sepharose chromatography, dialyzed against PBS, filter sterilized and stored at -20°C. Protein determinations are performed by a Bradford assay (Bio-Rad, Hercules, CA). A therapeutic monoclonal antibody or a cocktail comprising a mixture of individual monoclonal antibodies is prepared and used to treat mice receiving subcutaneous or orthotopic 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 fibroblast line NIH 3T3 (American Type Culture Collection) are maintained in RPMI and DMEM respectively, supplemented with L-glutamine and 10% FBS.

PC3-PSCA and 3T3-PSCA cell populations are generated by retroviral gene transfer as described in Hubert, R.S., et al., Proc Natl Acad Sci U S A, 1999. 96(25): 14523.

The LAPC-9 xenograft, which expresses a wild-type androgen receptor and produces prostate-specific antigen (PSA), is passaged into 6- to 8-week-old male ICR severe combined immunodeficiency (SCID) mice (Taconic Farms) by s.c. trocar implant (Craft, N., et al., Nat Med. 1999, 5:280). Single-cell suspensions of LAPC-9 tumor cells are prepared as described in Craft, et al.

Xenograft mouse models.

Subcutaneous (s.c.) tumors are formed by injecting 1 x 10<6> cancer cells mixed at a 1:1 dilution with Matrigel (Collaborative Research) into the right flank of male SCID mice. To test antibody effectiveness on tumor formation, i.e. antibody injections are started on the same day as tumor cell injections. As a control, mice are injected with either purified mouse IgG or PBS; or a purified monoclonal antibody that recognizes an irrelevant antigen not expressed in human cells. Tumor sizes are determined by krumpasser measurements and the tumor volume is calculated as length x width<2 >/ 2. Mice with subcutaneous tumors larger than 1.5 cm in diameter are euthanized.

Orthotopic injections are performed under anesthesia using ketamine/xylazine. For prostate orthotopic studies, an incision is made through the abdomen to expose the prostate and LAPC or PC3 tumor cells (2 x 10<6>) mixed with Matrigel are injected into the prostate capsule in a volume of 10 µl. To monitor tumor growth, mice are palpated and blood is collected on a weekly basis to measure PSA levels. The mice are separated into groups for the appropriate treatments, where anti-PSCA or control MAbs are injected i.p.

Anti-PSCA MAbs inhibit growth of PSCA-expressing xenograft cancer tumors

The effect of anti-PSCA MAbs on tumor formation is tested using HPAC and LAPC9 orthotopic models. Compared with s.c. tumor model, the orthotopic model, which requires injection of tumor cells directly into mouse pancreas or prostate, respectively, results in a local tumor growth, development of metastasis at distant sites, damage to mouse health and subsequent death (Saffran, D., et al., PNAS supra). These features make the orthotopic model more representative of human disease progression and allowed us to follow the therapeutic effect of MAbs on clinically relevant endpoints.

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

A major advantage of the orthotopic cancer models is the ability to study the development of metastases. Formation of metastasis in mice bearing established orthotopic tumors is examined by IHC analysis of lung sections using an antibody against a tumor-specific cell surface protein such as anti-CK19 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 is partly dependent on the development of new blood vessels. Although the capillary system and the developing blood network are 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 antibody and small molecule on neovascularization is studied according to procedures known in the art, such as by IHC analysis of tumor tissues and their surrounding microenvironment.

Mice bearing established orthotopic tumors are administered injections of either anti-PSCA MAb or control antibody over a 4-week period. Mice in both groups have a high tumor burden established, to ensure a high frequency of metastasis formation in mouse lungs. 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 a broad anti-tumor efficacy of anti-PSCA antibodies on the initiation and progression of prostate cancer in xenograft mouse models. Anti-PSCA antibodies inhibit tumorigenesis of tumors as well as slow the growth of already established tumors and prolong the survival of treated mice. Furthermore, the anti-PSCA MAb shows a dramatic inhibitory effect on the spread of local prostate tumor to distant sites, even in the presence of a large tumor burden. Accordingly, anti-PSCA MAb is effective for important clinically relevant endpoints (tumor growth), prolongation of survival and health.

Effect of PSCA MAbs on the growth of human prostate cancer in mice

Using the above methods, UGB-1, human androgen-independent prostate tumor cells (2.0 x 10 6 cells/mouse) were injected subcutaneously into male SCID mice. The mice were randomized into groups (n=10 mice in each group) and treatment initiated intraperitoneally (i.p.) at day 0 with H1-1.10, PBS or control human IgG1 MAb as indicated. Animals were treated twice weekly with a total of 5 doses until study day 16. Tumor growth was monitored using krumpasser measurements every 3 to 4 days as indicated. Tumor volume was calculated as Width<2 >x Length/2, where width is the smallest dimension and length is the largest. The results show that human anti-PCSA monoclonal antibody H1-1.10 significantly inhibited the growth of human prostate cancer xenografts implanted subcutaneously in SCID mice (p< 0.01, by Kruskal-Wallis test). (Figure 14).

In another experiment, PSCA MAb H1-1.10 inhibits subcutaneous growth of established human androgen-independent prostate cancer xenografts in SCID mice. UGB-1, human androgen-independent prostate tumor cells (1.5 x 10 6 cells/mouse) were injected subcutaneously into male SCID mice. When tumors reached approximately 70 mm3, mice were randomized into groups (n=10 mice in each group) and treatment initiated intraperitoneally (i.p.) at day 0 with H1-1.10 or with constituent control as indicated. Animals were treated twice weekly with a total of 7 doses until study day 21. Tumor growth was monitored using krumpasser measurements every 3 to 4 days as indicated. Tumor volume was calculated as Width<2 >x Length/2, where width is the smallest dimension and length is the largest. The results show that human anti-PCSA monoclonal antibody H1-1.10 significantly inhibited the growth of established human prostate cancer xenografts implanted subcutaneously in SCID mice (p< 0.05, by Student's t-test). (Figure 15).

In another experiment, PSCA MAb H1-1.10 inhibits subcutaneous growth of human androgen-dependent prostate cancer xenografts implanted in SCID mice. LAPC9-AD, human androgen-dependent tumor cells (2.0 x 10 6 cells/mouse) were injected subcutaneously into male SCID mice. The mice were randomized into groups (n=10 mice in each group) and treatment initiated intraperitoneally (i.p.) at day 0 with H1-1.10 or controls as indicated. Animals were treated twice weekly with a total of 6 doses until study day 18. Tumor growth was monitored using caliper measurements every 3 to 4 days as indicated. Tumor volume was calculated as Width<2 >x Length/2, where width is the smallest dimension and length is the largest. The results show that human anti-PCSA monoclonal antibody H1-1.10 significantly inhibited the growth of human prostate cancer xenografts implanted subcutaneously in SCID mice (p< 0.01, by Kruskal-Wallis test). (Figure 16).

Effect of PSCA MAbs on the growth of human pancreatic cancer in mice

In another experiment, HPAC, human pancreatic tumor cells (3.0 x 10 6 cells/mouse) were injected subcutaneously into male SCID mice. The mice were randomized into groups (n=10 mice in each group) and treatment initiated intraperitoneally (i.p.) at day 0 with H1-1.10 or control human IgG1 as indicated. Animals were treated twice weekly with a total of 5 doses until study day 18. Tumor growth was monitored using krumpasser measurements every 3 to 4 days as indicated. Tumor volume was calculated as Width<2 >x Length/2, where width is the smallest dimension and length is the largest. The results show that human anti-PCSA monoclonal antibody H1-1.10 significantly inhibited the growth of human pancreatic cancer xenografts implanted subcutaneously in SCID mice (p< 0.05, by Student's t-test). (Figure 17).

Effect of PSCA MAbs on growth of human bladder cancer in mice

In another experiment, human bladder cancer SW780 cells (2.0 x 10 6 cells/mouse) were injected subcutaneously into male SCID mice. The mice were randomized into groups (n=10 mice in each group) and treatment initiated intraperitoneally (i.p.) at day 0 with H1-1.10, PBS or control human IgG1 MAb as indicated. Animals were treated twice weekly with a total of 6 doses until study day 18. Tumor growth was monitored using krumpasser measurements every 3 to 4 days as indicated. Tumor volume was calculated as Width<2 >x Length/2, where width is the smallest dimension and length is the largest. The results show that human anti-PCSA monoclonal antibody H1-1.10 significantly inhibited the growth of SW780 human bladder cancer xenografts implanted subcutaneously in SCID mice (p< 0.05, by Dunnett test). (Figure 18).

The results of these trials demonstrate that PSCA MAbs can be used for therapeutic and diagnostic purposes to treat and manage the cancers listed in Table I.

Example 27

Therapeutic and diagnostic application of anti-PSCA antibodies in humans.

Anti-PCSA monoclonal antibodies are safely and effectively used for diagnostic, prophylactic, prognostic and/or therapeutic purposes in humans. Western blot and immunohistochemical analysis of cancer tissue and cancer xenografts with anti-PSCA MAb shows strong extensive labeling in carcinoma but significantly lower or undetectable levels in normal tissue. Detection of PSCA in carcinoma and in metastatic disease demonstrates the utility of MAb as a diagnostic and/or prognostic indicator. Anti-PSCA antibodies are therefore used in diagnostic applications such as immunohistochemistry of kidney biopsy samples to detect cancer in suspected patients.

As determined by flow cytometry, the anti-PSCA MAb binds specifically to carcinoma cells. Accordingly, anti-PSCA antibodies are used in diagnostic whole-body imaging applications, such as radioimmunoscintigraphy and radioimmunotherapy, (see, e.g., Potamianos S., et. al. Anticancer Res 20(2A):925-948 (2000)) for detection of localized and metastatic cancer showing expression of PSCA. Shedding or release of an extracellular domain of PSCA into the extracellular environment, as seen for alkaline phosphodiesterase B10 (Meerson, N.R., Hepatology 27:563-568 (1998)), allows diagnostic detection of PSCA by anti-PSCA antibodies in serum and/or urine samples from suspected patients.

Anti-PSCA antibodies that specifically bind PSCA are used in therapeutic applications for the treatment of cancers that express PSCA. Anti-PSCA antibodies are used as an unconjugated form and as a conjugated form where the antibodies are bound to one of various therapeutic or imaging forms well known in the art, such as a prodrug, enzymes or radioisotopes. In preclinical studies, unconjugated and conjugated anti-PSCA antibodies are tested for efficacy of tumor prevention and growth inhibition in the SCID mouse cancer xenograft models, e.g., kidney cancer models AGS-K3 and AGS-K6, (see e.g., the example designated “PSCA Monoclonal Antibody-Mediated Inhibition of Tumors In Vivo”).

Either conjugated or unconjugated anti-PSCA antibodies are used as a therapeutic form in human clinical trials either alone or in combination with other treatments as described in the following examples.

Example 28

Human clinical trials for the treatment and diagnosis of human carcinomas using human anti-PSCA antibodies in vivo

Antibodies are used in accordance with the present invention that recognize an epitope on PSCA and are used in the treatment of certain tumors, such as those listed in Table I. Based on several factors, including PSCA expression levels, tumors such as those listed in Table In currently preferred indications. In connection with each of these indications, three clinical methods have been successfully pursued.

I.) Additional treatment: In additional treatment, patients are treated with anti-PSCA antibodies in combination with a chemotherapeutic or anti-neoplastic agent and/or radiation therapy. Primary cancer targets, such as those listed in Table I, are treated under standard protocols by adding anti-PSCA antibodies to standard first- and second-line therapy. Protocol designs speak to efficacy as judged by reduction in tumor mass as well as the ability to reduce usual doses of standard chemotherapy. These dose reductions allow further and/or prolonged treatment by reducing dose-related toxicity of the chemotherapeutic or biological agent. Anti-PSCA antibodies are used in many additional clinical trials in combination with the chemotherapeutic or antineoplastic agents adriamycin (advanced prostrate carcinoma), cisplatin (advanced head and neck and lung carcinomas), taxol (breast cancer) and doxorubicin (preclinical).

II.) Monotherapy: In connection with the use of the anti-PSCA antibodies in the monotherapy of tumors, the antibodies are administered to patients without a chemotherapeutic or antineoplastic agent. In one embodiment, monotherapy is administered clinically in end-stage cancer patients with extensive metastatic disease.

Patients show a certain stabilization of the disease. Trials show an effect in resistant patients with cancerous tumors.

III.) Imaging agent: Through binding or a radionuclide (e.g., iodine or yttrium (I<131>, Y<90>) to anti-PSCA antibodies, the radioactively labeled antibodies are used as a diagnostic and/or imaging agent . In such a role, the labeled antibodies are localized to both solid tumors, as well as, metastatic lesions of cells expressing PSCA. In conjunction with the use of the anti-PSCA antibodies as imaging agents, the antibodies are used as an adjunct to the surgical treatment of solid tumors, as both a pre-surgical screening as well as a post-operative follow-up to determine which tumor remains and/or returns.In one embodiment, a (<111>In)-PSCA antibody is used as an imaging agent in a Phase In human clinical trials in patients who have a carcinoma expressing PSCA (analogously see, e.g., Divgi et al. J. Natl. Cancer Inst.83:97-104 (1991)), patients are followed with standard anterior and posterior gamma camera The results indic ers that primary lesions and metastatic lesions are identified.

Dose and route of administration

As understood by those skilled in the art, dosage considerations can be determined through comparison with the analogous products that are clinical. 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 anti-PSCA antibodies relative to the affinity of a known antibody for its target is one parameter used by those skilled in the art to determine analogous dosage regimens. Furthermore, anti-PSCA antibodies that are fully human antibodies, compared to chimeric antibody, have slower clearance; therefore, dosage for patients with such fully human anti-PSCA antibodies can be lower, perhaps in the range of 50 to 300 mg/m<2 >and still remain effective. Dosage in mg/m<2>, as opposed to the conventional measurement of dose in mg/kg, is a measurement based on surface area and is an appropriate dosage measure designed to encompass patients of all sizes from children to adults.

Three distinct delivery methods are applicable for delivery of anti-PSCA antibodies. Conventional intravenous delivery is one standard delivery technique for many tumors. However, in connection with tumors in the peritoneal cavity, such as tumors in the ovaries, bile ducts, other ducts and the like, intraperitoneal administration may prove advantageous to achieve a high dose of antibody at the tumor and also to reduce antibody excretion. Similarly, certain solid tumors have vasculature suitable for regional perfusion. Regional perfusion allows a high dose of antibody at the site of a tumor and limits short-term shedding of the antibody.

Clinical Development Plan (CDP)

Overview: CDP pursues and develops treatments of anti-PSCA antibodies in the context of adjunctive therapy, monotherapy and/or as an imaging agent. Trials initially show safety and then confirm effectiveness in repeated doses. The trial is an "open label" comparison of standard chemotherapy with standard treatment plus anti-PSCA antibodies. One criterion that may be used in enrolling patients is, as will be understood, PSCA expression levels in their tumors as determined by biopsy.

As with any protein or antibody infusion-based therapeutic agent, safety concerns are primarily related to (i) cytokine release syndrome, ie, hypotension, fever, chills, mild chills; (ii) development of an immunogenic response to the material (ie, development of human antibodies in the patient to the therapeutic antibody or HAHA response); and, (iii) toxicity to normal cells expressing PSCA. Standard tests and follow-up tests are used to monitor each of these safety concerns. anti-PSCA antibodies have been found to be safe for human administration.

Example 29

Human clinical trial: Monotherapy with human anti-PSCA antibody

Anti-PSCA antibodies are safe in connection with the additional trial described above, a Phase II human clinical trial confirms the effectiveness and optimal dosage for monotherapy. Such a trial is carried out and entails the same safety and outcome analyses, as the additional trial described above, with the exception that patients do not receive chemotherapy at the same time as receiving doses of anti-PSCA antibodies.

Example 30

Human clinical trial: Diagnostic imaging with anti-PSCA antibody

Again, as the adjunctive therapy described above is safe within the safety criteria described above, a human clinical trial is being conducted regarding the use of anti-PSCA antibodies as a diagnostic imaging agent. The protocol is designed in a mainly similar way to those described in the area, such as in Divgi et al. J. Natl. Cancer Inst. 83:97-104 (1991). The antibodies have been found to be both safe and effective when used as a diagnostic form.

Example 31

Human clinical trial adjunctive treatment with human anti-PSCA antibody and chemotherapeutic, radiation and/or hormone ablation therapy

A Phase I human clinical trial is being initiated to assess the safety of six intravenous doses of a human anti-PSCA antibody in the treatment of a solid tumor, e.g., cancer in a tissue listed in Table I. The study assesses the safety of single doses of anti-PSCA antibodies when used as an adjunctive therapy to an antineoplastic or chemotherapeutic or hormone ablation agent as defined herein, such as, without limitation: cisplatin, topotecan, doxorubicin, adriamycin, taxol, Lupron, Zoladex, Eulexin, Casodex, Anandron etc. The trial design comprises the delivery of approximately six single doses of an anti-PSCA antibody, where the dose of antibody is escalated from approximately 25 mg/m<2> to approximately 275 mg/m<2 >over the course of treatment according to the following or similar plan:

Day 0 Day 7 Day 14 Day 21 Day 28 Day 35 MAb Dose 25 mg/m<2 >75 mg/m<2 >125 mg/m<2 >175 mg/m<2 >225 mg/m<2 >275 mg/m<2 >Chemotherapy (standard dose)

Patients are followed closely for one week after each administration of antibody and chemotherapy. Specifically, patients are assessed for the safety concerns noted above: (i) cytokine release syndrome, ie, hypotension, fever, chills, mild chills; (ii) development of an immunogenic response to the material (ie, development of human antibodies in the patient against the therapeutic antibody or HAHA response); and, (iii) toxicity to normal cells expressing PSCA. Standard tests and follow-up are applied to monitor each of these safety concerns. Patients are also assessed for clinical outcome and especially reduction in tumor mass as evidenced by MRI or other imaging.

The anti-PSCA antibodies have been shown to be safe and effective. Phase II trials confirm effectiveness and develop optimal dosage.

Example 32

RNA interference (RNAi)

RNA interference (RNAi) technology has been implemented in a number of cell analyzes relevant to oncology. RNAi is a post-transcriptional gene silencing mechanism activated by double-stranded RNA (dsRNA). RNAi induces specific mRNA degradation which leads to changes in protein expression and subsequently in gene function. In mammalian cells, these dsRNAs termed small interfering RNAs (siRNAs) have the right composition to activate the RNAi pathway targeted for degradation, particularly 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).

Loss of cell proliferation control is a hallmark of cancerous cells; therefore, it is relevant to determine the role of PSCA in cell survival/proliferation experiments. Accordingly, RNAi was used to investigate the function of the PSCA antigen. To generate siRNA for PSCA, algorithms were used that predict oligonucleotides that exhibit the critical molecular parameters (G:C content, melting temperature, etc.) and have the ability to significantly reduce the expression levels of the PSCA protein when introduced into cells. According to this example, PSCA siRNA mixtures comprising siRNA (double-stranded small interfering RNA) corresponding to the nucleic acid ORF sequence of the PSCA protein or subsequences thereof are used. Thus, siRNA subsequences used in this way 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 consecutive RNA nucleotides in length. These siRNA sequences are complementary and non-complementary to at least part of the mRNA coding sequence. In a preferred embodiment, the subsequences are 19-25 nucleotides in length, most preferably 21-23 nucleotides in length. In preferred embodiments, these siRNAs achieve knockdown of PSCA antigen in cells expressing the protein and have functional effects as described below.

The selected siRNAs (PSCA.b oligo) were tested in a variety of cell lines in the survival/proliferation MTS experiment (measures cellular metabolic activity).

Tetrazolium-based colorimetric assay (ie, MTS) detects exclusively viable cells, since living cells are metabolically active and therefore can reduce tetrazolium salts to colored formazan compounds; which dead cells do not, however. Furthermore, this PSCA.b oligo achieved knockdown of PSCA antigen in cells expressing the protein and had functional effects as described below using the following methods.

Mammalian siRNA transfections: The day before siRNA transfection, the different cell lines were plated in media (RPMI 1640 with 10% FBS w/o antibiotics) at 2x10<3 >cells/well in 80 µl (96-well plate format) for survival/MTS -analysis. In parallel with the PSCA specific siRNA oligo, the following sequences were included in each experiment as controls: a) Mock transfected cells with Lipofectamin 2000 (Invitrogen, Carlsbad, CA) and hybridization buffer (no siRNA); b) Luciferase-4 specific siRNA (targeting sequence: 5'-AAGGGACGAAGACGAACACUUCTT-3') (SEQ ID NO: 20); and c) Eg5-specific siRNA (targeting sequence: 5'-AACTGAAGACCTGAAGACAATAA-3') (SEQ ID NO: 21). SiRNAs were used at 10nM and 1µg/ml Lipofectamin 2000 final concentration.

The procedure was as follows: the siRNAs were first diluted in OPTIMEM (serum-free transfection media, Invitrogen) at 0.1 µM µM (10-fold concentrated) and incubated 5-10 min RT. Lipofectamine 2000 was diluted at 10 µg/ml (10 times concentrated) for the total number of transfections and incubated 5-10 minutes at room temperature (RT). Appropriate amounts of diluted 10 times concentrated Lipofectamin 2000 were mixed 1:1 with diluted 10 times concentrated siRNA and incubated at RT for 20-30” (5 times concentrated transfection solution). 20 µl of the 5-fold concentrated transfection solutions were added to the respective samples and incubated at 37°C for 96 h before 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-(3carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS(b)] and an electron coupling reagent (phenazine ethosulfate; PES). Experiments were performed by adding a small amount of Solution Reagent directly to culture wells, incubating for 1-4 hours and then recording absorbance at 490nm with a 96-well plate reader.

The amount of colored formazan product as measured by the amount of 490nm absorbance is directly proportional to the mitochondrial activity and/or number of live cells in culture.

To address the function of PSCA in cells, PSCA is silenced by transfecting the endogenously expressing PSCA cell lines.

Another embodiment according to the invention is a method for analyzing PSCA-related cell proliferation is the measurement of DNA synthesis as a marker for proliferation. Labeled DNA precursors (ie 3H-Thymidine) are used and their incorporation into DNA is quantified. Incorporation of the labeled precursors into DNA is directly proportional to the amount of cell division occurring in the culture. Another method used to measure cell proliferation is the performance of "clonogenic" analysis. In these analyses, a defined number of cells are plated on a suitable matrix and the number of colonies formed after a period of growth after siRNA treatment is counted.

In PSCA cancer target validation, complementing the cell survival/proliferation assay with apoptosis and cell cycle profiling studies is considered. The biochemical hallmark of the apoptotic process is genomic DNA fragmentation, an irreversible event that causes the cell to die. One method for observing fragmented DNA in cells is the immunological detection of histone-complexed DNA fragments by an immunoassay (i.e. cell death detection ELISA) which measures the enrichment of histone-complexed DNA fragments (mono- and oligo-nucleosomes) in the cytoplasm of apoptotic cells . This assay does not require prelabeling of the cells and can detect DNA degradation in cells that do not proliferate in vitro (ie, freshly isolated tumor cells).

The most important effector molecules for triggering apoptotic cell death are caspases. Caspases are proteases that, when activated, cleave a number of substrates at the carboxy-terminal site of an aspartate residue, which mediates the very early stage of apoptosis upon activation. All caspases are synthesized as proenzymes and activation involves cleavage at aspartate residues. In particular, caspase 3 appears to play a central role in the initiation of cellular events in apoptosis. Caspase 3 activation assays detect early events of apoptosis. After RNAi treatments, Western blot detection of active caspase 3 presence or proteolytic cleavage products (ie, PARP) found in apoptotic cells further supports an active induction of apoptosis. Because the cellular mechanisms that result in apoptosis are complex, each has its advantages and limitations. Consideration of other criteria/endpoints such as cellular morphology, chromatin condensation, membrane blebbing, apoptotic bodies helps to further substantiate cell death as apoptotic. Since not all gene targets that regulate cell growth are antiapoptotic, the DNA content of permeabilized cells is measured to obtain the profile of DNA content or cell cycle profile. Nuclei of apoptotic cells contain less DNA due to leakage into the cytoplasm (sub-G1 population). In addition, the use of DNA labeling (ie, propidium iodide) also distinguishes between the different phases of the cell cycle in the cell population due to the presence of different amounts of DNA in G0/G1, S and G2/M. In these studies, the subpopulations can be quantified.

For the PSCA gene, RNAi studies facilitate the understanding of the contribution of the gene product in cancer response pathways. Such active RNAi molecules can be used in identification experiments to screen for MAbs that are active anti-tumor therapeutic agents. Furthermore, siRNAs are administered as therapeutic agents to cancer patients for the reduction of malignant growth of many cancer types, including those listed in Table 1. When PSCA has a function in cell survival, cell proliferation, tumorigenesis or apoptosis, it is used as a target for diagnostic, prognostic , preventive and/or therapeutic purposes.

Throughout the present application, various web page data content, publications, patent applications and patents are referenced. (Web sites are referenced by their Uniform Resource Locator, or URL, addresses on the World Wide Web.) The present invention shall not be limited in scope by the embodiments described herein, which are intended as simple illustrations of individual aspects of the invention and whatever is functional equivalent is within the scope of the invention. Various modifications of the models and methods according to the invention, in addition to those described here, will be apparent to those skilled in the art from the preceding description and theories and are accordingly intended to be included within the scope of the invention. Such modifications or other embodiments may be made without departing from the true scope and spirit of the invention.

Tables

Table I: PSCA-expressing tissue reaches malignancy.

Prostate

Pancreas

Bladder

Kidney

Colon

Lung

Ovary

Breast

TABLE II: Amino acid abbreviations

TABLE III: Amino acid substitution matrix

Adapted from GCG Software 9.0 BLOSUM62 amino acid substitution matrix (block substitution matrix). The higher the value, the more likely a substitution is found in related natural proteins. (See world wide web URL ikp.unibe.ch/manual/blosum62.html )

TABLE IV:

HLA Class I/II Motifs/Supermotifs

TABLE IV (A): HLA Class I Supermotifs/Motifs

Residues in bold are preferred, residues in italics are less preferred: A peptide is considered motif-bearing if it has primary anchors at each primary anchor position for a motif or supermotif as specified in the table above. TABLE IV (B): HLA Class II Supermotif

TABLE IV (C): HLA Class II Motifs

p

Residues in italics indicate less preferred or “tolerated” residues

TABLE IV (D): HLA Class I Supermotifs

Residues in italics indicate less preferred or “tolerated” residues TABLE IV (E): HLA Class I Motifs

TABLE IV (F):

Summary of HLA supertypes

( ) , , , , , ,

TABLE IV (G):

58

Motifs indicate the residues that define supertype specificities. The motifs comprise residues determined on the basis of published data to be recognized by multiple alleles within the supertype. Residues in brackets are additional residues also predicted to be tolerated by multiple alleles within the supertype.

Table VI: Exon boundaries of transcript PSCA v.1

Table VII: MFI values for each data point used for affinity calculation.

Table VIII: Affinity calculated from binding saturation curve using Graphpad Prism software: Single-site binding (hyperbola).

Table IX: FACS based affinity for fully human PSCA MAbs

FACS-Based Affinity

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

Table XI: PSCA: Epitope clustering by FACS analysis.

Explanation:

self-competition (100%), bkgd control for each biotinylated antibody none

color 100% competition

strong competition

no to weak competition

Claims (33)

PATENT CLAIMS 1. Antibody or antigen-binding fragment thereof, characterized in that it comprises a variable heavy chain region comprising an amino acid sequence according to SEQ ID NO:13 and a variable light chain region comprising an amino acid sequence according to SEQ ID NO:14, where the antibody or the antigen-binding fragment of which binds specifically to PSCA protein (SEQ ID NO:2). 2. Antibody or antigen-binding fragment thereof according to claim 1, characterized in that the antibody is a monoclonal antibody. 3. Antibody or antigen-binding fragment thereof according to claim 2, characterized in that the antibody is assigned A.T.C.C. Accession number: PTA-6697. 4. Antibody or antigen-binding fragment thereof according to claim 2, characterized in that the monoclonal antibody is a humanized antibody. 5. Antibody or antigen-binding fragment thereof according to claim 1, characterized in that the antigen-binding fragment is a Fab, F(ab′)2, Fv or scFv fragment. 6. Antibody or antigen-binding fragment thereof according to claim 1, characterized in that the antibody is a fully human antibody. 7. Antibody or antigen-binding fragment thereof according to claim 1, characterized in that the antibody or the antigen-binding fragment thereof comprises a murine antigen-binding domain. 8. Antibody or antigen-binding fragment thereof according to claim 1, characterized in that the antibody or the antigen-binding fragment is recombinantly produced. 9. Antibody or antigen-binding fragment thereof according to claim 8, characterized in that the antibody or antigen-binding fragment thereof is a chimeric antibody or antigen-binding fragment thereof. 10. Antibody or antigen-binding fragment thereof according to claim 1, characterized in that the antibody or the antigen-binding fragment thereof is linked to a detectable marker, a toxin, a therapeutic agent or a chemotherapeutic agent. 11. Antibody or antigen-binding fragment thereof according to claim 10, characterized in that the detectable marker is a radioisotope, a metal chelator, an enzyme, a fluorescent compound, a bioluminescent compound or a chemiluminescent compound. 12. Antibody or antigen-binding fragment thereof according to claim 11, characterized in that the radioisotope comprises <212>Bi, <131>I, <131>In, <90>Y, <186>Re, <211>At, <125>I, <188>Re, <153>Sm, <213>Bi, <32>P or Lu. 13. Antibody or antigen-binding fragment thereof according to claim 10, characterized in that the toxin comprises ricin, ricin A chain, doxorubicin, daunorubicin, a maytansinoid, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracindione, actinomycin, diphtheria toxin , Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, restrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, sapaonaria officinalis inhibitor, glucocorticoid, auristatin, auromycin, yttrium, bismuth, combrestatin, duocarmycins, dolostatin, cc1065 or a cisplatin. 14. Transgenic animal, characterized in that it produces the monoclonal antibody according to claim 2. 15. Hybridoma, characterized in that it produces the monoclonal antibody according to claim 2. 16. Vector, characterized in that it comprises a polynucleotide that codes for an antibody according to claim 2. 17. Vector according to claim 16, characterized in that the polynucleotide therein is a single-chain polynucleotide comprising regions that code for the variable heavy chain region and the variable light chain region of the monoclonal antibody. 18. Pharmaceutical mixture, characterized in that it comprises the antibody or the antigen-binding fragment thereof according to claim 1 in a human unit dosage form. 19. Analysis to detect the presence of a PSCA protein in a biological sample, characterized in that it comprises bringing the sample into contact with an antibody or an antigen-binding fragment thereof according to claim 1 and detecting the binding of PSCA protein (SEQ ID NO:2 ) in the sample. 20. Analysis according to claim 19, characterized in that the antibody is a monoclonal antibody. 21. Analysis according to claim 19, characterized in that the antibody or the antigen-binding fragment thereof is labeled with a detectable marker. 22. Vector according to claim 16 or 17, characterized in that it inhibits the growth of cells expressing PSCA (SEQ ID NO:2) in a subject. 23. Antibody or antigen-binding fragment thereof according to claim 1 for delivery of a cytotoxic agent or a diagnostic agent to a cell expressing a PSCA protein (SEQ ID NO:2), characterized in that the antibody or antigen-binding fragment thereof is conjugated to the cytotoxic the agent or the diagnostic agent. 24. Antibody or antigen-binding fragment thereof according to claim 23, characterized in that the cytotoxic agent or the diagnostic agent is selected from the group consisting of a detectable marker, a toxin and a therapeutic agent. 25. Antibody or antigen-binding fragment thereof according to claim 24, characterized in that the detectable marker is a radioisotope, a metal chelator, an enzyme, a fluorescent compound, a bioluminescent compound or a chemiluminescent compound. 26. Antibody or antigen-binding fragment thereof according to claim 25, characterized in that the radioisotope comprises <212>Bi, <131>I, <131>In, <90>Y, <186>Re, <211>At, <125>I, <188>Re, <153>Sm, <213>Bi, <32>P or Lu. 27. Antibody or antigen-binding fragment thereof according to claim 24, characterized in that the toxin comprises ricin, ricin A chain, doxorubicin, daunorubicin, a maytansinoid, taxol, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthracine dione, actinomycin , diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, sapaonaria officinalis inhibitor, glucocorticoid, auristatins, auromycin, yttrium, bismuth, combrestatin, duocarmycins, dolostatin, cc1065 or a cisplatin. 28. Method for detecting a PSCA protein (SEQ ID NO: 2) in a biological sample, characterized in that it comprises the steps of: providing the biological sample and a control sample; contacting the biological sample and the control sample with the antibody or antigen-binding fragment thereof according to claim 1, which specifically binds to the PSCA protein; and determining an amount of a complex of the substance with the PSCA protein and the antibody or antigen-binding fragment thereof present in the biological sample and the control sample. 29. Method according to claim 28, characterized in that it further comprises: take the biological sample and control sample from a patient who has or is suspected of having a cancer listed in Table I. 30. Antibody or antigen-binding fragment thereof according to claim 1 for use in inhibiting the growth of a cancer cell expressing PSCA (SEQ ID NO:2) in a subject. 31. Antibody or antigen-binding fragment thereof according to claim 30, where the subject suffers from a form of cancer selected from the group consisting of prostate, pancreatic, bladder, kidney, colon, lung, ovary and breast cancer. 32. Antibody or antigen-binding fragment thereof according to claim 31, where the antibody or antigen-binding fragment thereof is used in combination with a chemotherapeutic agent. 33. Antibody or antigen-binding fragment thereof according to claim 31, where the antibody or antigen-binding fragment thereof is used in combination with radiotherapy.