CN1997751B - Antibodies and related molecules that bind to PSCA proteins - Google Patents

Abstract

Antibodies and molecules derived therefrom, and variants thereof, that bind to novel PSCA proteins are described, wherein PSCA is expressed tissue-specifically in normal adult tissue and is aberrantly expressed in the cancers listed in table I. Thus, PSCA provides a target for cancer diagnosis, prognosis, prevention and/or treatment. The PSCA gene or fragments thereof, or proteins encoded thereby, or variants thereof, or fragments thereof, may be used to elicit a humoral or cellular immune response; antibodies or T cells reactive with PSCA may be used for active or passive immunization.

Description

Antibodies and related molecules that bind to PSCA protein

Cross References to Related Applications

This patent application is related to pending U.S. Patent Application 10/857,484 (filed May 28, 2004) claiming priority to U.S. Provisional Patent 60/475,064 (filed May 30, 2003); this application is related to U.S. Provisional Patent Application 60/616,381 (filed October 5, 2004), U.S. Provisional Patent Application 60/617,881 (filed October 12, 2004); U.S. Provisional Patent Application 60/621,310 (filed October 21, 2004); US Provisional Patent Application 60/633,077 (filed October 2, 2004); and an unassigned US Provisional Patent Application filed April 14, 2005. This invention is related to PCT patent application PCT/US2004/017231 (filed May 28, 2004). The contents of each application listed in this paragraph are hereby incorporated by reference in their entirety. the

Statement of Ownership Regarding Inventions from Government-Sponsored Research Projects

not applicable. the

technical field

The invention described herein relates to antibodies, binding fragments thereof, and molecules engineered therefrom that bind to a protein known as PSCA. The invention also relates to diagnostic, prognostic, prophylactic and therapeutic methods and compositions for the treatment of PSCA expressing cancers. the

Background of the invention

Cancer is the second leading cause of human death after coronary heart disease. Worldwide, millions of people die of cancer every year. In the United States alone, as reported by the American Cancer Society, cancer kills more than 500,000 people each year, while 1.2 million people are newly diagnosed with cancer each year. While the number of deaths from heart disease is clearly decreasing, the number of deaths from cancer is gradually increasing. At the beginning of the next century, cancer is projected to become the number one cause of death. the

Worldwide, several cancers are major killers. Cancers of the lung, prostate, breast, colon, pancreas, ovary, and bladder, in particular, are the leading causes of cancer death. These cancers, and virtually all others, share the same lethal features. Almost without exception, metastatic cancer disease is fatal. Furthermore, even for those cancer patients who survive their primary cancer, common experience shows that their lives are markedly altered. Many cancer patients experience intense anxiety due to fear of recurrence or treatment failure. Many cancer patients become weak after treatment. Additionally, many cancer patients experience relapses. the

Worldwide, prostate cancer is the fourth most prevalent cancer in men. In North America and Northern Europe, it is almost the most common cancer in men and the second leading cause of cancer death in men. In the United States alone, more than 30,000 men die from the disease each year—second only to lung cancer. In addition to these enormous numbers, there is no effective treatment for metastatic prostate cancer. Surgical prostatectomy, radiotherapy, hormone injection therapy, surgical castration, and chemotherapy remain the mainstays of treatment. Unfortunately, these treatments are not effective for many people and often have unpleasant results. the

In terms of diagnosis, the lack of prostate tumor markers that can accurately detect early localized tumors remains an important limitation in the diagnosis and treatment of this disease. Although serum prostate-specific antigen (PSA) testing has emerged as a useful tool, it is widely believed to lack specificity and generalizability in several important respects. the

The identification of additional prostate cancer-specific markers was facilitated by the generation of prostate cancer xenografts that reproducible different stages of the disease in mice. LAPC (Los Angeles Prostate Cancer) xenografts are prostate cancer xenografts that have survived passage in severe combined immunodeficiency (SCID) mice and were shown to mimic a transition from androgen-dependent to androgen-independent (Klein et al. 1997, Nat. Med. 3:402). Recently identified prostate cancer markers include PCTA-1 (Su et al., 1996, Proc.Natl.Acad.Sci.USA 93:7252), prostate-specific membrane (PSM) antigen (Pinto et al., Clin Cancer Res 1996-9-2 (9):1445-51), STEAP (Hubert et al., Proc Natl Acad Sci U S A.1999-12-7; 96(25):14523-8) and prostate stem cell antigen (PSCA) (Reiter et al., 1998, Proc. Natl. Acad. Sci. USA 95:1735). the

Although previously identified markers such as PSA, PSM, PCTA, and PSCA are helpful in the diagnosis and treatment of prostate cancer, there is a need to identify additional markers and therapeutic targets for prostate and related cancers to further facilitate diagnosis and treatment. the

Renal cell carcinoma (RCC) accounts for approximately 3% of adult malignancies. Once the adenoma reaches 2-3cm in diameter, it may deteriorate. In adults, the two main malignant renal tumors are renal cell adenocarcinoma and transitional cell carcinoma of the renal pelvis or ureter. The estimated incidence of renal cell adenocarcinoma in the United States is more than 29,000 cases, and in 1998 more than 11,600 patients died from this disease. Transitional cell carcinoma is relatively rare, with an incidence in the United States of approximately 500 cases per year. the

Surgery has been the mainstay of treatment for renal cell adenocarcinoma for decades. Until recently, metastatic disease was difficult to control with any systemic therapy. With the recent development of systemic therapies, especially immunotherapy, sustained responses may attack metastatic RCC in suitable patients. However, there remains a need for effective methods of treating these patients. the

Bladder cancer accounts for approximately 5 percent of all new cancer cases in the United States (the fifth most common neoplasm) in men and 3 percent in women (the eighth most common neoplasm). The incidence rate is increasing slowly, and this number is rising with the increase of the elderly population. In 1998, there were an estimated 54,500 cases, including 39,500 men and 15,000 women. In the United States, the age-adjusted incidence rate is 32 per 100,000 males and 8 per 100,000 females. The historical male/female ratio of 3:1 may be reduced by female smoking. An estimated 11,000 people (7,800 men and 3,900 women) died of bladder cancer in 1998. Bladder cancer incidence and mortality rise sharply with age and become a growing problem as the population ages. the

Most bladder cancers recur in the bladder. Bladder cancer is treated with a combination of transurethral cystectomy (TUR) and intravesical chemotherapy or immunotherapy. The multifocal and recurrent nature of bladder cancer points to the limitations of TUR. Most muscle-invasive cancers cannot be cured with TUR alone. Radical cystectomy and urinary diversion are the most effective treatments for this cancer, but have undeniable impacts on urinary and sexual function. There is also a clear need for therapeutic approaches that would benefit bladder cancer patients. the

An estimated 130,200 cases of colorectal cancer occurred in the United States in 2000, including 93,800 colon cancers and 36,400 rectal cancers. Colorectal cancer is the third most common cancer in both men and women. Its incidence decreased significantly (2.1% per year) between 1992-1996. The study showed that these decreases were due to increased inspections and polypectomy, which prevented polyps from developing into invasive cancer. There were an estimated 56,300 deaths in 2000 (47,700 from colon cancer and 8,600 from rectal cancer), accounting for approximately 11% of all cancer deaths in the United States. the

Surgery is currently the most common treatment for colorectal cancer and is often done for cancers that have not spread. Chemotherapy, or chemotherapy plus radiation, is usually given before or after surgery for most people with cancer that has deeply perforated the bowel wall or has spread to the lymph nodes. A permanent colostomy (an opening in the abdomen to remove body waste) is sometimes needed for cancer of the colon, and occasionally for cancer of the rectum. There remains a need for effective methods of diagnosing and treating colorectal cancer. the

An estimated 164,100 new cases of lung and bronchial cancer were diagnosed in 2000, accounting for 14% of all cancer diagnoses in the United States. The incidence of lung and bronchial cancers decreased significantly among men, from 86.5 per 100,000 in 1984 to 70.0 in 1996. In the 1990s, the increase in incidence among women began to slow. In 1996, the incidence among women was 42.3/100,000. the

Lung and bronchus cancers accounted for an estimated 156,900 deaths in 2000, accounting for 28% of all cancer deaths. Between 1992 and 1996, mortality from lung cancer decreased significantly among men (1.7% per year) but increased significantly among women (0.9% per year). Since 1987, more women have died each year from lung cancer than from breast cancer, the leading cause of cancer death in women for the past 40 years. Much of the reduction in lung cancer incidence and mortality is likely related to reductions in smoking rates over the past 30 years; however, reductions in smoking rates among women have lagged behind those among men. What needs to be concerned is that although the smoking rate of adults has gradually decreased, the smoking rate of young people has increased again. the

Treatment options for lung and bronchus 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 spreads from the time it is discovered, radiation and chemotherapy are often combined with surgery. Chemotherapy alone or in combination with radiation therapy is an option for small cell lung cancer; most patients experience remission, some of which is long-lasting, with this regimen. However, there remains a need for effective methods of treating and diagnosing lung and bronchial cancers. the

In 2000, there were an estimated 182,800 new cases of breast cancer among American women. In addition, an estimated 1,400 new cases of breast cancer were diagnosed in men in 2000. In the 1980s, the incidence of breast cancer among women increased by about 4% per year, while in the 1990s the rate was relatively flat at about 110.6 cases per 100,000 people. the

In the United States alone, an estimated 41,200 people (40,800 women, 400 men) died of breast cancer in 2000. Breast cancer is the second leading cause of cancer deaths in women. For both whites and blacks, the death rate fell significantly between 1992 and 1996, with the greatest reduction among young women, according to the latest data. These decreases may be the result of earlier detection and improved treatment. the

Depending on the medical setting and the patient's condition, breast cancer treatments may include lumpectomy (local removal of the tumor) with removal of lymph nodes under the arm; mastectomy (surgical removal of the breast) with removal of lymph nodes under the arm; radiation therapy; chemotherapy ; or hormone therapy. Usually two or more methods are used in combination. Numerous studies have shown that for early disease, long-term survival after lumpectomy plus radiation is similar to survival after modified radical mastectomy. Significant advances in reconstructive technology have provided some options for breast reconstruction after mastectomy. More recently, this reconstruction has been performed concurrently with mastectomy. the

Local resection of ductal carcinoma in situ (DCIS) and adequate surrounding normal breast tissue can prevent recurrence of DCIS. Breast irradiation and/or tamoxifen therapy can reduce the chance of developing DCIS in the remaining breast tissue. This is important because DCIS will develop into invasive breast cancer if left untreated. Moreover, these treatments have serious side effects or sequelae. Therefore, there is a need for effective methods of treating breast cancer. the

An estimated 23,100 new cases of ovarian cancer were diagnosed in the United States in 2000. This accounts for 4% of all gynecological cancers and is the second most common gynecological cancer. Between 1992 and 1996, the incidence of ovarian cancer decreased significantly. Ovarian cancer resulted in an estimated 14,000 deaths in 2000. Ovarian cancer kills more people than any other cancer of the women's reproductive system. the

Treatment options for ovarian cancer include surgery, radiation therapy, and chemotherapy. Surgery usually involves removing one or both ovaries, fallopian tubes (salpingo-oophorectomy), and uterus (hysterectomy). In some very early-stage tumors, only the ovaries are removed, especially in young women who wish to have children. In advanced disease, all diseased sites in the abdomen need to be removed to enhance the effect of chemotherapy. Effective methods of treating ovarian cancer remain an important need. the

An estimated 28,300 new cases of pancreatic cancer were diagnosed in the United States in 2000. The incidence of pancreatic cancer in men has decreased over the past 20 years. Incidence rates in females remain almost constant, but may also begin to decrease. Pancreatic cancer accounted for an estimated 28,200 deaths in the United States in 2000. Over the past 20 years, there has been a slight but significant decrease in mortality among men (approximately 0.9% per year), while the rate has slightly increased among women. the

Pancreatic cancer is treated with surgery, radiation therapy, and chemotherapy. These treatments prolong survival and/or relieve symptoms in most patients, but are unlikely to cure them all. There is an urgent need for alternative methods of treating and diagnosing cancer. These approaches include the use of antibodies, vaccines and small molecules as therapeutic modalities. In addition, there is a need for these modalities to be used as research tools for diagnosis, detection, monitoring, and state of the art in all areas of cancer treatment and research. the

The therapeutic use of monoclonal antibodies (MAbs) has been recognized (G. Kohler and C. Milstein, Nature 256:495-497 (1975)). Monoclonal antibodies are currently approved for use in the treatment of transplantation, cancer, infectious disease, cardiovascular disease, and inflammation. Different isotypes have different effector functions. These functional differences correspond to the distinct three-dimensional structures of the various immunoglobulin isotypes (P.M. Alzari et al., Annual Rev. Immunol., 6:555-580 (1988)). the

MAbs against human targets with therapeutic potential are usually of murine origin due to the convenience of mice for immunization and recognition of most heterogeneous human antigens. However, murine MAbs are inherently deficient in human therapy. Since MAbs have a shorter circulating half-life in humans than human antibodies, MAbs need to be administered more frequently. More critically, repeated administration of murine antibodies to the human immune system caused the human immune system to respond by recognizing the mouse protein as foreign and producing human anti-mouse antibodies (HAMAs). This HAMA response causes anaphylaxis and rapidly clears the murine antibody from the immune system, rendering therapy with the murine antibody ineffective. To avoid this effect, attempts have been made to establish a human immune system in mice. the

Initial attempts 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)) were limited. The amount of DNA that can be stably maintained by an available cloning vector. The use of yeast artificial chromosome (YAC) cloning vectors has opened the way to introduce large germline fragments of the human Ig locus into transgenic mammals. Essentially most of the human V, D and J region genes are arranged at the same intervals found in the human genome, and YAC was used to introduce human constant regions into mice. One such transgenic mouse strain is known as the XenoMouse(r) mouse and is commercially available from Abgenix, Inc. (Fremont CA). the

Summary of the invention

The present invention provides antibodies binding to PSCA protein and PSCA protein polypeptide fragments, their binding fragments and molecules obtained through engineering. The invention includes polyclonal and monoclonal antibodies, murine and other mammalian antibodies, chimeric antibodies, humanized and fully human antibodies, and antibodies labeled with detectable labels or therapeutic agents. In certain embodiments, the premise is that the nucleic acid sequence of Figure 3 is not entirely encoded and/or the amino acid sequence of Figure 2 is not entirely prepared. In certain embodiments, the entire nucleic acid sequence of Figure 3 is encoded and/or the entire amino acid sequence of Figure 2 is produced, either in a respective human unit dosage form. the

The present invention also provides methods for detecting the presence and status of PSCA polynucleotides and proteins in various biological samples, and methods for identifying PSCA-expressing cells. One embodiment of the invention provides a method of monitoring PSCA gene product in a tissue or blood sample containing or suspected of having certain forms of growth disorder, such as cancer. the

The invention also provides various immunogenic or therapeutic compositions and methods of treating PSCA-expressing cancers, such as cancers of tissues listed in Table I, including therapies aimed at inhibiting the transcription, translation, processing or function of PSCA , and cancer vaccines. In one aspect, the invention provides compositions and methods comprising the compositions for treating a patient with a PSCA-expressing cancer, wherein the compositions comprise a vector suitable for human use and a human unit dose of one or more compounds that inhibit PSCA production or function. reagents. Preferably said vector is a single human vector. In another aspect of the invention, the agent is a moiety that is immunoreactive with the 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 (including naturally occurring or synthetic), and combinations thereof . The antibodies can be conjugated to diagnostic or therapeutic moieties. In another aspect, said agent is a small molecule as defined above. the

Description of drawings

figure 1. The cDNA and amino acid sequence of PSCA (also known as PSCA v.1" or "PSCA variant 1") is shown in Figure 1A. The starting methionine is underlined. The open reading frame extends from amino acid 18 to amino acid 389 amino acids, including stop codons. 

The cDNA and amino acid sequence of PSCA variant 2 (also referred to as "PSCA v.2") is shown in Figure 1B. The codon for the initiation methionine is underlined. The open reading frame extends from amino acid 56 to amino acid 427, including the stop codon. the

The cDNA and amino acid sequence of PSCA variant 3 (also referred to as "PSCA v.3") is shown in Figure 1C. The codon for the initiation methionine is underlined. The open reading frame extends from amino acid 423 to amino acid 707, including the stop codon. the

The cDNA and amino acid sequence of PSCA variant 4 (also referred to as "PSCA v.4") is shown in Figure ID. The codon for the initiation methionine is underlined. The open reading frame extends from amino acid 424 to amino acid 993, including the stop codon. the

The cDNA and amino acid sequence of PSCA variant 5 (also referred to as "PSCA v.5") is shown in Figure 1E. The codon for the initiation methionine is underlined. The open reading frame extends from amino acid 910 to amino acid 1479, including the stop codon. the

The cDNA and amino acid sequence of PSCA variant 6 (also referred to as "PSCA v.6") is shown in Figure 1F. The codon for the initiation methionine is underlined. The open reading frame extends from amino acid 83 to amino acid 427, including the stop codon. the

Figure 1G. SNP variants of PSCA v.2, PSCA v.7 to v.18. The PSCA v.7 to v.18 proteins contain 123 amino acids. Variants PSCA v.7 to v.18 are variants that differ from PSCA v.2 by one nucleotide and encode the same protein as v.2. Although these SNP variants are shown individually, they can also be present in any combination and in any transcript variant listed in Figures 1A-1F above. the

Figure 1H. SNP variants of PSCA v.4, PSCA v.19 to v.30. The PSCA v.19 to v.30 proteins contain 189 amino acids. Variants PSCA v.19 to v.30 are variants that differ from PSCA v.4 by one nucleotide. PSCA v.9, v.10, v.11, v.24 and v.25 proteins differ from PSCA v.1 by only one amino acid. PSCA v.23, v.28, v.29 and v.30 encode the same proteins as v.4. Although these SNP variants are shown separately, they can also be present in any combination and in any transcript variants v.3 and v.4. the

Figure 1I. Expression of PSCA variants. (1I(a)) Primers were designed to differentiate PSCA v.1/v.2/v.4, PSCAv.3 and PSCA v.5. In the figure, the small arrows above the exons are primers A and B, through which the 425bp PCR product of PSCA v.1/v.2/v.4, the 300bp PCR product of PSCA v.3 and 910 bp PCR product of PSCA v.5. (II(b)) First-strand cDNA prepared from: normal bladder, brain, heart, kidney, liver, lung, prostate, spleen, skeletal muscle, testis, pancreas, colon, stomach, prostate cancer mixture, bladder cancer, kidney cancer, colon cancer, lung cancer, ovarian cancer, breast cancer, metastatic cancer and pancreatic cancer. Normalization was performed by PCR with primers for actin. Semi-quantitative PCR was performed with 30 rounds of amplification using variant-specific primers. The results showed that PSCA v.5 was mainly expressed in breast cancer, metastatic cancer and pancreatic cancer, while the expression level was lower in colon cancer and lung cancer. PCR products of PSCA v.1/v.2/v.4 were detected in prostate cancer, bladder cancer, kidney cancer, colon cancer, lung cancer, ovarian cancer, breast cancer, metastatic cancer and pancreatic cancer. Among normal tissues, PCR products of PSCA v.1/v.2/v.4 were detected only in prostate and stomach, and at lower levels in kidney and lung, while PSCA v. 5. The PCR detection product of PSCA v.3 was not detected in any of the samples tested. the

Figure 1J. Expression of PSCA v.4 and PSCA v.5. 1J(a) Design primers labeled B and C as indicated by the arrows in the figure to distinguish PSCA v.4 and PSCA v.5. The PSCA v.4 specific primers produced a PCR product of 460bp, while the PSCA v.5 specific primers produced a PCR product of size 945bp. 1J(b) Prepared first-strand cDNA from: normal bladder, brain, heart, kidney, liver, lung, prostate, spleen, skeletal muscle, testis, pancreas, colon, stomach, prostate cancer mixture, bladder cancer and multiple Xenograft mixture (prostate, kidney and bladder cancer xenografts). Normalization was performed by PCR with primers for actin. Semi-quantitative PCR was performed with 30 rounds of amplification using variant-specific primers. Results showed that PSCA v.4 was expressed in prostate cancer, bladder cancer, and a mixture of multiple xenografts, normal kidney, and prostate. PSCA v.5 was only detected in normal prostate and bladder cancers. the

figure 2. Amino acids of PSCA antibody. Figure 2A. Amino acid sequence of Ha1-4.117 VH. The heavy chain constant region is underlined. Figure 2B. Amino acid sequence of Ha1-4.117VL. The light chain constant region is underlined. Figure 2C. Amino acid sequence of Ha1-4.120 VH. Figure 2D. Ha1-4.120VL amino acid sequence. The light chain constant region is underlined. Figure 2E. Amino acid sequence of Ha1-5.99 VH. The heavy chain constant region is underlined. Figure 2F. Amino acid sequence of Ha1-5.99VL. The light chain constant region is underlined. Figure 2G. Amino acid sequence of Ha1-4.121 VH. The heavy chain constant region is underlined. Figure 2H. Amino acid sequence of Ha1-4.121VLc.5. The light chain constant region is underlined. Figure 2I. Amino acid sequence of Ha1-4.121VLc.26. The light chain constant region is underlined. Figure 2J. Amino acid sequence of Ha1-1.16 VH. The heavy chain constant region is underlined. Figure 2K. Amino acid sequence of Ha1-1.16VL. The light chain constant region is underlined. Figure 2L. Amino acid sequence of Ha1-4.5 VH. The heavy chain constant region is underlined. Figure 2M. Amino acid sequence of Ha1-4.5VL. The light chain constant region is underlined. Figure 2N. Amino acid sequence of Ha1-4.40 VH. The heavy chain constant region is underlined. Figure 20. Amino acid sequence of Ha1-4.40VL. The light chain constant region is underlined. Figure 2P. Amino acid sequence of Ha1-4.37 VH. The heavy chain constant region is underlined. Figure 2Q. Amino acid sequence of Ha1-4.37VL. The light chain constant region is underlined. Figure 2R. Amino acid sequence of Ha1-1.43 VH. The heavy chain constant region is underlined. Figure 2S. Amino acid sequence of Ha1-1.43VL. The light chain constant region is underlined. Figure 2T. Amino acid sequence of Ha1-1.152 VH. The heavy chain constant region is underlined. Figure 2U. Amino acid sequence of Ha1-1.152VL. The light chain constant region is underlined. the

image 3. Nucleotide and amino acid sequences of PSCA antibodies. Figure 3A. cDNA and amino acid sequence of Ha1-4.117 VH. The heavy chain constant region is underlined. Figure 3B. cDNA and amino acid sequence of Ha1-4.117VL. The light chain constant region is underlined. Figure 3C. cDNA and amino acid sequence of Ha1-4.120 VH. The heavy chain constant region is underlined. Figure 3D. cDNA and amino acid sequence of Ha1-4.120VL. The light chain constant region is underlined. Figure 3E. cDNA and amino acid sequence of Ha1-5.99VH. The heavy chain constant region is underlined. Figure 3F. cDNA and amino acid sequence of Ha1-5.99VL. The light chain constant region is underlined. Figure 3G. cDNA and amino acid sequence of Ha1-4.121 VH. The heavy chain constant region is underlined. Figure 3H. cDNA and amino acid sequence of Ha1-4.121VLc.5. The light chain constant region is underlined. Figure 3I. cDNA and amino acid sequence of Ha1-4.121VLc.26. The light chain constant region is underlined. Figure 3J. cDNA and amino acid sequence of Ha1-1.16 VH. The heavy chain constant region is underlined. Figure 3K. cDNA and amino acid sequence of Ha1-1.16VL. The light chain constant region is underlined. Figure 3L. cDNA and amino acid sequence of Ha1-4.5 VH. The heavy chain constant region is underlined. Figure 3M. cDNA and amino acid sequence of Ha1-4.5VL. The light chain constant region is underlined. Figure 3N. cDNA and amino acid sequence of Ha1-4.40 VH. The heavy chain constant region is underlined. Figure 30. cDNA and amino acid sequence of Ha1-4.40VL. The light chain constant region is underlined. Figure 3P. cDNA and amino acid sequence of Ha1-4.37VH. The heavy chain constant region is underlined. Figure 3Q. cDNA and amino acid sequence of Ha1-4.37VL. The light chain constant region is underlined. Figure 3R. cDNA and amino acid sequence of Ha1-1.43 VH. The heavy chain constant region is underlined. Figure 3S. cDNA and amino acid sequence of Ha1-1.43VL. The light chain constant region is underlined. Figure 3T. cDNA and amino acid sequence of Ha1-1.152 VH. The heavy chain constant region is underlined. Figure 3U. cDNA and amino acid sequence of Ha1-1.152VL. The light chain constant region is underlined. the

Figure 4. Alignment of PSCA antibodies to germline V-D-J sequences. Figure 4A. Alignment of Ha1-4.117 VH (SEQ ID NO: 13) with human VH4-31. Figure 4B. Alignment of Ha1-4.117VL (SEQ ID NO: 14) with human L19. Figure 4C. Alignment of Ha1-4.120 VH (SEQ ID NO: 15) with human VH4-31. Figure 4D. Alignment of Ha1-4.120VL (SEQID NO: 16) with human 02. Figure 4E. Alignment of Ha1-5.99 VH (SEQ ID NO: 17) with human VH4-34. Figure 4F. Alignment of Ha1-5.99VL (SEQ ID NO: 18) with human A27. Figure 4G. Alignment of Ha1-4.121 VH (SEQ ID NO: 19) with human VH4-34. Figure 4H. Alignment of Ha1-4.121c.5VL (SEQ ID NO: 20) with human 08. Figure 4I. Alignment of Ha1-4.121c.26VL (SEQ ID NO: 21) with human A3. Figure 4J. Alignment of Ha1-1.16 VH (SEQ ID NO: 22) with human VH6-1. Figure 4K. Alignment of Ha1-1.16VL (SEQ ID NO: 23) with human B3. Figure 4L. Alignment of Ha1-4.37 VH (SEQ ID NO: 28) with human VH4-31. Figure 4M. Alignment of Ha1-4.37VL (SEQ ID NO: 29) with human 02. the

Figure 5. Expression of PSCA protein in recombinant mouse, rat and human cell lines. The indicated mouse, rat and human cell lines were infected with retroviruses carrying the human PSCA cDNA and the neomycin resistance gene or with a control virus carrying only the neomycin resistance gene. Stable recombinant cell lines were screened in the presence of G418. Expression of PSCA was determined by FACS stained with 1G8 anti-PSCA MAb (5 μg/ml). Shown are FACS profiles in each cell line, showing that the fluorescence shift is only present in PSCA-infected cell lines, indicating that PSCA is expressed on the cell surface. These cell lines can be used as sources of immunity in MAb development, MAb screening reagents, and in functional assays. the

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

Figure 7. Purification of recombinant glycosylated PSCA protein expressed by 293T cells. 293T cells were transfected with the psec Tag2 vector encoding PSCA cDNA28-100 amino acids. A stable recombinant PSCA-secreting cell line was established by drug selection with hygromycin B. PSCA protein present in conditioned media was purified by affinity chromatography using 1G8 MAb. Shown is a Coomassie blue-stained SDS-PAGE gel of the low pH eluted fraction. The broad molecular weight protein tail observed in endogenously expressed PSCA indicates glycosylation of the recombinant PSCA protein. the

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). Induction of GST-PSCA protein with isopropyl-β-D-thiogalactopyranoside (IPTG) in log-phase cultures and affinity chromatography with glutathione-dextran matrix from lysed bacteria method to purify the protein. Shown is an SDS-PAGE Coomassie blue-stained gel of the glutathione-eluted fraction containing GST-PSCA. It indicated the presence of intact GST-PSCA fusion protein and a small amount of GST-containing degradation products. This protein is useful as a MAb and pAb immunogen and as an Ab screening reagent. the

Figure 9. Human PSCA antibody screening by FACS. The antibody concentration in the supernatant was determined by ELISA. (Natural) 50 μl/well was added to a 96-well FACS plate and serially diluted. Cells expressing PSCA (endogenous or recombinant, 50,000 cells/well) were added and the mixture was incubated at 4°C for 2 hours. After incubation, cells were washed with FACS buffer and then incubated in 100 μl of detection antibody (anti-hlgG-PE) for 45 minutes at 4°C. After incubation, the cells were washed with FACS buffer, fixed with formaldehyde, and analyzed with FACScan. Data were analyzed using CellQuestPro software. Filled histograms represent data obtained from negative control cells, open histograms represent data obtained from PSCA positive cells. the

Figure 10. PSCA MAb relative affinity rank determined by FACS. Twenty-one serial 1 :2 dilutions of each PSCA antibody were incubated with SW780 cells (50,000 cells/well) overnight at 4°C (final MAb concentration 40 nM-0.038 pM). After incubation, cells were washed and incubated with anti-hIgG-PE detection antibody. After washing away the unbound secondary antibody, the cells were analyzed by FACS, and the average fluorescence intensity of each point was obtained by using CellQuest Pro software. Affinity was calculated with Graphpad Prism software using the sigmoid dose-response (variable slope) formula. Shown is a representative FACS analysis depicting PSCA MAb 4.121 binding titers. the

Figure 11. Expression of mouse and macaque PSCA in 293T cells and recognition with anti-human PSCA MAb. 293T cells were transiently transfected with pCDNA3.1 vector carrying mouse PSCA cDNA, simian PSCA cDNA or empty vector (neo, neo). Two days after transfection, cells were harvested and stained with human anti-PSCA MAb Ha1-4.117 or mouse MAb 1G8 (5 μg/ml). Shown are FACS profiles showing binding of MAbHal-4.117 produced from human PSCA protein to mouse and simian PSCA protein expressed in 293T cells. Mouse 1G8 MAb binds to simian PSCA, but not mouse PSCA. This result demonstrates the ability of selected MAbs to cross-react with antigens from other species. Cross-reactive MAbs can be used in expression and toxicity studies in those species. the

Figure 12. PSCA internalization after co-incubation with MAb 4.121. PSCA MAb 4.121 was incubated with PC3-PSCA for 90 minutes at 4°C to allow the antibody to bind to the cell surface. Cells were then divided into two aliquots and incubated at 37°C (to allow antibody internalization) or 4°C (no internalization control). After incubation at 37°C or 4°C, acid wash was used to remove residual PSCA MAb 4.121 bound to the cell surface. Subsequent detection secondary antibody permeabilization and incubation allowed detection of internalized PSCA MAb 4.121. Cells were analyzed by FACS or observed under a fluorescence microscope. About 30% of PSCA MAb 4.121 was internalized after 2 hours of incubation at 37°C. the

Figure 13. Antibodies to PSCA in PSCA-expressing cells mediate saporin-dependent killing. B300.19 cells (750 cells/well) were seeded into 96-well plates on the first day. The next day, an equal volume of the indicated primary antibody containing 2× concentration and a 2-fold excess of anti-human (Hum-Zap) or anti-goat (Goat-Zap) polyclonal antibody linked to saporin toxin was added to each well ( Advanced Targeting Systems, San Diego, CA). Cells were incubated at 37°C for 5 days. After the incubation period, MTS (Promega) was added to each well and incubation was continued for 4 hours. An OD of 450 nM was determined. The results in Figure 13(A) show the presence of saporin-dependent cytotoxicity mediated by PSCA antibodies HA1-4.121 and HA1-4.117 in B300.19-PSCA cells, but no effect in control non-specific IgG1 antibodies. The results in Figure 13(B) show that the addition of a saporin-conjugated secondary antibody that does not recognize human Fc does not mediate cytotoxicity. the

Figure 14. Complement-mediated cytotoxicity of PSCA MAb. PSCA antibody (0-50 μg/ml) was diluted with RHB buffer (RPMI 1640, GibcoLife Technologies, 20 mM HEPES). Wash B300.19-PSCA expressing cells in RHB buffer and resuspend at a density of ¹⁰⁶ cells/ml. In a typical assay, 50 μl of PSCA antibody, 50 μl of diluted rabbit complement serum (Cedarlane, Ontario, Can) and 50 μl of cell suspension were simultaneously added to a flat bottom 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 was continued at 37° C. for 4-5 hours. Use a 96-well fluorometer to read the fluorescence value of each well with excitation at 530nm and emission at 590nm. The results show that PSCA antibodies with IgG1 (HA1-4.121) or IgG2 isotype (HA1-5.99.1) but not IgG4 isotype (HA1-6.46) mediate complement-dependent lysis of target cells.

Figure 15. The F(ab')2 fragment of MAb Ha1-4.121 was generated by pepsin digestion. 20 mg of MAb Ha1-4.121 in 20 mM sodium acetate buffer (pH 4.5) were incubated with and without immobilized pepsin (Pierce. Rockford IL) for the indicated times. Intact MAb and digested Fc fragments were removed by protein A chromatography. Shown are PAGE Coomassie-stained gels of intact undigested, unreduced MAb, unreduced aliquots of digested material taken at the indicated times, and reduced samples of the final digested F(ab')2 product . the

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

Figure 17. Detection of PSCA protein by immunohistochemistry. The expression of PSCA protein in tumor specimens obtained from cancer patients was detected using antibody HA1-4.117. Formalin-fixed, paraffin-embedded tissues were sectioned at 4 microns and mounted on glass slides. Sections were deparaffinized, rehydrated and treated at high temperature with Antigen Retrieval Citra Solution (BioGenex, 4600 Norris Canyon Road, San Ramon, CA, 94583). Sections were then incubated in fluorescein-conjugated human monoclonal anti-PSCA antibody Ha1-4.117 for 16 hours at 4°C. The slides were washed 3 times in buffer and incubated with rabbit anti-fluorescein for 1 hour. After washing in buffer, the slides were immersed in DAKO EnVision+ ^TM peroxidase-conjugated goat anti-rabbit immunoglobulin secondary antibody (DAKO Corporation, Carpenteria, CA) for 30 minutes. Sections were then washed in buffer, developed with a DAB kit (SIGMA Chemicals), counterstained with hematoxylin, and analyzed by brightfield microscopy. The results showed that PSCA was expressed in tumor cells of prostate cancer (Panel A, Panel B), bladder transitional cell carcinoma (Panel C) and pancreatic ductal adenocarcinoma (Panel D). These results indicate that PSCA is expressed in human cancers and that antibodies raised against this antigen can be used as diagnostic reagents.

Figure 18. Inhibition of growth of subcutaneous prostate cancer xenografts by PSCA MAb Ha1-4.120. LAPC-9AI tumor cells (2.0×10 ⁶ cells) were injected subcutaneously into male SCID mice. Mice were randomized into groups (n=10 in each group) and treatment was initiated on day 0 by intraperitoneal injection (ip) of HA1-4.120 or isotype MAb control as indicated. Animals were treated twice a week for a total of 7 doses until day 28 of the study. Tumor growth was measured with calipers every 3-4 days as indicated. The results showed that the human anti-PSCA monoclonal antibody Ha1-4.120 significantly inhibited the growth of subcutaneously implanted human prostate cancer xenografts in SCID mice (p<0.05).

Figure 19. Inhibition of growth of established prostate cancer xenografts in SCID mice by PSCA MAb Ha1-5.99. LAPC-9AI tumor cells (2.0×10 ⁶ cells) were injected subcutaneously into male SCID mice. When tumor volumes reached 50 ^mm3 , mice were randomized (n=10 in each group) and treatment was initiated with intraperitoneal (ip) injection of HA1-5.99.1 or isotype MAb control as indicated. Animals were treated twice a week for a total of 5 doses until day 14 of the study. Tumor growth was measured with calipers every 3-4 days as indicated. The results showed that fully human anti-PSCA monoclonal antibody Ha1-5.99 significantly inhibited the growth of established androgen-independent human prostate cancer xenografts implanted subcutaneously in SCID mice (p<0.05).

Figure 20. Inhibition of growth of established androgen-dependent human prostate cancer xenografts by PSCA MAb HA1-4.121. LAPC-9AD tumor cells (2.5×10 ⁶ cells) were injected subcutaneously into male SCID mice. When tumor volumes reached 40 ^mm3 , mice were randomized (n=10 in each group) and treatment was initiated with intraperitoneal (ip) injection of high concentrations of HA1-4.121 or isotype MAb control as indicated. Animals were treated twice a week for a total of 7 doses until day 21 of the study. Tumor growth was measured with calipers every 3-4 days as indicated. The results of the present study show that Ha1-4.121 inhibits the growth of established human androgen-dependent prostate cancer xenografts implanted subcutaneously in SCID mice. The results were statistically significant on days 14, 17 and 21 (p<0.05, Kruskal-Wallis test, two-tailed α=0.05) of the 300 μg dose group and 10, 14, 17 and 21 days of the 700 μg dose group ( p<0.05, Kruskal-Wallis test, two-tailed α=0.05).

Figure 21. Androgen-dependent LAPC-9AD tumor cells (2.0 x ¹⁰⁶ cells) obtained from a patient were injected into the dorsal lobe of the prostate of male SCID mice. Tumors were allowed to grow for approximately 10 days and mice were randomized into groups. Treatment was initiated 10 days after tumor implantation with 500 μg of human HA1-4.117, HA1-4.121 or isotype control. Antibody was delivered intraperitoneally twice a week for a total of 7 doses. Four days after the last dose, animals were sacrificed and primary tumors were removed and weighed. The results showed that human anti-PSCA monoclonal antibodies Ha1-4.121 (p<0.01) and Ha1-4.117 (p<0.05) significantly inhibited the growth of LAPC-9AD prostate cancer xenografts orthotopically transplanted into SCID mice.

Figure 22. PSCA MAb HA1-4.121 Prolongs Survival Time of SCID Mice Bearing Established Orthotopic Human Androgen-Dependent Prostate Tumors. Androgen-dependent LAPC-9AD tumor cells (2.0 x ¹⁰⁶ cells) obtained from a patient were injected into the dorsal flap of the prostate of male SCID mice. Tumors were allowed to grow for approximately 9 days and mice were randomized into groups. Animals randomized into the survival group included 11 mice in the isotype MAb control group and 12 mice in the HA1-4.121 treated group. Animals were treated intraperitoneally with 1000 μg Ha1-4.121 or 1000 μg isotype MAb control twice weekly for a total of 9 doses. The results showed that HA1-4.121 significantly (log-graded test: p<0.01) prolonged the survival time of SCID mice bearing human androgen-dependent prostate tumors. 110 days after the last treatment, 2 mice in the HA1-4.121 treated group remained free of palpable tumors.

Figure 23. Enhanced inhibition of prostate tumor growth by combined treatment with HA1-4.21 and taxotere. LAPC-9AI tumor cells (2×10 ⁶ cells/animal) were injected subcutaneously into male SCID mice. When the tumor volume reached 65 ^mm3 , animals were randomized into 4 different groups (n=10 in each group) as indicated. Ha1-4.121 or the isotype MAb control were dosed ip twice a week at a dose of 500 μg starting on day 0 for a total of 6 doses. The last dose was given on day 17. Taxotere was administered intravenously at a dose of 5 mg/kg on days 0, 3 and 7. Tumor growth was measured with calipers every 3-4 days. The results of this study showed that Ha1-4.121 as a single agent inhibited the growth of androgen-independent prostate cancer xenografts in SCID mice by 45% compared with control antibody alone at day 28 (ANOVA/Tukey Test: p<0.05). Administration of the isotype MAb control plus taxotere inhibited tumor growth by 28%, which was not statistically significant, compared to control antibody treatment alone. The combined administration of HA1-4.121 and taxotere had a potentiated effect and resulted in a 69% inhibition of tumor growth compared to the control antibody alone (ANOVA/Tukey test: p<0.01). When the combination group of HA1-4.121 and taxotere was compared with the HA1-4.121 or isotype MAb control plus taxotere group, both showed statistically significant differences (ANOVA/Tukey test: p < 0.05).

Figure 24. Inhibition of pancreatic cancer xenograft growth by human PSCA MAb in SCID mice/human HPACs. Pancreatic cancer cells (2×10 ⁶ cells/mouse) were injected subcutaneously into immunodeficient ICR SCID mice (Taconic Farm, Germantown, NY). Mice were randomized into groups (n=10 animals/group) and treatment with indicated human PSCA monoclonal antibodies was initiated on the same day. Antibody (500 mg/mouse) was delivered intraperitoneally twice a week for a total of 8 doses. The results showed that human anti-PSCA monoclonal antibodies Ha1-4.121, Ha1-4.117 and Ha1-1.16 significantly inhibited the growth of human pancreatic cancer xenografts implanted subcutaneously in SCID mice. Statistical analysis was performed using t-test (two-tailed, α=0.05).

Figure 25. Inhibition of PSCA MAb HA1-4.121 on the growth of orthotopically transplanted pancreatic tumors in SCID mice. HPAC cells (3.0 x ¹⁰⁶ cells) were ordinarily implanted into the pancreas of SCID mice. Mice were randomly assigned into 3 groups (n=9 for each group) as indicated. Treatment with HA1-4.121 (250 μg or 1000 μg) or isotype MAb control (1000 μg) was initiated on the day of transplantation. Antibodies were administered intraperitoneally twice a week for a total of 10 administrations. Thirteen days after the last dose, animals were sacrificed and primary tumors were removed and weighed. The results of the present study showed that both dose levels of HA1-4.121 tested significantly inhibited the orthotopic growth of human pancreatic cancer xenografts in SCID mice. 250 μg and 1000 μg AGS-PSCA inhibited tumor growth by 66% and 70%, respectively (Kruskal-Wallis/Tukey test: p<0.01 and p<0.01, respectively).

Figure 26. Inhibition of metastases by PSCA MAb HA1-4.121. At autopsy, visible metastases to lymph nodes and distant organs were observed in the control antibody-treated group. No visible metastases were observed in either HA1-4.121-treated group. Lymph nodes, lungs and livers were removed from all animals and examined histologically for the presence of transitional tumors. Sections from the lungs and lymph nodes of each animal were stained with human cytokeratin, and the number of metastases was determined microscopically. Results of histological analysis showed a significant reduction in lymph node (LN) metastases in animals treated with HA1-4.121 (p=0.0152, as measured by Fishers exact test). The incidence of metastasis and invasion was also significantly reduced in animals treated with both concentrations of HA1-4.121 (p=0.0152, as measured by Fishers exact test). The number of lung metastases was significantly reduced in mice treated with only 1.0 mg dose of HA1-4.121 (p=0.0498, as measured by Fishers exact test). the

Figure 27. Inhibition of SW780 bladder tumor growth by human PSCA MAb in SCID mice. Human SW780 bladder cancer cells (2×10 ⁶ cells/mouse) were injected subcutaneously into immunodeficient ICR SCID mice (Taconic Farm, Germantown, NY). Mice were randomized into groups (n=10 animals/group) and treatment with the indicated human PSCA MAbs was initiated on the same day. Antibody (250 mg/mouse) was delivered intraperitoneally twice a week for a total of 7 doses. The results showed that HA1-4.117 (p=0.014), HA1-4.37 (p=0.0056), HA1-1.78 (p=0.001), Ha1-5.99 (p=0.0002) and HA1-4.5 (p=0.0008) significantly inhibited SCID Growth of subcutaneously implanted SW780 bladder tumors in mice. Statistical analysis was performed by t-test (two-tailed, α=0.05).

Detailed description of the invention

chapter overview

I.) Definition

II.) PSCA polynucleotide

II.A.) Uses of PSCA polynucleotides

II.A.1.) Monitoring for genetic abnormalities

II.A.2.) Examples of antisense

II.A.3.) Primers and primer pairs

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

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

III.) PSCA-related protein

III.A.) Examples of proteins with motifs

III.B.) Expression of PSCA-related proteins

III.C.) Modification of PSCA-related proteins

III.D.) Uses of PSCA-related proteins

IV.) Antibody to PSCA

V.) PSCA cellular immune response

VI.) PSCA transgenic animals

VII.) Methods for detecting PSCA

VIII.) Methods of Monitoring the State of PSCA-Associated Genes and Their Products

IX.) Identification of molecules that interact with PSCA

X.) Treatment methods and compositions

X.A.) Anticancer vaccines

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

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

X.C.1. Small gene vaccine

X.C.2. Combinations of CTL peptides and helper peptides

X.C.3. Combinations of CTL peptides and T cell elicitors

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

X.D.) Adoptive immunotherapy

X.E.) Vaccinations for therapeutic or prophylactic purposes

XI.) Diagnosis and prognosis implementation of PSCA

XII.) Inhibit the function of PSCA protein

XII.A.) Inhibition of PSCA by Intrabody

XII.B.) Inhibition of PSCA with recombinant proteins

XII.C.) Inhibition of PSCA transcription or translation

XII.D.) General considerations of treatment methods

XIII.) Identification, Characterization and Use of PSCA Modulators

XIV.) Therapeutic uses of RNAi and small interfering RNAs (siRNAs)

XV.) Production of kits/products

I.) Definition:

Unless otherwise stated, all technical terms, symbols and other scientific terms or terms used herein have the meanings commonly understood by those skilled in the art to which this invention belongs. In some cases, commonly understood terms are defined here for clarification and/or for reference, and such definitions herein should not be construed as being substantially different from the commonly understood meanings in the art. Many of the techniques and methods described or referenced herein are well understood and can generally be performed by those skilled in the art using routine methods, such as those widely used in Sambrook et al., Molecular Cloning: A Laboratory Manual ) (Second Edition, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) as described in Molecular Cloning. Unless otherwise indicated, methods using commercially available kits and reagents should generally be performed according to the procedures and/or parameters specified by the manufacturer. the

The terms "advanced prostate cancer", "locally advanced prostate cancer", "advanced disease" and "locally advanced disease" refer to prostate cancer that has extended beyond the prostate capsule, including stage C disease as defined by the American Urological Association (AUA) system , C1-C2 stage disease defined by Whitmore-Jewett system and T3-T4 stage and N+ stage disease defined by TNM (Tumor, Nodule, Metastasis) system. Surgery is generally not recommended for patients with locally advanced disease, who generally have poorer surgical outcomes than patients with clinically localized (organ-confined) prostate cancer. Locally advanced disease is clinically identified by palpable sclerosis on the lateral border of the prostate or asymmetry or sclerosis at the base of the prostate. Locally advanced prostate cancer is currently diagnosed by pathological examination following radical prostatectomy if the tumor invades or penetrates the prostate capsule, extends to the surgical margin, or invades the seminal vesicles. the

"Altering the native glycosylation pattern" here refers to the deletion of one or more sugar moieties found on the native PSCA sequence (either by removing potential glycosylation sites or by chemical and/or enzymatic means ), and/or add one or more glycosylation sites that do not exist in the native PSCA sequence. Furthermore, the phrase includes alterations in the nature of native protein glycosylation, including alterations in the nature and ratio of the various sugar moieties present. the

The term "analogue" refers to a molecule that is structurally similar to another molecule (eg, PSCA-related protein) or that shares similar or corresponding properties. For example, analogs of PSCA proteins can be specifically bound by antibodies or T cells that specifically bind PSCA. the

Unless otherwise stated, the term "antibody" is used in the broadest sense. Thus, an "antibody" may be naturally occurring or man-made, such as a monoclonal antibody produced by conventional hybridoma technology. Anti-PSCA antibodies include monoclonal and polyclonal antibodies, as well as fragments comprising the antigen binding domain and/or one or more complementarity determining regions of these antibodies. The term "antibody" as used herein refers to any form of antibody or fragment thereof that specifically binds PSCA and/or has desired biological activity, and this definition specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies , multispecific antibodies (eg, bispecific antibodies) and antibody fragments, so long as they specifically bind PSCA and/or exhibit the desired biological activity. Any specific antibody can be used in the methods and compositions provided herein. Thus, in one embodiment, the term "antibody" includes molecules comprising at least one variable region of a light chain immunoglobulin and at least one variable region of a heavy chain molecule associated with each other to form an antibody against a target antigen. specific binding sites. In one embodiment, the antibody is an IgG antibody. For example, the antibody is an IgGl, IgG2, IgG3 or IgG4 antibody. Antibodies useful in the methods and compositions of the invention can be produced in cell culture, phage, or in various animals including, but not limited to: cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep , dog, cat, monkey, orangutan, ape. Thus, in one embodiment, the antibodies of the invention are mammalian antibodies. Phage technology can be used to isolate original antibodies or to generate variants with altered specificity or affinity properties. These techniques are conventional and well known in the art. In one embodiment, the antibody is produced using recombinant methods known in the art. For example, recombinant antibodies can be produced by transfecting host cells with a vector comprising a DNA sequence encoding the antibody. One or more vectors can be used to transfect host cells with DNA sequences expressing at least one VL and one VH region. Exemplary descriptions of recombinant methods for antibody generation and production include: Delves, ANTIBODIES PRODUCTION: ESSENTIALTECHNIQUES (Wiley, 1997); Shephard et al., MONOCLONAL ANTIBODIES (Oxford University Press, 2000); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES ANDPRACTICE (Academic Press, 1993); CURRENT PROTOCOLS IN IMMUNOLOGY (John Wiley & Sons, recent editions). Antibodies of the invention can be modified by recombinant means to increase the greater effectiveness of the antibodies in mediating a desired function. Accordingly, it is also within the scope of the invention to modify the antibodies of the invention by substitution using recombinant means. Typically, such substitutions are conservative substitutions. For example, at least one amino acid in the constant region of an antibody may be substituted with a different residue. See, eg, US Patent 5,624,821, US Patent 6,194,551, patent application WO 9958572; and Angal et al., Mol. Immunol. 30:105-08 (1993). Modifications to amino acids include deletions, insertions, and substitutions of amino acids. In some instances, these changes are made to reduce undesirable activities, eg, complement dependent cytotoxicity. Antibodies are often covalently or non-covalently linked to a substrate that provides a detectable signal. A wide variety of labeling and linking techniques are known and widely reported in the scientific and patent literature. These antibodies can be screened for binding to normal or defective PSCA. See, eg, ANTIBODY ENGINEERING: APRACTICAL APPROACH (Oxford University Press, 1996). Suitable antibodies with the desired biological activity can be identified by in vitro assays including, but not limited to: proliferation, migration, adhesion, soft agar growth, angiogenesis, cell-cell communication, apoptosis, transport, signaling lead, as well as following in vivo assays, such as tumor growth inhibition. Antibodies provided herein can also be used in diagnostic applications. As capture or non-neutralizing antibodies, they can be screened for their ability to bind a specific antigen without inhibiting receptor binding or the biological activity of that antigen. As neutralizing antibodies, the antibodies can be used in competitive binding assays. They can also be used for the quantification of PSCA or its receptors. the

An "antibody fragment" is defined as at least a portion of the variable region, ie, the antigen-binding region, of an immunoglobulin molecule that binds to its target molecule. In one embodiment, it specifically includes single anti-PSCA antibodies and clones thereof (including agonistic, antagonistic and neutralizing antibodies) and anti-PSCA antibody compositions with polyepitope specificity. Antibodies in the methods and compositions herein can be monoclonal or polyclonal. Antibodies may be antigen-binding antibody fragments, including Fab fragments, F(ab')2 fragments, single chain variable regions, and the like. Fragments of intact molecules can be generated using methods well known in the art, including enzymatic digestion and recombinant methods. the

As used herein, any form of "antigen" can be used to generate antibodies specific for PSCA. Thus, the eliciting antibody can be a single epitope, multiple epitopes, or a whole protein alone or in combination with one or more immunogenicity enhancing agents known in the art. The eliciting antigen can be an isolated full-length protein, a cell surface protein (eg, to immunize cells transfected with at least a portion of the antigen), or a soluble protein (eg, to immunize only a portion of the extracellular region of the protein). The antigens can be produced in genetically engineered cells. The DNA encoding the antigen may be genomic or nongenic (eg, cDNA) and encodes at least part of the extracellular region. The term "portion" as used herein refers to the exact minimum number of amino acids or nucleic acids used to construct an immunogenic epitope of an antigen of interest. Any genetic vector suitable for transforming the cells of interest may be used, including but not limited to: adenoviral vectors, plasmids, and non-viral vectors such as cationic liposomes. In one embodiment, the antibodies in the methods and compositions herein specifically bind at least a portion of the extracellular domain of PSCA of interest. the

The antibodies or antigens and binding fragments thereof provided herein can be conjugated to a "bioactive agent". The term "bioactive agent" as used herein refers to any synthetic or naturally occurring compound that binds to an antigen and/or promotes or mediates a desired biological effect to enhance cytotoxicity. the

In one embodiment, the binding fragments used in the present invention are biologically active fragments. The term "biologically active" as used herein refers to an antibody or antibody fragment capable of binding to a desired epitope and conferring a biological effect directly or indirectly. Direct effects include, but are not limited to: modulating, stimulating and/or inhibiting growth signaling, modulating, stimulating and/or inhibiting anti-apoptotic signaling, modulating, stimulating and/or inhibiting apoptotic or necrotic signaling, modulating, stimulating and/or inhibiting ADCC cascade, and modulating, stimulating and/or inhibiting the CDC cascade. the

"Bispecific" antibodies may also be used in the methods and compositions of the invention. As used herein, the term "bispecific antibody" refers to an antibody, usually a monoclonal antibody, that 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 of making bispecific antibodies are known in the art. For example, bispecific antibodies can be produced by co-expressing two immunoglobulin heavy chain/light chain pairs. See, eg, Milstein et al., Nature 305:537-39 (1983). Alternatively, bispecific antibodies can be prepared by chemical linkage. See, eg, Brennan et al., Science 229:81 (1985). Bispecific antibodies include bispecific antibody fragments. See, eg, Hollinger et al., Proc. Natl. Acad. Sci. U.S.A. 90:6444-48 (1993), Gruber et al., J. Immunol. 152:5368 (1994). the

The monoclonal antibodies herein specifically include "chimeric" antibodies and fragments of the antibodies, as long as they specifically bind to the target antigen and/or have the desired biological activity, part of the heavy chain and/or light chain in the chimeric antibody and The corresponding sequence of an antibody derived from a specific species or belonging to a specific antibody family or subfamily is identical or homologous to the corresponding sequence of an antibody derived from another specific species or belonging to another antibody family or subfamily, while the remainder of the chain is identical or homologous (US Patent 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). the

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 such as nitrogen mustards, aziridines, and alkylsulfonates; antimetabolites such as folic acid, purine or pyrimidine antagonists; mitotic inhibitors such as vinca Alkaline and podophyllotoxin derivatives, cytotoxic antibiotics, compounds that disrupt or interfere with DNA expression, and growth factor receptor antagonists. In addition, chemotherapeutic agents include cytotoxic agents (as defined herein), antibodies, biomolecules and small molecules. the

The term "codon-optimized sequence" refers to a nucleotide sequence optimized by substituting codons whose usage frequency is less than about 20% in a particular species of host. In addition to codon optimization, optimization in a given host by removing pseudopolyadenylation sequences, removing exon/intron splicing signals, removing transposon-like repeats, and/or optimizing GC content The expressed nucleotide sequence is referred to herein as an "expression enhancing sequence". the

A "combinatorial library" is a collection of diverse compounds produced by combining many chemical "building blocks" (eg, reagents) by chemical synthesis or biosynthesis. For example, a linear combinatorial chemical library, such as a polypeptide (e.g., mutein) library, is formed by combining a set of chemical building blocks called amino acids into a given compound length (i.e., the number of amino acids in a polypeptide compound) using all possible methods. Most compounds are synthesized by combinatorial mixing of such chemical building blocks (Gallop et al., J. Med. Chem. 37(9):1233-1251 (1994)). the

(美国专利号5,288,514)、diversomers如乙内酰脲、苯并二氮杂 

和二肽(Hobbs等，Proc.Nat.Acad.Sci.USA90：6909-6913(1993))、插烯多肽(Hagihara等，J.Amer.Chem.Soc.114：6568(1992))，具有β-D-葡萄糖支架的非肽的肽模拟物(Hirschmann等，J.Amer.Chem.Soc.114：9217-9218(1992))、小化合物的类似有机综合体文库(Chen等，J.Amer.Chem.Soc.116：2661(1994))、oligocarbarnate(Cho等，Science261：1303(1993))，和/或肽酰膦酸酯(Campbell等，J.Org.Chem.59：658(1994))。通常可见Gordon等，J.Med.Chem.37：1385(1994)，核酸文库(见例如Stratagene，Corp.)、肽核酸文库(见例如美国专利5,539,083)、抗体文库(见例如Vaughn等，Nature Biotechnology 14(3)：309-314(1996)和PCT/US96/10287)、糖类文库(见例如Liang等，Science 274：1520-1522(1996)和美国专利号5,593,853)，以及小有机分子文库(见例如苯并二氮杂 

，Baum，C & EN，1-18，第33页(1993)；类异戊二烯，美国专利号5,569,588；噻唑烷酮(thiazolidinone)和间噻嗪烷酮(metathiazanone)，美国专利号5,549,974；吡咯烷，美国专利号5,525,735和5,519,134；吗啉代化合物，美国专利号5,506,337；苯并二氮杂 ，美国专利号5,288,514；等等)。  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. )), peptoids (PCT publication WO 91/19735), encoded peptides (PCT publication WO 93/20242), random biooligomers (PCT publication WO 92/00091), benzodiazepines

(US Patent No. 5,288,514), diversomers such as hydantoins, benzodiazepines

And dipeptide (Hobbs etc., Proc.Nat.Acad.Sci.USA90: 6909-6913 (1993)), insertion polypeptide (Hagihara etc., J.Amer.Chem.Soc.114: 6568 (1992)), have β - Non-peptidic peptide mimetics of D-glucose scaffolds (Hirschmann et al., J.Amer.Chem.Soc.114:9217-9218 (1992)), similar organic synthesis libraries of small compounds (Chen et al., J.Amer. Chem.Soc.116:2661 (1994)), oligocarbarnate (Cho et al., Science 261:1303 (1993)), and/or peptidyl phosphonate (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 eg benzodiazepines

, Baum, C & EN, 1-18, p. 33 (1993); Isoprenoids, U.S. Patent No. 5,569,588; Thiazolidinone and metathiazanone, U.S. Patent No. 5,549,974; Pyrrolidines, U.S. Patent Nos. 5,525,735 and 5,519,134; Morpholino compounds, U.S. Patent No. 5,506,337; Benzodiazepines , US Patent No. 5,288,514; etc.).

Devices for preparing combinatorial libraries are commercially available (see, e.g., 357NIPS, 390NIPS, Advanced Chem Tech, Louisville KY; Symphony, Rainin, Woburn, MA; 433A, Applied Biosystems, Foster City, CA; 9050, Plus, Millipore, Bedford, NIA ). A number of well-known robotic systems have also been developed to solve phase chemistry problems. These systems include automated workstations, such as the automated synthesizer developed by Takeda Chemical Industries, LTD. (Osaka, Japan), and robotic systems that employ robotic arms that mimic the manual synthesis operations of chemists (Zymate H, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif.). Any of the devices described above are suitable for use with the present invention. It will be within the skill of the art to modify, if necessary, the nature and mode of operation of these devices to enable them to function as described herein. In addition, many combinatorial libraries themselves are 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.). the

The term "conservative substitution" as used herein refers to amino acid substitutions known to those skilled in the art and which are made without generally altering the biological activity of the resulting molecule. Those skilled in the art recognize that often single amino acid substitutions in non-essential regions of polypeptides 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)). These exemplary substitutions are preferably made as described in Table III(a-b). For example, these changes may include substitution of isoleucine (I), valine (V) and leucine (L) for other such hydrophobic amino acids; substitution of aspartic acid (D) for glutamic acid (E) , and vice versa; replace asparagine (N) with glutamine (Q) and vice versa; replace threonine (T) with serine (S) and vice versa. Other substitutions may also be considered conservative, depending on the context of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) are often interchangeable, as are alanine (A) and valine (V). The relatively hydrophobic methionine (M) is often interchangeable with leucine, isoleucine, and sometimes valine. Lysine (K) and arginine (R) are often interchangeable at positions where the distinguishing feature of the amino acid residue is its charge and the differing pK of the two amino acid residues is not significant. There are also other changes that may be considered "conservative" under certain circumstances (see, e.g., Table III(a) herein; "Biochemistry" 2nd ed. pp. 13-15. Lubert Stryer ed. (Stanford University); Henikoff et al. , PNAS 1992, Vol. 89, 10915-10919; Lei et al., J Biol Chem 1995 May 19;270(20):11882-6). Other substitutions are permitted and may be determined empirically or based on known conservative substitutions. the

The term "cytotoxic agent" refers to a substance that inhibits or prevents cellular expression activity, cellular function, and/or causes cellular destruction. The term includes radioisotopic chemotherapeutic agents, as well as 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, auristatin, aureomycin, maytansinoid, yttrium, bismuth, ricin, ricin A-chain, combrestatin, doca Duocarmycin, dolostatins, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc 1065, ethidium bromide, mitomycin, etoposide, podophylloside, vinca Neosine, vinblastine, colchicine, dihydroxyanthraxin diketone, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, capsule Lotus root toxin chain A, α-sarcinina, gelonin, mitogen, retstrictocin, phenomycin, enomycin, curicin, crotonin, thorn Sapaonaria officinalis inhibitors and glucocorticoids and other chemotherapeutic agents, and radioisotopes such as At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, P32 and radioisotopes of Lu, including Lu177 . Antibodies can also be conjugated to anticancer prodrug activating enzymes capable of converting prodrugs to their active forms.

The term "diabody" as used herein refers to a small antibody fragment with two antigen-binding sites comprising a heavy chain variable region (VL) linked to a light chain variable region (VL) in the same polypeptide chain (VH-VL). District (VH). By using a linker that is too short to allow pairing between two regions in the same chain, the regions are forced to pair with complementary regions in another chain and two antigen-binding sites are created. Dimers are more fully described in, eg, European Patent 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-48 (1993). the

"Gene product" refers herein to peptide/protein or mRNA. For example, "gene product of the present invention" is sometimes referred to herein as "amino acid sequence of cancer", "oncoprotein", "protein of cancer listed in Table I", "mRNA of cancer", "anticancer protein of cancer listed in Table I". mRNA" and so on. In one embodiment, an oncoprotein is encoded by the nucleic acid of FIG. 1 . The oncoprotein may be a fragment, or may be a fragment of the full-length protein encoded by the nucleic acid of FIG. 1 . In one embodiment, cancer amino acid sequences are used to determine sequence identity or similarity. In another embodiment, the sequence is an allelic variant of the naturally occurring protein encoded by the nucleic acid of Figure 1 . In another embodiment, the sequence is a sequence variant as further described herein. the

"Heteroconjugated" antibodies may be used in the methods and compositions of the invention. The term "heteroconjugate antibody" as used herein refers to two covalently linked antibodies. These antibodies can be prepared by methods known in synthetic protein chemistry, including the use of cross-linking agents. See, eg, US Patent 4,676,980. the

"High-throughput screening" assays for determining the presence, absence, quantification or other properties of particular nucleic acid or protein products are well known to those skilled in the art. Similarly, binding assays and reporter gene assays are also well known. Thus, for example, U.S. Patent No. 5,559,410 discloses high-throughput screening methods for proteins; U.S. Patent No. 5,585,639 discloses high-throughput screening methods for detecting nucleic acid binding (i.e., using array detection); High-throughput screening method for body/antibody binding. the

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 the entire process, including all sample and reagent aspiration, liquid dispensing, timed incubations, and finally microplate reading on a detector appropriate for the assay. These configurable systems provide high throughput and enable rapid start-up, and are highly flexible and customizable. Manufacturers of such systems provide detailed protocol instructions for various high-throughput systems. Thus, for example, Zymark Corp. provides technical note booklets describing screening systems for detecting modulation of gene transcription, ligand binding, and the like. the

The term "homologue" refers to a molecule that exhibits homology to another molecule, eg, by having a sequence of chemically identical or similar residues at corresponding positions. the

In one embodiment, the antibodies provided herein are "human antibodies". The term "human antibody" as used herein refers to an antibody in which substantially the entire sequence of the light and heavy chain sequences, including complementarity determining regions (CDRs), is derived from human genes. In one embodiment, the EBV transformation technology (see, for example, Cole et al., MONOCLONAL ANTIBODY AND CANCER THERAPY 77-96 (1985)) or using phage display (see eg, Marks et al., J. Mol. Biol. 222:581 (1991)) to prepare human monoclonal antibodies. In a specific embodiment, said human antibodies are produced in transgenic mice. Techniques for making these partially human antibodies to fully human antibodies are known in the art, and any such technique can be used. According to a preferred embodiment, full-length human antibody sequences are produced in transgenic mice engineered to express human heavy and light chain antibody genes. An exemplary description of making transgenic mice is found in patent application WO 02/43478 and US Patent 6,657,103 (Abgenix) and its successors. Transgenic mouse-derived B cells producing the desired antibody can then be fused to produce a hybridoma cell line that continuously produces the antibody. See, eg, U.S. Patents 5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,545,806; and Jakobovits, Adv. 1998). the

"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 edition, Lange Publishing, Los Altos, CA (1994) .

As used herein, the term "humanized antibody" refers to a form of antibody that contains antibody sequences from a non-human (eg, mouse and human antibodies). These antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Typically, the humanized antibody will comprise substantially all of at least one and usually two variable domains corresponding to all or substantially all hypervariable loops and all or Essentially all FR regions are those in human immunoglobulin sequences. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, eg, U.S. Patent 4,816,567 to Cabilly; Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86:10029-10033; and ANTIBODY ENGINEERING: A PRACTICAL APPROACH (Oxford University Press 1996). the

The term "hybridization" etc. refers to conventional hybridization conditions when used for polynucleotides, preferably such as hybridization in 50% formamide/6×SSC/0.1% SDS/100 mg/ml ssDNA, wherein the hybridization temperature is higher than 37 ° C, and at the same time The temperature for washing with 0.1 x SSC/0.1% SDS was above 55°C. the

The phrase "isolated" or "biologically pure" refers to a composition that is substantially or completely free from materials that normally accompany it in its native state. Thus, an isolated peptide as described herein is preferably free of materials that normally accompany the peptide in its natural environment. For example, a polynucleotide is said to be "isolated" when it is substantially separated from contaminating polynucleotides corresponding to or complementary to genes other than the PSCA gene or genes encoding polypeptides other than the PSCA gene product or fragments thereof. ". The skilled artisan can conveniently employ nucleic acid isolation methods to obtain isolated PSCA polynucleotides. For example, a PSCA protein is said to be "isolated" when physical, mechanical, or chemical methods are used to remove proteins that normally accompany the PSCA protein in cellular components. The skilled artisan can readily employ standard purification methods to obtain isolated PSCA protein. Alternatively, isolated proteins can be prepared chemically. the

Suitable "labels" include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents that teach the use of these markers include: US Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; In addition, the antibodies provided herein can be used as antigen-binding components of fluorophores. See, eg, Zeytun et al., Nat. Biotechnol. 21:1473-79 (2003). the

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

The terms "metastatic prostate cancer" and "metastatic disease" refer to prostate cancer that has spread to regional lymph nodes or to distant sites, including stage D disease in the AUA system and stage TxNxM+ disease in the TNM system. In cases of locally advanced prostate cancer, surgery is usually not performed on patients with metastatic disease, and hormonal (androgen ablation) therapy is the preferred treatment. Patients with metastatic prostate cancer eventually develop androgen refractory status within 12-18 months of treatment initiation. Almost half of these androgen-refractory patients die within 6 months of developing this stage. The most common site of prostate cancer metastasis is the bone. Prostate cancer bone metastases are usually osteoblastic rather than osteolytic (ie, result in net bone formation). Bone metastases are most common in the spine, followed by the femur, pelvis, rib cage, skull, and humerus. Other common metastatic sites include lymph nodes, lung, liver, and brain. Metastatic prostate cancer is usually diagnosed by surgical incision or laparoscopic pelvic lymphadenectomy, whole-body radionuclide scan, bone radiography, and/or biopsy of bone lesions. the

The terms "modulator" or "detection compound" or "drug candidate" or grammatical equivalents herein describe substances that are to be tested for directly or indirectly altering the phenotype of a cancer or a cancer sequence (e.g., a nucleic acid or protein sequence). Any molecule, such as a protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., that has the ability to express, or the effect of a cancer sequence (eg, signal generation, gene expression, protein interaction, etc.). In one aspect, a modulator will neutralize the effect of an oncoprotein of the invention. "Neutralization"means that the activity of the protein and its subsequent effects on the cell are inhibited or suppressed. In another aspect, normalization of the protein levels by the modulator neutralizes the efficacy of the gene of the invention and its corresponding protein. In preferred embodiments, a modulator alters the expression profile, or expression profile, of a nucleic acid or protein provided herein, or alters a downstream effector pathway. In one embodiment, the modulator suppresses a cancer phenotype, such as a normal tissue fingerprint. In another embodiment, the modulator induces a cancer phenotype. Often multiple mixtures are tested in parallel with different reagent concentrations to obtain differential responses to the different concentrations. Typically, one of these concentrations, zero or below the level of detection, serves as a negative control. the

Modulators, drug candidates or test compounds include various classes of chemical agents, but they are generally organic molecules, preferably small organic compounds with molecular weights 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 500D. Candidate reagents contain functional groups structurally necessary to interact with the protein, specifically hydrogen bonding, and typically contain at least one amine, carbonyl, hydroxyl or carboxyl group, preferably at least two chemical functional groups. Such candidate agents typically comprise carbocyclic or heterocyclic structures and/or aromatic or aromatic polymeric structures substituted with one or more of the aforementioned functional groups. Modulators also include biomolecules such as peptides, carbohydrates, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof. Especially preferred are peptides. One class of modulators are peptides, eg, peptides containing about 5-35 amino acids, preferably about 20 amino acids, more preferably about 7-15 amino acids. Preferably the cancer regulatory protein is soluble and includes a non-transmembrane region and/or an N-terminal Cys to aid in dissolution. In one embodiment, the C-terminus of the fragment is retained as a free acid while the N-terminus is a free amine to facilitate coupling, ie to cysteine. In one embodiment, an oncoprotein of the invention is conjugated to an immunogenic agent as described herein. In one embodiment, the oncoprotein is conjugated to BSA. Peptides of the invention, such as peptides of preferred length, can be combined with each other or with other amino acids to form longer peptides/proteins. Such regulatory peptides can be digested into naturally occurring proteins (as described above), random peptides, or "biased" random peptides. In a preferred embodiment, peptide/protein based modulators are antibodies and fragments thereof as defined herein. the

Regulators of cancer can also be nucleic acids. Nucleic acid modulators can be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids. For example, digestion products of prokaryotic or eukaryotic genomes can be used in the corresponding analogs of the above-mentioned proteins. the

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, an antibody comprising a population of identical antibodies but with the possibility of minor naturally occurring mutations present. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations generally include multiple antibodies directed against (or specific for) different epitopes. In one embodiment, the polyclonal antibody is a plurality of monoclonal antibodies having different epitope specificities, affinities or avidities for a single antigen containing multiple antigenic epitopes. The modifier "monoclonal" means that the properties of the antibody are obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies useful in the present invention can be prepared using hybridoma technology first described by Kohler et al., Nature 256:495 (1975), or can be prepared by recombinant DNA methods (see, e.g., U.S. Patent 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using techniques such as those described by Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991). . These monoclonal antibodies typically bind with a Kd of at least about 1 μM, more usually at least about 300 nM, usually at least about 30 nM, preferably at least about 10 nM, more preferably at least about 3 nM or better, typically by ELISA. the

"Motif", as in the biological motif of a PSCA-related protein, refers to any pattern of amino acids that forms part of the protein's primary sequence and that is associated with a specific function (e.g., protein-protein interaction, protein-DNA interaction, etc.) or modification ( Such as phosphorylation, glycosylation or amidation modification), or localization (such as secretory sequence, nuclear localization sequence, etc.) or related to the sequence related to the production of humoral or cellular immunity. Motifs may be contiguous or capable of being arranged in a certain position generally associated with a certain function or property. In HLA motifs, "motif" refers to a pattern of residues in a peptide of defined length, typically a peptide of about 8-13 amino acids for class I HLA motifs, and typically for class II HLA motifs It is a peptide of about 6-25 amino acids, which can be recognized by specific HLA molecules. Individual proteins encoded by respective human HLA alleles generally differ in their HLA-binding peptide motifs, as well as in the pattern of primary and secondary anchor residues. Common base sequences are listed in Table V. the

"Pharmaceutical excipients" include adjuvants, carriers, pH regulators and buffers, tonicity regulators, wetting agents, preservatives and other substances. the

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

The term "polynucleotide" means a polymeric form of ribonucleotides or deoxynucleosides or modified forms of both nucleotide types of at least 10 bases or base pairs in length, and refers to a single polynucleotide including DNA and/or RNA chain and double chain forms. In the art, the term is often used interchangeably with "oligonucleotide." A polynucleotide may include the nucleotide sequences disclosed herein, as shown in Figure 1, where thymine (T) may also be uracil (U); this definition relates to the distinction between the chemical structures of DNA and RNA, specifically Say, one of the four main bases in RNA is uracil (U) instead of thymine (T). the

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

An HLA "primary anchor residue" is an amino acid at a specific position along a peptide sequence that is thought to provide a point of contact between the immunogenic peptide and the HLA molecule. 1-3, usually 2, major anchor residues within a peptide of defined length define a "motif" of the immunogenic peptide. These residues are thought to fit into tight contact with the binding groove of the HLA molecule while inserting their side chains into specific pockets of the binding groove. For example, in one embodiment, the primary anchor residue of the HLA class I molecule is at position 2 (from the amino-terminal position) and at position 8, 9, 10, 11 or 12 carboxy-terminal to the peptide epitope of the invention. residue position. Alternatively, in another embodiment, the primary anchor residues of the HLA class II binding peptide are spaced apart from each other rather than at the end of the peptide, where the peptide is usually at least 9 amino acids in length. The major anchor positions for each motif and supermotif are listed in Table IV(a). For example, analog peptides can be generated by altering the presence or absence of specific residues in the primary and/or secondary anchor positions shown in Table IV. Such analogs are used to modulate the binding affinity and/or population coverage of peptides containing specific HLA motifs or supermotifs. the

"Radioisotopes" include, but are not limited to, the following (non-limiting exemplary uses are also listed in Table IV(I)). the

"Random" or the grammatical equivalent is used herein for nucleic acids and proteins to indicate that each nucleic acid and peptide is composed of substantially random (linked) nucleotides and amino acids, respectively. These random peptides (or nucleic acids as discussed herein) can incorporate any nucleotide or amino acid at any position. Synthetic methods can be designed to generate random proteins or nucleic acids to form all or most possible combinations over the length of the sequence to form a random library of candidate biologically active protein agents. the

In one embodiment, the library is "fully randomized" with no preferred or constant sequences at any position. In another embodiment, the library is a "biased random" library. That is, some positions in the sequence are either held constant or selected from a limited number of possible residues. For example, nucleotide or amino acid residues can be randomly selected among defined classes (e.g., hydrophobic amino acids, hydrophilic residues, sterically biased (small or large) residues) to form a nucleic acid binding domain, forming an intersection Cysteine for the combination, proline for the SH-3 domain, serine, threonine, tyrosine or histidine for the phosphorylation site, etc., or for purines, etc. the

A "recombinant" DNA or RNA molecule is a DNA or RNA molecule produced in vitro. the

The term "single-chain Fv" or "scFv" or "single-chain" antibody as used herein refers to an antibody fragment comprising the VH and VL regions of the antibody, wherein these regions are present in one polypeptide chain. Typically, the Fv polypeptide also comprises a polypeptide linker between the VH and VL regions that allows the sFv to form the structure required for antigen binding. For sFv see Pluckthun, THE PHARMACOLOGY OF MONOCLONAL ANTIBODY, Vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994). the

Non-limiting examples of "small molecules" include compounds that bind or interact with PSCA, ligands (including hormones, neuropeptides, chemokines, odorants, phospholipids, and their functions that bind to and preferably inhibit PSCA protein function). equivalent). Such non-limiting small molecules preferably have a molecular weight of less than about 10 kDa, more preferably less than about 9, about 8, about 7, about 6, about 5 or about 4 kDa. In certain embodiments, small molecules are physically associated with or bind PSCA proteins; are not found in naturally occurring metabolic pathways; and/or are more soluble in aqueous solutions than in non-aqueous solutions. the

The term "specificity" as used herein refers to the selective binding of an antibody to a target antigenic epitope. The binding specificity of an antibody can be tested by comparing the binding of the antibody to the appropriate antigen compared to the binding of the antibody to an unrelated antigen or mixture of antigens under the given conditions. The antibody is considered specific if it binds to the appropriate antigen at least 2, 5, 7 and more preferably 10 times more than it binds to an unrelated antigen or mixture of antigens. In one embodiment, a specific antibody is one that binds only to a PSCA antigen and not to an unrelated antigen. In another embodiment, the specific antibody is one that binds to a human PSCA antigen but does not have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% binding to the PSCA antigen. %, 95%, 96%, 97%, 98%, 99% or more amino acid homology to a non-human PSCA antigen-binding antibody. In another embodiment, the specific antibody is one that binds a human PSCA antigen and binds a mouse PSCA antigen, but to a greater extent human antigen. In another embodiment, the specific antibody is one that binds a human PSCA antigen and binds a primate PSCA antigen, but to a greater extent, a human antigen. In another embodiment, the specific antibody binds human PSCA antigen and any non-human PSCA antigen but binds to a greater extent human antigen or any combination thereof. the

"Stringency" of a hybridization reaction is readily determined by one of ordinary skill in the art and can generally be calculated empirically based on probe length, washing temperature and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally depends on the ability of denatured nucleic acid sequences to re-anneal when complementary strands are present in an environment below the melting temperature. The higher the desired degree of homology between the probe and the hybridizable sequence the higher the temperature can be used. As a result, relatively higher temperatures make the reaction conditions more stringent, while lower temperatures make the reaction conditions less stringent. Additional details on the stringency of hybridization reactions are explained in Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995). the

"Stringent conditions" or "highly stringent conditions" as defined herein can be identified by, but are not limited to, the following characteristics, which are: (1) low ionic strength and high temperature for washing, e.g. 0.015M sodium chloride/0.0015M lemon Sodium lauryl sulfate/0.1% sodium lauryl sulfate, the temperature is 50 ℃; (2) Use formamide and other denaturants during the hybridization process, such as 50% (v/v) formamide and 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer (pH6.5) and 750mM sodium chloride, 75mM sodium citrate at 42°C; or (3) use 50% formamide, 5x SSC (0.75M NaCl , 0.075M sodium citrate), 50mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50mg/ml), 0.1% SDS and 10% dextrose sulfate Sugar at 42°C, washed with 0.2x SSC (sodium chloride/sodium citrate) at 42°C and 50% formamide at 55°C, then washed with 0.1x SSC containing EDTA at 55°C at high stringency washing. "Intermediately stringent conditions" are described in, but not limited to, Sambrook et al. in "Molecular Cloning: A Laboratory Manual" (Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989), and include the use of stringent conditions lower than those described above. Conditioned wash solutions and hybridization conditions (eg, temperature, ionic strength, and % SDS). An example of moderately stringent conditions is an overnight incubation at 65°C in a solution containing: 1% bovine serum, 0.5% sodium phosphate pH 7.5, 1.25mM EDTA and 7% SDS 5×SSC (150mM NaCl, 15mM citric acid Trisodium), then wash the filter membrane with 2×SSC/1% SDS at about 50°C and with 0.2×SSC/0.1% SDS at 50°C. A skilled artisan will know how to adjust temperature, ionic strength, etc. to accommodate factors such as probe length. the

An HLA "supermotif" is a peptide binding specificity shared by HLA molecules encoded by two or more HLA alleles. All phenotypic frequencies of HLA-supertypes in different ethnic populations are listed in Table IV(f). A non-limiting composition of the various supertypes is 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)

The calculated population coverage for different HLA-supertype combinations is presented in Table IV(g). the

"Treatment" or "therapeutic" and grammatically related terms herein refer to any improvement in the outcome of any disease, such as increased survival time, reduced morbidity and/or reduced side effects, which are by-products of changing modes of treatment; in It is well understood in the art that while complete eradication of the disease is not required, it is a preferred outcome. the

A "transgenic animal" (eg, a mouse or a rat) is an animal that has cells containing a transgene that was introduced into the animal or into an ancestor of the animal before birth (eg, at the embryonic stage). A "transgene" is DNA that is integrated into the genome of a cell to produce a transgenic animal. the

HLA or cellular immune response "vaccine" means herein a composition containing or encoding one or more peptides of the invention. There are many examples of such vaccines, such as mixtures of one or more peptides; one or more peptides of the invention contained in polyepitopic peptides; or nucleic acids encoding such individual peptides or polypeptides, e.g. Small genes encoding polyepitopic peptides. "One or more peptides" may comprise any integer from 1 to 150 or more, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 ,41,42,43,44,45,46,47,48,49,50,55,60,65,70,75,80,85,90,95,100,105,110,115,120,125 , 130, 135, 140, 145 or 150 or more peptides of the invention. The peptide or polypeptide may optionally be modified, such as by lipidation, addition of targeting sequences or other sequences. The HLA class I peptides of the present invention can be mixed or linked with HLA class II peptides to facilitate activation of cytotoxic T lymphocytes and activation of helper T lymphocytes. HLA vaccines may contain peptide-pulsed antigen-presenting cells, such as dendritic cells. the

The term "variant" refers to a molecule showing a change from the stated or normal type, such as containing one or more different amino acid residues at corresponding positions in a well-described protein (PSCA protein as shown in Figure 1) of protein. An example of a variant protein is an analog. Splice isoforms and single nucleotide polymorphisms (SNPs) are other examples of variants. the

"PSCA-associated proteins" of the present invention include those specifically described herein, as well as allelic variants, conservative substitution variants, analogs and homologues, which can be modified without undue experimentation by methods set forth herein or readily available in the art. It can then be isolated/produced and characterized. Also included are fusion proteins produced by combining portions of different PSCA proteins or fragments thereof, as well as fusion proteins of PSCA proteins and heterologous polypeptides. Such PSCA proteins are collectively referred to as PSCA-related proteins, proteins of the invention, or PSCA. The term "PSCA-associated protein" means that there are 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25 or more 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, Polypeptide fragments of 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 or PSCA protein sequence. the

II.) PSCA polynucleotide

One aspect of the present invention provides polynucleotides corresponding to or complementary to all or part of the PSCA gene, mRNA and/or coding sequence, preferably in isolated form, including polynucleotides, DNA, RNA encoding PSCA-related proteins and fragments thereof , DNA/RNA hybrids and related molecules, polynucleotides or oligonucleotides complementary to PSCA gene or mRNA sequence or part thereof, and PSCA gene, mRNA or polynucleotides encoding PSCA (collectively referred to as "PSCA polynucleotide") hybridized polynucleotides or oligonucleotides. T can also be U in Figure 1 in all cases mentioned in the chapter. the

Examples of PSCA polynucleotides include: PSCA polynucleotides with the sequence shown in Figure 1, the nucleotide sequence of PSCA shown in Figure 1, wherein T is replaced by U; the polynucleotide with the sequence shown in Figure 1 At least 10 contiguous nucleotides; or at least 10 contiguous nucleotides of a polynucleotide having the sequence shown in Figure 1 but substituting T for U. the

Polynucleotides encoding relatively long portions of PSCA proteins are also within the scope of the invention. For example, about amino acid 1 (or 20 or 30 or 40, etc.) to about amino acid 20 (about 30 or 40 or 50, etc.) polynucleotides. These polynucleotide fragments can include any portion of the PSCA sequence shown in Figure 1. the

II.A.) Uses of PSCA polynucleotides

II.A.1. Monitoring for Genetic Abnormalities

The polynucleotides described above have many different uses. The chromosomal map of the human PSCA gene is presented in the Example entitled "Chromosomal Mapping of PSCA". For example, due to the PSCA gene map of this chromosome, polynucleotides encoding different regions of the PSCA protein are used to determine cytogenetic abnormalities at that chromosomal site, such as those identified to be associated with various cancers. Multiple chromosomal abnormalities, including rearrangements in certain genes, have been identified as frequent cytogenetic abnormalities in many different cancers (see, eg, Krajinovic et al., Mutat. Res. 382(3-4): 81-83 (1998); Johansson et al., Blood 86(10):3905-3914 (1995) and Finger et al., P.N.A.S. 85(23):9158-9162 (1988)). Thus, polynucleotides encoding specific regions of the PSCA protein are most likely new tools to delineate cytogenetic abnormalities in chromosomal regions encoding PSCA that may be responsible for malignant phenotypes more precisely than previously possible. Herein, these polynucleotides meet the need in the art to increase the sensitivity of chromosomal screening to identify more refined and less common chromosomal abnormalities (see, e.g., Evans et al., Am. J. Obstet. Gynecol 171(4): 1055-1057 (1994)). the

In addition, PSCA has been shown to be highly expressed in prostate and other cancers, and PSCA polynucleotides are used in methods for assessing the status of PSCA gene products in normal and cancerous tissues. Typically, polynucleotides encoding specific regions of the PSCA protein are used to assess whether specific regions of the PSCA gene (eg, regions containing one or more motifs) are disturbed (eg, deletions, insertions, point mutations, or changes that result in loss of antigenicity) . Exemplary assays include RT-PCR assays and single-strand conformation polymorphism (SSCP) analysis (see, e. Region-specific polynucleotides to detect these regions within proteins. 

II.A.2. Antisense Embodiments

Other specific nucleic acids relevant to the embodiments of the invention described herein are genomic DNA, cDNA, ribozymes, and antisense molecules, as well as nucleic acid molecules based on alternative backbones or containing alternative bases, which may be derived from natural sources or are synthetic and include molecules capable of inhibiting the expression of RNA or PSCA protein. For example, an antisense molecule can be RNA or other molecules including peptide nucleic acid (PNA) or non-nucleic acid molecules such as phosphorothioate derivatives that specifically bind DNA or RNA in a base pair dependent manner. These types of nucleic acid molecules can be readily obtained by the skilled artisan using the PSCA polynucleotides and polynucleotide sequences described herein. the

Antisense technology requires the use of exogenous oligonucleotides that bind to the target polynucleotide inside the cell. The term "antisense" means that such oligonucleotides are complementary to their intracellular target (e.g. PSCA). See, eg, Jack Cohen, Oligodeoxynucleotides, Antisense Inhibitors of Gene Expression, CRCPress, 1989; and Synthesis 1:1-5 (1988). The PSCA antisense oligonucleotides of the present invention include derivatives, such as S-oligonucleotides (phosphorothioate derivatives or S-oligo, see Jack Cohen, supra), which have enhanced inhibition of cancer cell growth activity. S-oligo (nucleoside phosphorothioate) is an isoelectronic analog of an oligonucleotide (O-oligo) in which the non-bridging oxygen atom of the phosphate group is replaced by a sulfur atom. The S-oligo of the present invention can be prepared by treating the corresponding O-oligo with the sulfur atom transfer agent ³ H-1,2-benzodithiol-3-one-1,1-dioxide. See eg Iyer, RP et al., J. Org. Chem. 55:4693-4698 (1990); and Iyer, RP et al., J. Am. Chem. Soc. 112:1253-1254 (1990). Other PSCA antisense oligonucleotides of the invention include morpholino antisense oligonucleotides known in the art (see, eg, Partridge et al., 1996, Antisense & Nucleid Acid Drug Development 6:169-175).

The PSCA antisense oligonucleotide of the present invention can usually be RNA or DNA that is complementary or stably hybridized to the first 100 5' codons and the last 100 3' codons of the PSCA genome sequence or corresponding mRNA. Full complementarity is not required, although a high degree of complementarity is preferred. The use of oligonucleotides complementary to this region enables selective hybridization to PSCA mRNA but not to mRNA of other regulatory subunits of protein kinases. In one embodiment, a PSCA antisense oligonucleotide of the invention is a 15 to 30 residue fragment of an antisense DNA molecule that contains a sequence that hybridizes to PSCA mRNA. Optionally, the PSCA antisense oligonucleotide is a 30-residue oligonucleotide that is complementary to the first 10 5' codons or the last 10 3' codons of PSCA. Alternatively, the antisense molecules are modified to inhibit PSCA expression with ribozymes, see e.g. L.A. Couture & D.T. Stinchcomb; Trends Genet 12:510-515 (1996). the

II.A.3. Primers and primer pairs

Other specific embodiments of these nucleotides of the invention include primers and primer pairs that are capable of specifically amplifying a polynucleotide of the invention, or any specific portion thereof, and are selective or specific for a nucleic acid molecule of the invention, or any portion thereof. hybridized probes. Probes can be labeled with detectable labels, such as radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelators, or enzymatic labels. The probes and primers are used to detect the presence of PSCA polynucleotides in samples and as a tool for detecting cells expressing PSCA protein. the

Examples of such probes include polypeptides containing all or part of the human PSCA cDNA sequence shown in Figure 1. Examples of primer pairs capable of specifically amplifying PSCA mRNA are also described in the Examples. The skilled artisan will appreciate that a number of different primers and probes can be prepared based on the sequences provided herein and used to efficiently amplify and/or detect PSCA mRNA. the

The PSCA polynucleotides of the present invention can be used for many purposes, including but not limited to their use as probes and primers for the amplification and/or detection of PSCA genes, mRNA or fragments thereof; as diagnostic and/or predictive of prostate cancer and other As an agent for cancer; as a coding sequence capable of directing the expression of a PSCA polypeptide; as a means of modulating or inhibiting PSCA gene expression and/or translation of PSCA transcripts; and as a therapeutic agent. the

The present invention includes the use of any of the probes described herein to identify and isolate PSCA or PSCA-related nucleic acid sequences from natural sources such as humans or other mammals, as well as the isolated nucleic acid sequences themselves, which sequences may be included in the probes used All or most of the sequences found. the

II.A.4. Isolation of PSCA-encoded nucleic acid molecules

The PSCA cDNA sequences described herein enable the isolation of other polynucleotides encoding PSCA gene products, as well as the isolation of polynucleotides encoding PSCA gene product homologs, alternatively spliced isoforms, allelic variants, and mutated forms of PSCA gene products, and polynucleotides encoding PSCA-related protein analogs. Various molecular cloning methods (see for example Sambrook, J. et al., "Molecular Cloning Laboratory Manual" (Second Edition), Cold Spring Harbor Press, New York, 1989; Ausubel Current Methods in Molecular Biology, eds. Wiley and Sons, 1995). For example, lambda phage cloning is conveniently performed using commercially available cloning systems (e.g., Lambda ZAP Express, Stratagene). The labeled PSCA cDNA or its fragments can be used as a probe to identify phage clones containing PSCA gene cDNA. For example, in one embodiment, PSCA cDNA (eg, Figure 1 ) or portions thereof can be synthesized and used as probes to retrieve overlapping and full-length cDNAs corresponding to the PSCA gene. The PSCA gene itself can be isolated by screening genomic DNA libraries, bacterial artificial chromosome libraries (BAC), yeast artificial chromosome libraries (YAC), etc. with PSCA DNA probes or primers. the

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

The present invention also provides recombinant DNA or RNA molecules containing PSCA polynucleotides, fragments, analogs or homologues thereof, including but not limited to phages, plasmids, phagemids, cosmids, YACs, BACs, and well-known in the art Various viral and non-viral vectors, and cells transformed or transfected with such recombinant DNA or RNA molecules. Methods for producing such molecules are well known (see eg Sambrook et al., 1989, supra). the

The present invention also provides host-vector systems comprising recombinant DNA comprising PSCA polynucleotides, fragments, analogs or homologues thereof capable of (expressing) in suitable prokaryotic or eukaryotic host cells. Examples of suitable eukaryotic host cells include yeast cells, plant cells or animal cells, such as mammalian cells or insect cells (eg, cells that can be infected by baculovirus, such as Sf9 or HighFive cells). Examples of suitable mammalian cells include various prostate cancer cell lines, such as DU145 and TsuPr1, other transfectable or transducible prostate cancer cell lines, primary cells (PrEC), and many cells commonly used to express recombinant proteins. Mammalian cells (such as COS, CHO, 293, 293T cells). More specifically, any of the host-vector systems commonly used and widely understood in the art can be used to produce fragments of PSCA protein residues using polynucleotides comprising PSCA coding sequences or fragments, analogs or homologues thereof. the

A number of host-vector systems suitable for expressing PSCA proteins or fragments thereof are available, see eg Sambrook et al., 1989, supra; Current Methods in Molecular Biology, 1995, supra). Preferred vectors for mammalian expression include, but are not limited to, pcDNA 3.1myc-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 several prostate cancer and non-prostate cell lines including, for example, 293, 293T, rat-1, NIH 3T3 and TsuPr1 cells. The host-vector system of the present invention can be used to produce PSCA protein or fragments thereof. This host-vector system can be used to study the functional properties of PSCA and PSCA mutants or analogs. the

Recombinant human PSCA protein or an analog or homologue or fragment thereof can be produced by mammalian cells transfected with a construct encoding a PSCA-related nucleotide. For example, 293T cells can be transfected with an expression plasmid encoding PSCA or a fragment, analog, or homologue thereof such that PSCA-related proteins are expressed in 293T cells and purified using standard purification methods (e.g., affinity purification with an anti-PSCA antibody). Isolation of recombinant PSCA protein. In another embodiment, the PSCA coding sequence was subcloned into the retroviral vector pSR MSVtkneo 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 encoding a leader peptide linked in frame to the PSCA coding sequence can be used to produce secreted forms of recombinant PSCA protein. the

As discussed herein, the redundancy of the genetic code allows for variations in the PSCA gene sequence. Specifically, it is known in the art that a particular species of host generally has a particular codon preference, so sequences known to be preferred by the desired host can be adopted. For example, preferred analog codon sequences typically have high frequency codons in place of rare codons (ie, codons that are used less than 20% of the time in known sequences of the desired host). For example, preferred codons for a specific species can be calculated using a codon usage table available on the Internet (URL: dna.affrc.go.jp/~nakamura/cocon.html). the

Other sequence modifications are known to enhance protein expression within a cellular host. These modifications include deletion of sequences encoding spurious polyadenylation signals, exon/intron splice site signals, transposon-like repeats, and/or other such sequences that are well known to be detrimental to gene expression. The GC content of a sequence can be adjusted to an average level for a given cellular host, which can be calculated with reference to known genes expressed within the cellular host. Where possible, sequence modifications were made to avoid predicted mRNA hairpin secondary structures. Other useful modifications include the addition of a translation initiation consensus sequence at the beginning of the open reading frame as described by Kozak, Mol. Cell Biol., 9:5073-5080 (1989). The skilled artisan is aware of the general principle that eukaryotic ribosomes invariably initiate translation from the AUG codon closest to the 5' end, which only in rare cases fails (see e.g. Kozak PNAS 92(7): 2662 -2666, (1995), and Kozak NAR 15(20):8125-8148(1987)). the

III.) PSCA-related protein

Other aspects of the invention provide PSCA-related proteins. Specific embodiments of the PSCA protein include polypeptides having all or part of the amino acid sequence of human PSCA shown in Figure 1, preferably Figure 1A. Alternatively, embodiments of PSCA proteins include variants, homologues or analog polypeptides with changes in the amino acid sequence of PSCA shown in FIG. 1 . the

Embodiments of PSCA polynucleotides include: a PSCA polypeptide having the sequence shown in Figure 1, the peptide sequence of PSCA shown in Figure 1, wherein T is replaced by U; at least 10 contiguous cores of the polypeptide having the sequence shown in Figure 1 or at least 10 contiguous nucleotides of a polypeptide having the sequence shown in Figure 1 but with T replaced by U. the

Table II provides amino acid abbreviations. Conservative amino acid substitutions can often be made within a protein without altering the conformation or function of the protein. A protein of the invention may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 conservative substitutions. the

Embodiments of the invention described herein include a number of art-accepted variants or analogs of PSCA proteins, such as polypeptides with amino acid insertions, deletions and substitutions. PSCA variants can be produced by 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 cloned DNA to generate PSCA variant DNA. the

Scanning amino acid analysis can also be used to identify one or more amino acids in contiguous sequences that are associated with a particular biological activity, such as protein-protein interactions. Preferred scanning amino acids are relatively small neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is usually the preferred scanning amino acid in this group because it has no side chain on its beta carbon and is less likely to change the main-chain conformation of the variant. Alanine is also generally preferred because it is the most common amino acid. Furthermore, it is commonly found in both buried and exposed locations (Creighton, Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)). Isosteric amino acids can be used if alanine substitutions do not yield an appropriate amount of variants. the

As defined herein, at least one identifiable attribute of a PSCA variant, analog or homologue is the possession of an epitope that "cross-reacts" with a PSCA protein comprising the amino acid sequence of Figure 1 . In this sentence, "cross-reactive" means that an antibody or T cell that specifically binds a PSCA variant also specifically binds a PSCA protein having the amino acid sequence shown in Figure 1 . A polypeptide is no longer a variant of the protein shown in Figure 1 when it no longer contains any epitopes recognized by antibodies or T cells that specifically bind the starting PSCA protein. Those skilled in the art know that antibodies recognizing proteins bind epitopes of varying sizes, from a set of about 4 or 5 contiguous or noncontiguous amino acids considered to be the typical number of amino acids in the smallest epitope. See, eg, Nair et al., J. Immunol 2000 165(12):6949-6955; Hebbes et al., Mol Immunol (1989) 26(9):865-73; Schwartz et al., J Immunol (1985) 135(4):2598- 608. the

Other types of PSCA-related protein variants have 70%, 75%, 80%, 85% or 90% or more similarity to the amino acid sequence of Figure 1 or fragments thereof. Other specific types of PSCA protein variants or analogs comprise one or more of the PSCA biological motifs described herein or currently known in the art. Accordingly, the invention includes analogs (nucleic acid or amino acid) of PSCA fragments that have altered functional (eg, immunogenic) properties compared to the starting fragment. It is also known that such motifs or motifs known in the art will be used for the nucleic acid or amino acid sequences of Figure 1 . the

As discussed herein, embodiments of the invention include polypeptides comprising less than the full-length amino acid sequence of the PSCA protein shown in FIG. 1 . For example, exemplary embodiments of the invention include peptides/proteins comprising any 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids of the PSCA protein shown in Figure 1 . the

PSCA-related proteins are produced using standard peptide synthesis techniques or by chemical splicing methods well known in the art. Alternatively, recombinant methods can be used to produce nucleic acid molecules encoding PSCA-related proteins. In one embodiment, nucleic acid molecules can be used to generate defined fragments of PSCA proteins (or variants, homologs or analogs thereof). the

III.A.) Embodiments of proteins with motifs

Other exemplary embodiments of the invention disclosed herein include PSCA polypeptides comprising amino acid residues of one or more biological motifs contained in the PSCA polypeptide sequence shown in FIG. 1 . Various motifs are known in the art and can be accessed through a number of publicly available Internet sites (e.g., the following URLs: pfam.wustl.edu/; searchlauncher.bcm.tmc.edu/seq-search/struc-predict.html; psort.ims.u-tokyo.ac.jp/; cbs.dtu.dk/; ebi.ac.uk/interpro/scan.html; expasy.ch/tools/scnpsitl.html; Epimatrix ^TM and Epimer ^TM , BrownUniversity, brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html; and BIMAS, bimas.dcrt.nih.gov/.) to assess the presence of such motifs.

Subsequences with motifs for all PSCA variant proteins are listed and identified in Tables V-XVIII and XXII-LI. the

Table IV(h) lists some common motifs based on pfam search (see URL pfam.wustl.edu/). Column (1) of Table IV(h) lists: (1) the abbreviation of the motif name, (2) the percent identity between different members of the motif family, (3) the motif name or description, and ( 4) Most common function; position information is also included if the motif is related to localization. the

Given that the aforementioned PSCA motifs are associated with growth dysregulation and because PSCA is overexpressed in certain cancers (see, eg, Table I), polypeptides containing one or more of the aforementioned PSCA motifs are useful for elucidating specific features of malignant phenotypes. For example, casein kinase II, cAMP and cAMP-dependent protein kinases, and protein kinase C are known enzymes associated with the development of malignant phenotypes (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, glycosylation and myristoylation are also protein modifications associated with cancer and cancer development (see for example 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 that has also been implicated in cancer and cancer development (see eg Treston et al., J. Natl. Cancer Inst. Monogr. (13): 169-175 (1992)). the

In another embodiment, a protein of the invention comprises one or more immunoreactive epitopes identified using art-accepted methods (eg, methods listed in Tables V-XVIII and XXII-LI). CTL epitopes can be determined using specific algorithms to identify peptides within the PSCA protein that best bind to specific HLA alleles (e.g. Table IV; Epimatrix ^™ and Epimer ^™ , Brown University, URLbrown.edu/Research/TB-HIV_Lab/epimatrix /epimatrix.html; and BIMAS, URL bimas.dcrt.nih.gov/). Furthermore, methods for identifying peptides of sufficient binding affinity to HLA molecules and associated with being immunogenic epitopes are well known in the art and can be performed without undue experimentation. Furthermore, methods for identifying peptides that are immunogenic epitopes are well known in the art and can be performed in vitro or in vivo without undue experimentation.

The principle of generating analogs of such epitopes to modulate immunogenicity is also known in the art. For example, we can start with epitopes with CTL or HTL motifs (see e.g. HLA class I and HLA class II motifs/supermotifs of Table IV). Similar epitopes can be generated by substituting an amino acid at a given position and substituting it with another amino acid designated for that position. For example, based on the bases defined in Table IV, we can substitute any other residue, such as a preferred residue, for a detrimental residue; a preferred residue for a less preferred residue; or another preferred residue. Residues are substituted for preferred residues initially generated. Substitutions may occur at the primary anchor position of a peptide or at other positions within the peptide; see, eg, Table IV. the

Many references reflect techniques for identifying and generating epitopes in proteins of interest and their analogs. See eg WO 97/33602 to Chesnut et al; Sette, Immunogenetics 1999 50(3-4):201-212; Sette et al, J. Immunol.2001 166(2):1389-1397; Sidney et al, Hum. Immunol.1997 58(1):12-20; Kondo et al., Immunogenetics 1997 45(4):249-258; Sidney et al., J. Immunol. 1996 157(8):3480-90; and Falk et al., Nature 351:290-6 (1991); Hunt et al., Science 255: 1261-3 (1992); Parker et al., J. Immunol. 149: 3580-7 (1992); Parker et al., J. Immunol. 152: 163-75 (1994)); Kast et al., 1994152(8):3904-12; Borras-Cuesta et al., Hum. Immunol.2000 61(3):266-278; Alexander et al., J.Immunol.20001 64(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 1(9):751-761, and Alexander et al. , Immunol. Res. 1998 18(2):79-92. the

Related embodiments of the present invention include the different motifs listed in Tables IV(a), IV(b), IV(c), IV(d) and IV(h), and/or one or more of Tables V- Predicted CTL epitopes of XVIII and XXII-LI, and/or one or more predicted HTL epitopes of Table XLVIII-LI, and/or combinations of one or more T cell binding motifs known in the art peptide. Preferred embodiments contain no insertions, deletions or substitutions within the motif or intervening sequences of the polypeptide. In addition, instances where there are multiple N-terminal and/or C-terminal amino acid residues on either side of these motifs are also contemplated (e.g., comprising a larger portion of the polypeptide structure comprising the motif). Typically, the number of N-terminal and/or C-terminal amino acid residues on either side of the motif is about 1-100 amino acid residues, preferably 5-50 amino acid residues. the

Examples of PSCA-related proteins can be in many forms, preferably in isolated form. A purified PSCA protein molecule will be substantially free of other proteins or molecules that would impair the binding of PSCA to antibodies, T cells or other ligands. The type and degree of isolation and purification will depend on the desired use. Examples of PSCA-related proteins include purified PSCA-related proteins and functionalized soluble PSCA-related proteins. In one embodiment, the functionalized soluble PSCA protein or fragment thereof retains the ability to be bound by antibodies, T cells or other ligands. the

The present invention also provides a PSCA protein containing a biologically active fragment of the PSCA amino acid sequence shown in FIG. 1 . This protein has the characteristics of the original PSCA protein, for example, it can induce the production of antibodies that can specifically bind to the relevant epitope of the original PSCA protein; can be bound by this antibody; can induce the activation of HTL or CTL; and/or can be similarly specific HTL or CTL recognition of sexually bound protoproteins. the

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, Chou-Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson -Wolf's analytical method, or an immunogenicity-based method. Fragments containing this structure are particularly useful for generating subunit-specific anti-PSCA antibodies or T cells, or for identifying cytokines that bind PSCA. For example, the method of Hopp, T.P. and Woods, K.R. (1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828) can be used to generate hydrophilicity maps and identify immunogenic peptide fragments. The method of Kyte, J. and Doolittle, R.F. (1982, J. Mol. Biol. 157:105-132) can be used to generate hydrophilicity profiles and identify immunogenic peptide fragments. The method of Janin J. (1979, Nature 277:491-492) can be used to generate a percent (%) map of accessible residues and identify immunogenic peptide fragments. The method of Bhaskaran R., Ponnuswamy P.K. (1988, Int. J. Pept. Protein Res. 32: 242-255) was used to generate mean flexural profiles and identify immunogenic peptide fragments. The method of Deleage, G., Roux B. (1987, Protein Engineering 1:289-294) can be used to generate β-turn maps and identify immunogenic peptide fragments. the

Specific algorithms can be used to determine CTL epitopes to identify peptides within the PSCA protein that best bind to a particular HLS allele (e.g., by using the SYFPEITHI site, whose World Wide Web URL is syfpeithi.bmi-heidelberg.com/; Table IV(A) - Algorithms listed for (E); Epimatrix ^™ and Epimer ^™ , Brown University, URL (brown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix.html); and BIMAS, URL bimas.dcrt.nih.gov/ ). Peptide epitopes from PSCA, such as HLA-A1, A2, A3, A11, A24, B7, and B35 described in the Human MHC Class I Molecules section herein, can be predicted using the algorithm described above (see, e.g., Tables V-XVIII, XXII-LI ). Specifically, the entire amino acid sequence of the PSCA protein and relevant portions of other variants that correspond to the predicted HLA class I point mutation or the nine flanking residues on either side of the exon , and predicted HLA class II point mutations or 14 flanking residues on either side of the exon were used in the HLA peptide motif retrieval algorithm available at the aforementioned Bioinformatics and Molecular Analysis Section (BIMAS) website found in ; there is also the SYFPEITHI site at the URL syfpeithi.bmi-heidelberg.com/.

The HLA peptide motif search algorithm was established by Dr. Ken Parker based on the binding of specific peptide sequences in HLA class I molecules, especially the HLA-A2 groove (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)). The algorithm is capable of mapping and ranking 8-, 9-, and 10-peptides from complete protein sequences for predictive binding to HLA-A2 and many other HLA class I molecules. Many HLA class I binding peptides are 8-, 9-, 10 or 11-peptides. For example, for class I HLA-A2, the epitope preferably contains leucine (L) or methionine (M) at position 2 and valine (V) or leucine ( L) (see eg Parker et al., J. Immunol. 149:3580-7 (1992)). Selected PSCA predicted binding peptide results are shown in Tables V-XVIII and XXII-LI herein. The selected candidates (9-peptide and 10-peptide) for each family member are shown in Tables V-XVIII and XXII-XLVIII along with their position, the amino acid sequence of each specific peptide and the estimated binding score. The selected candidate 15 peptides for each family member are shown in Table XLVIII-LI along with their positions, the amino acid sequence of each specific peptide and the estimated binding score. The binding score corresponds to the expected half-life of dissociation of the peptide-containing complex at 37°C, pH 6.5. Peptide cell surface HLA class I molecules with the highest binding scores are expected to bind the most tightly and have the longest half-lives, thus providing the best immunogenic targets for T cell recognition. the

Actual binding of peptides to HLA alleles can be assessed by the stabilization of HLA expression on the antigen processing deficient cell line T2 (see for example Xue et al., Prostate 30:73-8 (1997) and Peshwa et al., Prostate 36:129-38 (1998)). The immunogenicity of a particular peptide can be assessed in vitro by the activation of CD8+ cytotoxic T lymphocytes (CTLs) in the presence of antigen presenting cells such as dendritic cells. the

Preferably, each epitope predicted by the BIMAS site, Epimer ^™ and Epimatrix ^™ site, or specified by an HLA class I or class II motif will be "used" in the PSCA protein of the present invention, said HLAI Class or class II motifs can be obtained or become part of the described techniques, as listed in Table IV (or with the World Wide Web site URL syfpeithi.bmi-heidelberg.com/, or BIMAS, bimas.dcrt.nih.gov/ Sure). "Used to" means herein to evaluate the PSCA protein, for example by visual evaluation or by computer-based pattern-finding methods, which can be conveniently performed by a person skilled in the relevant art. Each subsequence of 8, 9, 10, or 11 amino acid residues with an HLA class I motif, or 9 or more amino acid residue subsequences with an HLA class II motif, of the PSCA protein is fall within the scope of the present invention.

III.B.) Expression of PSCA-related proteins

In one embodiment described in the Examples below, PSCA can be conveniently expressed in cells (such as 293T cells) transfected with a commercially available expression vector, such as a CMV-driven encoding containing six C-terminal Expression vectors for histidine and MYC-tagged PSCA (pcDNA3.1/mycHIS, Invitrogen or Tag5, GenHunter Corporation, Nashville TN). The Tag5 vector provides an IgGK secretion signal that can be used to promote the production of secreted PSCA protein by transfected cells. For example, HIS-tagged PSCA secreted into the culture medium can be purified using nickel columns using standard techniques. the

III.C.) Modification of PSCA-related proteins

Modifications of PSCA-related proteins, such as covalent modifications, are within the scope of the present invention. One type of covalent modification involves reacting targeted amino acid residues of the PSCA polypeptide with organic derivatizing agents capable of reacting with selected side chains or N- or C-terminal residues of the PSCA protein. Another type of covalent modification of a PSCA polypeptide within the scope of the invention involves altering the native glycosylation pattern of the protein of the invention. Another type of covalent modification of PSCA involves linking the PSCA polypeptide to one of a variety of non-proteinaceous polymers such as polyethylene glycol in the manner enumerated in U.S. Patents 4,640,835; 4,496,689; alcohol (PEG), polypropylene glycol or polyoxyalkylene. the

The PSCA-related proteins of the invention can be modified to form chimeric molecules containing PSCA fused to other heterologous polypeptide or amino acid sequences. Such chimeric molecules can be synthesized chemically or recombinantly. Chimeric molecules may contain proteins of the invention fused to other tumor-associated antigens or fragments thereof. Alternatively, the proteins of the invention may contain fusions of fragments of the PSCA sequence (amino acid or nucleic acid sequence), thus forming molecules that are not directly homologous in their length to the amino acid or nucleic acid sequence shown in Figure 1 . Such chimeric molecules may contain multiple identical PSCA subsequences. A chimeric molecule may comprise a fusion of a PSCA-related protein with a polyhistidine epitope tag (providing a fixed nickel-selectable binding epitope) with a cytokine or with a growth factor. The epitope tag is usually placed at the amino- or carboxyl-terminus of the PSCA protein. In another embodiment, a chimeric molecule may comprise a fusion of a PSCA-related protein to an immunoglobulin or a specific region of an immunoglobulin. For the bivalent form of this chimeric molecule (also called "immunoadhesin"), the fusion may be the Fc region of an IgG molecule. Ig fusions preferably comprise a substitution of at least one variable region of the Ig molecule with a soluble (transmembrane domain deleted or inactivated) form of the PSCA polypeptide. In a preferred embodiment, the immunoglobulin fusion comprises the hinge, CH2 and CH3 regions, or the hinge, CHI, CH2 and CH3 regions of an IgGl molecule. See, eg, US Patent No. 5,428,130, issued June 27, 1995, for making immunoglobulin fusions. the

III.D.) Uses of PSCA-related proteins

The proteins of the invention have many different uses. Since PSCA is highly expressed in prostate and other cancers, PSCA-related proteins are used in methods to assess the status of PSCA gene products in normal and cancerous tissues to determine the malignant phenotype. Typically, polypeptides from specific regions of the PSCA protein are used to assess whether there is a disorder (eg, deletion, insertion, point mutation, etc.) in those regions (eg, a region containing one or more motifs). Exemplary assays utilize antibodies or T cells targeting PSCA-related proteins containing amino acid residues of one or more biological motifs contained in the PSCA polypeptide sequence to evaluate the properties of this region in normal and cancerous tissues, or An immune response to the epitope is elicited. Alternatively, PSCA-related proteins containing amino acid residues from one or more biologically active motifs of PSCA proteins are used to screen for factors that interact with this region of PSCA. the

PSCA protein fragments/subsequences are particularly useful for generating or characterizing domain-specific antibodies (e.g., antibodies that recognize extracellular or intracellular epitopes of PSCA protein) to identify self or cytokines that bind PSCA or specific domains thereof, and in They are particularly useful in a variety of therapeutic and diagnostic applications including, but not limited to, diagnostic assays, cancer vaccines, and methods of making such vaccines. the

Proteins encoded by the PSCA gene or analogs, homologues or fragments thereof have many uses including, but not limited to, the production of antibodies and the identification of ligands and other reagents and cellular components that bind to PSCA gene products. Antibodies raised against PSCA proteins or fragments thereof are useful in diagnostic and prognostic assays and in imaging methods for those cancers listed in Table I characterized by expression of PSCA proteins in humans. Such antibodies can be expressed intracellularly and used in methods of treating such cancer patients. PSCA-associated nucleic acids or proteins are also used to generate HTL or CTL responses. the

Various immunoassays effective for detecting PSCA protein are used, including but not limited to various types of radioimmunoassays, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunofluorescence assay (ELIFA), immunocytochemistry, etc. . Antibodies can be labeled and used as immunoimaging agents capable of detecting cells expressing PSCA (eg, in radioscintigraphy). PSCA proteins are also particularly useful for generating cancer vaccines, as described further herein. the

IV.) Antibody to PSCA

Another aspect of the invention provides antibodies that bind PSCA-related proteins. Preferred antibodies are peptides or proteins that specifically bind PSCA-related proteins but do not bind (or bind weakly) to proteins that are not PSCA-related under physiological conditions. Herein, examples of physiological conditions include: 1) phosphate-buffered saline; 2) Tris-buffered saline containing 25 mM Tris and 150 mM NaCl; or physiological saline (0.9% NaCl); 4) animal serum such as human serum; or 5) 1)- 4) the combination of any one; These reactions should occur at pH7.5, or occur in the scope of pH7.0-8.0, or occur in the scope of pH6.5-8.5; Simultaneously, these reactions occur at 4- Occurs at a temperature of 37°C. For example, an antibody that binds PSCA can bind a PSCA-related protein, such as a homolog or analog thereof. the

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

The invention also provides various immunoassays for detecting and quantifying OSCA and mutant PSCA-related proteins. Such assays may include one or more PSCA antibodies that appropriately recognize and bind PSCA-related proteins. These assays can be performed in various immunoassay formats well known in the art, including various types of radioimmunoassays, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunofluorescent assay (ELIFA), and the like. the

Non-antibody immunoassays of the invention may also include T cell immunogenicity assays (inhibition or stimulation) as well as major histocompatibility complex (MHC) binding assays. the

In addition, the present invention provides immunoimaging methods capable of detecting prostate cancer and other PSCA-expressing cancers, including but not limited to radioscintigraphy using labeled PSCA antibodies. This assay is used clinically for the detection, monitoring and prediction of PSCA expressing cancers such as prostate cancer. the

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

Various methods of making antibodies are well known in the art. For example, antibodies can be prepared by immunizing a mammalian host with 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 may also be used, such as PSCAGST-fusion proteins. In a specific embodiment, a GST fusion protein comprising all or most of the amino acid sequence of Figure 1 is produced and then used as an immunogen to generate suitable antibodies. In another embodiment, PSCA-related proteins are synthesized and used as immunogens. the

In addition, naked DNA immunization techniques known in the art were used (with or without purified PSCA-related protein or PSCA-expressing cells) to generate an immune response to the encoded immunogen (see Donnelly et al., 1997, Ann. Rev. Immunol. 15:617-648). the

The amino acid sequence of the PSCA protein shown in Figure 1 can be analyzed to select specific regions of the PSCA protein that produce antibodies. For example, hydrophobicity and hydrophilicity analysis of the PSCA amino acid sequence can be used to identify hydrophilic regions in the PSCA structure. Regions of the PSCA protein having immunogenic structures, 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 maps can be generated by the method of Hopp, T.P. and Woods, K.R. (1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824-3828). Hydrophilicity profiles can be generated by the method of Kyte, J. and Doolittle, R.F. (1982, J. Mol. Biol. 157:105-132). A map of the percent (%) accessible residues can be generated using the method of Janin J. (1979, Nature 277:491-492). Mean buckling maps were generated by the method of Bhaskaran R., Ponnuswamy P.K. (1988, Int. J. Pept. Protein Res. 32:242-255). Beta-turn maps can be generated by the method of Deleage, G., Roux B. (1987, Protein Engineering 1:289-294). Accordingly, regions identified by any of these procedures or methods are within the scope of the invention. Preferred methods for generating PSCA antibodies are further illustrated herein by way of examples. Methods for preparing proteins or polypeptides for use as immunogens are well known in the art. Methods for preparing immunogenic conjugates of proteins with carriers such as BSA, KLH or other carrier proteins are also well known in the art. In some cases, direct conjugation is employed, for example using carbodiimide reagents; in other cases, conjugation reagents such as those supplied by Pierce Chemical Co. (Rockford, IL) are effective. The PSCA immunogen is often administered by injection with a suitable adjuvant at an appropriate time, as is known in the art. During the immunization process, the antibody titer can be used to determine whether sufficient antibodies are formed. the

Monoclonal antibodies to PSCA can be produced by various methods well known in the art. For example, it is well known to immortalize antibody-producing B cells using the standard hybridoma technique of Kohler and Milstein or a modification thereof to produce immortal cell lines that secrete the desired monoclonal antibody. Immortal cell lines secreting the desired antibody were screened by immunoassay using PSCA-related protein as antigen. When suitable immortal cell cultures are identified, cells can be expanded and antibodies produced from in vitro cultures or from ascitic fluid. the

Antibodies or fragments of the invention can be produced by recombinant methods. Domains that specifically bind desired regions of the PSCA protein can also be produced in multi-species chimeric or complementarity determining region (CDR) grafted antibody formats. Humanized or human PSCA antibodies can also be made and are advantageously used in therapy. Methods for preparing humanized murine or other nonhuman antibodies by substituting one or more CDRs of a nonhuman antibody for the corresponding human antibody sequences are well known (see, e.g., 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. the

Methods for making fully human monoclonal antibodies include phage display and transgenic methods (see, eg, Vaughan et al., 1998, Nature Biotechnology 16:535-539). Fully human PSCA monoclonal antibodies can be generated by cloning techniques (i.e., phage display) using large combinatorial libraries of human Ig genes (Griffiths and Hoogenboom, "Building an in vitro immune system: Human antibodies from a phage display library" system: human antibodies from phagedisplay libraries). Quoted from: "Protein Engineering of Antibody Molecules for Prophylactic and Therapeutic Applications in Man" written by Clark, M., Nottingham Academic, pp. 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 loci, as described in PCT Patent Application WO 98/24893, published December 3, 1997 by Kucherlapati and Jakobovits et al. (See also Jakobovits, 1998, Exp. Opin. Invest. Drugs 7(4):607-614; U.S. Pat. 6,114598 published on the 5th). This method avoids the in vitro manipulation required by phage display technology and can efficiently produce high-affinity true human antibodies. the

The reactivity of PSCA antibodies with PSCA-related proteins can be established by a number of well-known methods, including Western blotting, immunoprecipitation, ELISA and FACS analysis using appropriate PSCA-related proteins, PSCA-expressing cells or extracts thereof. The PSCA antibody or fragment thereof can be labeled with a detectable label or conjugated to a second molecule. Suitable detectable labels include, but are not limited to, radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelators, or enzymes. In addition, bispecific antibodies to two or more epitopes of PSCA are generated using methods generally understood 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). the

In a preferred embodiment, the monoclonal antibodies known as Ha1-1.16, Ha1-5.99, Ha1-4.117, Ha1-4.20, Ha1-4.121, Ha1-4.37 provided by the present invention were produced on May 4, 2005 They were sent to the American Type Culture Collection (ATCC) and assigned accession numbers PTA-6698, PTA-6703, PTA-6699, PTA-6700, PTA-6701 and PTA-6702, respectively. the

V.) PSCA cellular immune response

The mechanism by which T cells recognize antigens has been elucidated. Effective peptide epitope vaccine compositions of the present invention are capable of inducing therapeutic and prophylactic immune responses in most populations worldwide. To understand the value and efficacy of the conjugates of the invention in inducing cellular immune responses, a brief review of immunology-related techniques is provided. the

HLA-restricted T cells recognize complexes of HLA molecules and peptide antigens as ligands (Buus, S. et al., Cell 47:1071, 1986; Babbitt, B.P. et al., Nature 317:359, 1985; Townsend, A. and Bodmer, H., Annu. Rev. Immunol. 7:601, 1989; Germain, R.N., Annu. Rev. Immunol. 11:403, 1993). By studying the sequence determination of single amino acid substituted antigen analogs and naturally processed endogenous binding peptides, key residues corresponding to the motifs required for specific binding of HLA antigen molecules have been identified, listed in Table IV (also See, e.g., Southwood et al., J. Immunol. 160:3363, 1998; Rammensee et al., Immunogenetics 41:178, 1995; Rammensee et al., SYFPEITHI, Access the World Wide Web URL site (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 Gray, H.M., Curr . Opin. Immunol. 4:79, 1992; Sinigaglia, F. and Hammer, J.Curr.Biol. 4307-4312, 1995; Sidney et al., J. Immunol. 157:3480-3490, 1996; Sidney et al., Immunol. 45:79-93, 1996; Sette, A. and Sidney, J. Immunogenetics 1999-11; 50 (3-4): 201-12, Review). the

Furthermore, X-ray crystallographic analysis of HLA-peptide complexes revealed that pockets within the peptide-binding cleft/groove of HLA molecules can accommodate residues of peptide ligands in an allele-specific manner; Ability of peptides to bind HLA. (see for example 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; 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). the

Thus, the determination of class I and class II allele-specific HLA binding motifs, or class I or class II supermotifs, enables the identification of relevant regions within proteins that bind specific HLA antigens. the

Thus, epitope-based vaccine candidates can be identified by identifying HLA motifs; such candidates can also be further evaluated by HIA-peptide binding assays to determine the binding affinity and/or binding affinity associated with the epitope and its corresponding HLA molecule. Combine cycle. Additional validation work can be performed to select among these vaccine candidates epitopes with better properties in terms of population coverage and/or immunogenicity. the

A variety of methods can be used to assess the immunogenicity of cells, 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). The procedure involves stimulating peripheral blood lymphocytes (PBL) of normal humans with a test peptide for several weeks in vitro in the presence of antigen presenting cells. The peptide-specific T cells will be activated during this period, which can be detected, for example, by a lymphokine- or 51Cr-release assay of peptide-sensitized target cells. the

2) Immunization of HLA transgenic mice (see for example Wentworth, P.A. et al., J.Immunol. 159:4753, 1997). For example, in this method, Hla transgenic mice are subcutaneously administered the peptide in Freund's incomplete adjuvant. Splenocytes were harvested several weeks after immunization and cultured in vitro for approximately 1 week in the presence of the test peptide. Peptide-specific T cells are detected using, for example, 51Cr-release assays of peptide-sensitized target cells and target cells expressing endogenously produced antigens. the

3) Demonstration of memory T cell responses in immunized individuals who have been effectively vaccinated and/or in 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; 1997). Thus, memory responses can be tested by culturing PBLs from persons who have been exposed to the antigen due to the disease and thus mounted a "natural" immune response, or who have been vaccinated against this antigen. The patient's PBLs were cultured in vitro for 1-2 weeks in the presence of the test peptide and antigen-presenting cells (APCs) to activate "memory" T cells, compared to "naive" T cells. At the end of the culture period, T cell activity was measured using assays including 51Cr release from peptide-sensitized target cells, T cell proliferation, or lymphokine release. the

VI.) PSCA transgenic animals

Those encoding PSCA-related proteins can also be used to generate transgenic or "knockout" animals, which can then be used in the development and screening of therapeutically useful agents. According to prior art, cDNA encoding PSCA can be used to clone genomic DNA encoding PSCA. The cloned genomic sequence can then be used to generate transgenic animals containing cells expressing DNA encoding PSCA. Methods of making transgenic animals, especially animals such as mice or rats, have become routine in the art and are described, for example, in U.S. Patent No. 4,736,866, issued April 12, 1988 and U.S. Patent No. 4,736,866, issued September 26, 1989. 4,870,009 published today. Typically, specific cells will be targeted for incorporation of the PSCA transgene with a tissue-specific enhancer. the

Transgenic animals containing a copy of the transgene encoding PSCA can be used to examine the effect of enhanced expression of DNA encoding PSCA. Such animals can also be used as experimental animals for agents believed to confer protection, for example, from pathological conditions associated with their overexpression. According to this aspect of the invention, animals treated with the agent have a reduced incidence of the pathological condition compared to untreated animals having the transgene, indicating that the agent has a potential therapeutic effect on the pathological condition. the

Alternatively, a non-human homolog of PSCA can be used to construct a PSCA "knockout" animal that results in the encoding of The gene for PSCA is defective or altered. For example, cDNA encoding PSCA can be used to clone genomic DNA encoding PSCA according to prior art. A portion of the genomic DNA partially encoding PSCA can be deleted or replaced with another gene (eg, a gene encoding a selectable marker that can be used to monitor integration). Typically, thousands of bases 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 homologous recombination vectors) . The vector is introduced into an embryonic stem cell line (eg, by electroporation) and cells are selected for which the introduced DNA has been homologously recombined with endogenous DNA (see, eg, Li et al., Cell, 69:915 (1992)). The selected cells are then injected into blastocysts of animals such as mice or rats to form aggregated chimeras (see e.g. Bradley in Teratocarcinomas and Embryonic Stem Cells Actual Progress by E.J. Robertson). Cells: A Practical Approach) (IRL, Oxford, 1987), pp. 113-152). The chimeric embryos can then be implanted into a suitable pseudopregnant female foster animal, which is then brought to term to produce a "knockout" animal. Progeny carrying the homologously recombined DNA in their germ cells can be identified by standard techniques and used to reproduce animals in which all of the animal's cells contain the homologously recombined DNA. Knockout animals can be characterized for their ability to resist disease or develop disease due to a lack of PSCA polypeptide. the

VII.) Method for detecting PSCA

Another aspect of the invention relates to methods of detecting PSCA polynucleotides and PSCA-related proteins, and methods of identifying cells expressing PSCA. The expression profile of PSCA makes it a diagnostic marker for metastatic disease. Thus, the status of PSCA gene products provides useful information for predicting various factors including susceptibility to develop advanced disease, rate of progression, and/or tumor aggressiveness. As discussed in detail herein, the status of the PSCA gene product in a patient sample can be analyzed by various methods well known in the art, including immunohistochemical analysis, various Northern blot techniques including in situ hybridization, RT-PCR analysis ( Examples include RT-PCR, Western blot analysis, and tissue array analysis on laser capture microdissected samples. the

More specifically, the invention provides assays for detecting PSCA polynucleotides in biological samples (eg, serum, bone, prostate, and other tissues, urine, semen, cell preparations, etc.). Detectable PSCA polynucleotides include, for example, the PSCA gene or fragments thereof, PSCA mRNA, alternate splice variants of PSCA mRNA, and recombinant DNA or RNA molecules comprising PSCA polynucleotides. A number of methods for amplifying and/or detecting the presence of PSCA polynucleotides are well known in the art and can be used in the practice of this aspect of the invention. the

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

In another embodiment, a method for detecting a PSCA gene in a biological sample comprises first 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 combinations of suitable sense and antisense probes can be designed from the PSCA nucleotide sequence (see eg, Figure 1) and used for this purpose. the

The present invention also provides assays for detecting the presence of PSCA protein in tissues or other biological samples (eg, serum, semen, bone, prostate, urine, cell preparations, etc.). Methods for detecting PSCA-related proteins are also well known and include, for example, immunoprecipitation, immunohistochemical analysis, Western blot analysis, molecular binding assays, ELISA, ELIFA, and the like. For example, a method of detecting the presence of a PSCA-associated protein in a biological sample comprises first contacting the sample with a PSCA antibody, a PSCA-reactive fragment of an antibody, or a recombinant protein comprising an antigen-binding region of the PSCA antibody; and then detecting binding of the PSCA-associated protein in the sample. the

Methods of identifying cells expressing PSCA are also within the scope of the invention. In one embodiment, the assay for identifying cells expressing a PSCA gene comprises detecting the presence or absence of PSCA mRNA in the cells. Methods for detecting specific mRNA in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization with labeled PSCA riboprobes, Northern blotting, and related techniques) and various nucleic acid amplifications Assays (such as RT-PCR with specific complementary primers for PSCA, and other types of amplification assays such as eg branched DNA, SISBA, TMA, etc.). Alternatively, assays to identify cells expressing a PSCA gene include detecting the presence of PSCA-related proteins within or secreted by the cells. Various methods for detecting proteins are well known in the art and are used to detect PSCA-related proteins and cells expressing PSCA-related proteins. the

PSCA expression analysis is also used as a tool to identify and evaluate agents that modulate PSCA gene expression. For example, PSCA expression is significantly upregulated in prostate cancer and expressed in cancers of the tissues listed in Table I. Identifying molecules or biological agents that inhibit the expression or overexpression of PSCA in cancer cells has therapeutic value. For example, screening methods that quantify PSCA expression by RT-PCR, nucleic acid hybridization, or antibody binding can be used to identify such agents. the

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

Tumor formation is known to be a multistep process in which cell growth is progressively deregulated while cells progress from a normal physiological state to a precancerous state and then to a 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)). Here, examining biological samples for evidence of dysregulated cell growth, such as aberrant PSCA expression in cancer, enables early detection of abnormal physiological states, such as cancer, before they progress to a stage where treatment options are more limited or prognosis is poorer. In such embodiments, the state of PSCA in a biological sample of interest can be compared to, for example, the state of PSCA in a corresponding normal sample (eg, a sample from an individual or other individual not affected by the pathology). Altered PSCA status in biological samples (compared to normal samples) provides evidence of unregulated cell growth. Instead of using a biological sample not affected by a lesion as a normal sample, we can also use a predetermined standard value, such as a predetermined normal mRNA expression level (see, for example, Grever et al., J. Comp. Neurol. 1996-12-9; 376(2 ): 306-14 and US Patent 5,837,501) were compared to the PSCA status of the samples. the

Herein, the term "state" has an art-accepted meaning and refers to the condition or state of a gene and its product. In general, a number of parameters are available to the skilled artisan to assess the condition or state of a gene and its product. These parameters include, but are not limited to, the location (including location of PSCA expressing cells) and level of expressed gene products, biological activity of expressed gene products (such as PSCA mRNA, polynucleotides and polypeptides). Typically, changes in PSCA status include changes in PSCA and/or cellular localization of PSCA expression, and/or increased expression of PSCA mRNA and/or protein. the

The PSCA status of a sample can be analyzed by a number of methods well known in the art, including but not limited to immunohistochemical analysis, in situ hybridization, RT-PCR analysis on laser capture microdissected samples, Western blot analysis, and tissue array analysis. Typical methods for assessing the status of the PSCA gene and gene products can be found, for example, in: Ausubel et al. ) and 18 (PCR analysis) units. Accordingly, the skilled artisan can assess the PSCA status of a biological sample using a variety of methods including, but not limited to, genomic Southern analysis (to detect, for example, disturbances within the PSCA gene), Northern analysis of PSCA mRNA, and/or PCR analysis (using For example, to detect the change of polynucleotide sequence or PSCA mRNA expression level), and Western and/or immunohistochemical analysis (used to detect, for example, the change of polypeptide sequence, the change of polypeptide localization in the sample, the change of PSCA protein expression level and/or the combination of PSCA protein and polypeptide binding partner). Detectable PSCA polynucleotides include, for example, the PSCA gene or fragments thereof, PSCA mRNA, alternative splice variants, PSCA mRNA, and recombinant DNA or RNA molecules comprising PSCA polynucleotides. the

The expression profile of PSCA makes it a diagnostic marker for localized and/or metastatic disease and provides information on the growth or oncogenic potential of biological samples. Specifically, PSCA status provides useful information for predicting susceptibility to specific disease stages, progression and/or tumor aggressiveness. The present invention provides methods and assays for determining PSCA status and diagnosing PSCA-expressing cancers, such as cancers of tissues listed in Table I. For example, because PSCA mRNA is so highly expressed in prostate and other cancers compared to normal prostate tissue, assays that evaluate PSCA mRNA transcript or protein levels in biological samples can be used to diagnose diseases associated with PSCA dysregulation and can be used in Provides useful predictive information when determining appropriate treatment. the

The expression status of PSCA provides information including the presence, status and localization of dysplastic, precancerous and cancerous cells; predicting susceptibility to various disease stages and/or measuring tumor aggressiveness. Furthermore, this expression profile makes it useful as an imaging agent for metastatic disease. Accordingly, one aspect of the invention pertains to various molecular prognostic and diagnostic methods for detecting PSCA status in biological samples, such as samples from individuals suffering from or suspected of having a disorder characterized by unregulated cell growth, such as cancer. the

As noted above, the PSCA status of a biological sample can be detected by a number of methods well known in the art. For example, the PSCA status of a biological sample taken from a particular part of the body can be detected by assessing the presence or absence of PSCA expressing cells (eg, cells expressing PSCA mRNA or PSCA protein) in the sample. For example, when PSCA-expressing cells are found in a biological sample that does not normally contain such cells, such as a lymph node, since such changes in PSCA status in the biological sample are often associated with dysregulated cell growth, this assay may provide Evidence of unregulated cell growth. Specifically, one indicator of unregulated cell growth is the metastasis of cancer cells from the organ of origin (such as the prostate) to other areas of the body (such as the lymph nodes). Evidence of dysregulated cell growth is important here because, for example, occult lymph node metastases can be detected in a proportion of prostate cancer patients and 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 1995-8154(2 Pt 1): 474-8). the

In one aspect, the invention provides a method of monitoring PSCA gene product by determining the state of PSCA gene product expressed by the cells of an individual suspected of having a disease (such as hyperplasia or cancer) associated with dysregulation of cell growth, which will then The determined status is compared to the status of the corresponding normal tissue PSCA gene product. The presence of an abnormal PSCA gene product in a test sample relative to normal tissue is indicative of unregulated cell growth in the individual's cells. the

In another aspect, the invention provides an assay useful for determining the presence of cancer in an individual, the method comprising detecting a significant increase in the expression of PSCA mRNA or protein in a test cell or tissue sample relative to the expression level in a corresponding normal cell or tissue . The presence of PSCA mRNA can be assessed in tissues such as, but not limited to, those listed in Table I. Significant PSCA expression in any of these tissues can be used to indicate the presence, presence, and/or severity of cancer, since corresponding normal tissues do not express PSCA mRNA or express it at lower levels. the

In related embodiments, PSCA status is determined at the protein level rather than the nucleic acid level. For example, such methods include determining the level of PSCA protein expressed by cells of a test tissue sample, and comparing the level so determined to the level of PSCA expressed in a corresponding normal sample. In one embodiment, the presence of PSCA protein is assessed, eg, by immunohistochemistry. PSCA antibodies or binding partners capable of detecting PSCA protein expression are used in various assay formats well known in the art for this purpose. the

In another embodiment, we can evaluate the status of PSCA nucleotide and amino acid sequences in biological samples to identify disturbances within these molecular structures. These perturbations may include insertions, deletions, substitutions, and the like. This evaluation is useful because disturbances in nucleotide and amino acid sequences have been observed in a large number of proteins associated with growth disorder phenotypes (see e.g. Marrogi et al., 1999, J. Cutan. Pathol. 26(8): 369- 378). For example, mutations in the PSCA sequence can indicate the presence or promotion of tumors. Thus, mutations in PSCA indicate underlying loss of function or accelerated tumor growth, and this assay has diagnostic and predictive value. the

A number of assays for observing disturbances within nucleotide and amino acid sequences are well known in the art. For example, the size and structure of the PSCA gene product nucleic acid or amino acid sequence can be visualized by Northern, Southern, Western, PCR and DNA sequencing methods described herein. In addition, other methods of observing disturbances within nucleotide and amino acid sequences, such as single-strand conformation polymorphism analysis, are well known in the art (see, e.g., U.S. Patent No. 5,382,510 issued September 7, 1999, and 5,952,170 published on . the

In addition, we can detect the methylation status of the PSCA gene in biological samples. Aberrant demethylation and/or hypermethylation of CpG islands in the 5' regulatory regions of genes is frequently observed in immortal and transformed cells and can lead to altered expression of various genes. For example, promoter hypermethylation of π-glutathione-like S-transferase (a protein expressed in normal prostate but not in more than 90% of prostate cancers) will permanently silence transcription of this gene, and is the most frequently detected genomic alteration in prostate cancer (De Marzo et al., Am. J. Pathol. 155(6):1985-1992 (1999)). Furthermore, this change occurs in at least 70% of high-grade prostatic intraepithelial neoplasia (PIN) (Brooks et al., Cancer Epidemiol. Biomarkers Prev., 1998, 7:531-536). In another embodiment, deoxy-azacytidine induces the expression of the LAGE-I tumor-specific gene (which is not expressed in normal prostates but is expressed in 25-50% of prostate cancers) 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 detecting the methylation status of genes are well known in the art. For example, we can use restriction enzymes that are sensitive to methylation but cannot cleave sequences containing methylated CpG sites in the Southern hybridization method to understand the methylation status of CpG islands. In addition, MSP (Methylation-Specific PCR) quickly reveals the methylation status of all CpG sites present in the CpG islands of a given gene. The process involves first modifying the DNA with sodium bisulfite (which will convert all unmethylated cytosines to uracil), followed by amplification with specific primers for methylated and non-unmethylated DNA. An approach to methylation disorders can also be found, for example, in Current Methods in Molecular Biology (1995), Unit 12, eds. Frederick M. Ausubel et al. the

Gene amplification is another method of obtaining PSCA status. Direct determination of gene amplification in samples, e.g. by conventional Southern or Northern blotting to quantify mRNA transcription (Thomas, 1980, Proc. Natl. Acad. Sci. USA, 77: 5201 5205), dot blot (DNA analysis) or In situ hybridization using appropriately labeled probes according to the sequences provided here. Alternatively, antibodies that recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA RNA hybrid duplexes or DNA protein duplexes, can be used. The antibodies are then labeled and assayed in which the duplexes bind to the surface so that duplexes are formed on the surface and thus the presence or absence of antibodies bound to the duplexes can be detected. the

Biopsies or peripheral blood are typically tested for the presence of cancer cells using, for example, Northern blot, dot blot, or RT-PCR analysis to detect PSCA expression. The presence of RT-PCR amplifiable PSCA mRNA indicates the presence of cancer. RT-PCR assays are well known in the art. RT-PCR assays of peripheral blood tumor cells are currently being evaluated for use in the diagnosis and management of a variety of human solid tumors. In the field of prostate cancer, these include RT-PCR assays to detect cells expressing PSA and PSM (Verkaik et al., 1997, Urol. Res. 25:373-384; Ghossein et al., 1995, J. Clin. Oncol. 2000; Heston et al., 1995, Clin. Chem. 41: 1687-1688). the

Another aspect of the invention is the assessment of an individual's susceptibility to cancer. In one embodiment, the method of predicting susceptibility to cancer comprises detecting PSCA mRNA or PSCA protein in a tissue sample, the presence of which is indicative of susceptibility to cancer, wherein the degree of expression of PSCA mRNA corresponds to the degree of susceptibility. In a specific embodiment, the presence of PSCA in the prostate or other tissue is detected, and the presence of PSCA in the sample indicates susceptibility to prostate cancer (or the presence or presence of a prostate tumor). Similarly, we can assess the integrity of PSCA nucleotide and amino acid sequences within biological samples to identify disturbances in these molecular structures, such as insertions, deletions, substitutions, and the like. The presence of one or more disturbances in a sample PSCA gene product is indicative of cancer susceptibility (or the presence or presence of a tumor). the

The invention also includes methods of determining tumor aggressiveness. In one embodiment, the method of determining tumor aggressiveness comprises determining the level of PSCA mRNA or PSCA protein expressed by tumor cells, comparing the level so determined to the level of PSCA mRNA expressed in corresponding normal tissue taken from the same individual or a reference sample of normal tissue. The expression level of PSCA mRNA or PSCA protein in tumor samples relative to normal samples indicates the degree of invasiveness. In a specific embodiment, tumor aggressiveness is evaluated by determining the expression level of PSCA in tumor cells, with higher expression levels indicating more aggressive tumors. Other embodiments evaluate the integrity of PSCA nucleotide and amino acid sequences in biological samples to identify disturbances within these molecular structures, such as insertions, deletions, substitutions, and the like. The presence of one or more disorders indicates a more aggressive tumor. the

Another embodiment of the invention relates to a method of observing the development of a malignancy in an individual over a period of time. In one embodiment, a method of observing the development of a malignancy in an individual over time comprises determining the level of PSCA mRNA or PSCA protein expressed by cells within a tumor sample, comparing the levels so determined to the same tissue sample taken at different times from the same individual. The levels of PSCA mRNA or PSCA protein expressed over a period of time are compared, where the extent of PSCA mRNA or PSCA protein expressed in a tumor sample over a period of time provides information on cancer development. In a specific embodiment, cancer progression is assessed by measuring expression of PSCA in tumor cells over a period of time where increased expression is indicative of cancer progression. At the same time, we can evaluate the integrity of PSCA nucleotide and amino acid sequences in biological samples to identify disturbances within these molecular structures, such as insertions, deletions, substitutions, etc., the presence of one or more of which is indicative of cancer development . the

The diagnostic methods described above may also be combined with any of a variety of prognostic and diagnostic methods known in the art. For example, another embodiment of the present invention relates to a method for observing the expression (or disturbance in PSCA gene and PSCA gene product) of PSCA gene and PSCA gene product consistent with the factors associated with malignancy, which is used as a diagnostic and prognostic tissue sample state means. A variety of factors associated with malignancy can be used, such as expression of malignancy-associated genes (e.g., expression of PSA, PSCA, and PSM in prostate cancer, etc.) and gross cytological observations (see, e.g., Bocking et al., 1984, Anal. Quant.Cytol.6(2):74-88; Epstein, 1995, Hum.Pathol.26(2):223-9; Thorson et al., 1998, Mod.Pathol.11(6):543-51; Baisden et al. , 1999, Am. J. Surg. Pathol. 23(8):918-24). For example, because of the presence of a specific set of factors consistent with disease, which provides important information for diagnosis and prediction of tissue sample status, observation of expression of (or disturbances in) PSCA genes and PSCA gene products is associated with A consistent approach to other malignancy-associated factors would be useful. the

In one embodiment, the method of observing expression (or disturbances in PSCA genes and PSCA gene products) of PSCA genes and PSCA gene products consistent with other malignancy-associated factors entails detection of overexpression of PSCA mRNA or protein in tissue samples, Detect the overexpression of PSA mRNA or protein (or the expression of PSCA or PSM) in the tissue sample, and observe the consistency of the overexpression of PSCA mRNA or protein and PSA mRNA or protein (or the expression of PSCA or PSM). In a specific embodiment, the expression of PSCA and PSA mRNA in prostate tissue is detected, and the overexpression of PSCA and PSA mRNA in the sample is consistent with the presence of prostate cancer, susceptibility to prostate cancer, or recurrence or status of prostate tumors . the

Methods for detecting and quantifying expression of PSCA mRNA or protein are described herein, and standard nucleic acid and protein detection and quantification techniques are well known in the art. Standard methods to detect and quantify PSCA mRNA include in situ hybridization using labeled PSCA riboprobes, Northern blotting and related techniques using PSCA polynucleotide probes, RT-PCR analysis using PSCA-specific primers, and other amplifications. Types of detection methods, such as branched DNA, SISBA, TMA, etc. In a specific embodiment, semi-quantitative RT-PCR is used to detect and quantify the expression of PSCA mRNA. 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 wild-type PSCA protein are used in immunohistochemical assays of biopsies. the

IX.) Identification of Molecules Interacting with PSCA

A skilled artisan can use the PSCA protein and nucleic acid sequences disclosed herein to identify proteins, small molecules and other agents that interact with PSCA by any of a variety of methods accepted in the art. For example, we can use a system called interaction traps (also called "two-hybrid assays"). In this system, the expression of a reporter gene can be detected by interacting with and reconstituting a molecule that directs the expression of a reporter gene. Other systems identify protein-protein interactions in vivo by reconstitution of eukaryotic transcriptional activators, see for example U.S. Patents 5,955,280 issued September 21, 1999, 5,925,523 issued July 20, 1999, and December 8, 1998 5,846,722 and 6,004,746 published on December 21, 1999. Genome-based algorithms for predicting protein function also exist in the art (see, eg, Marcotte et al., Nature 402:4 Nov. 4, 1999, 83-86). the

Alternatively, we can screen peptide libraries to identify molecules that interact with the PSCA protein sequence. In this method, PSCA-binding peptides are identified by screening libraries encoding random or controlled collections of amino acids. Peptides encoded by this library are expressed as fusion proteins of phage coat proteins, and phage particles against PSCA proteins can then be selected. the

Thus, peptides with various uses, such as therapeutic, prognostic or diagnostic peptides, were identified without any prior knowledge of the structure of the 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 US Patent Nos. 5,723,286, issued March 3,1998 and 5,733,731, issued March 31,1998. the

Alternatively, cell lines expressing PSCA were used to identify PSCA-mediated protein-protein interactions. This interaction can be detected using immunoprecipitation techniques (see eg Hamilton B.J. et al. Biochem. Biophys. Res. Commun. 1999, 261:646-51). PSCA protein can be immunoassayed from PSCA expressing cell lines with an anti-PSCA antibody. Alternatively, anti-His-tag antibodies can be employed in cell lines engineered to express fusions of PSCA and His-tag (vectors described above). Protein binding of immunoprecipitated complexes can be detected by methods such as Western blotting, 35S-methionine-labeled proteins, protein microsequencing, silver staining, and two-dimensional gel electrophoresis. the

Small molecules and ligands that interact with PSCA can be identified by embodiments related to such screening assays. For example, small molecules that disrupt protein function, including PSCA's ability to mediate phosphorylation and dephosphorylation, interact with DNA or RNA molecules, can be identified as indicators of cell cycle regulation, second messenger signaling, or tumorigenesis . Similarly, small molecules that modulate the function of PSCA-associated ion channels, protein pumps, or cellular communication can be identified and used to treat patients with PSCA-expressing cancers (see, e.g., Hille, B., Ion Channels of Excitable Membranes, Ionic Channels of Excitable Membranes), Second Edition, Sinauer Assoc., Sunderland, MA, 1992). In addition, ligands that modulate PSCA function can be identified based on their ability to bind PSCA and activate receptor constructs. Exemplary methods are described, for example, in US Patent 5,928,868, issued July 27, 1999, and include methods for forming hybrid ligands in which at least one ligand is a small molecule. In an exemplary embodiment, cells engineered to express PSCA and DNA-binding protein fusion proteins are used to co-express hybrid ligand/small molecule fusion proteins and cDNA library transcriptional activator proteins. The cell also contains a reporter gene whose expression brings the first and second fusion proteins into proximity, which only occurs when the hybrid ligand binds the target sites on the two hybrid proteins. Those cells expressing the reporter gene are selected and unknown small molecules or unknown ligands are identified. This method can identify modulators that activate or inhibit PSCA. the

One embodiment of the invention includes a method of screening for interactions with the PSCA amino acid sequence shown in Figure 1, the method comprising contacting a population of molecules with the PSCA amino acid sequence, allowing the population of molecules to interact with the PSCA amino acid sequence under conditions that facilitate interaction, determining There is a step of separating the molecules that interact with the PSCA amino acid sequence and then the molecules that do not interact with the PSCA amino acid sequence from the molecules that interact with the PSCA amino acid sequence. In a specific embodiment, the method further comprises purifying, characterizing and identifying molecules that interact with the amino acid sequence of PSCA. The identified molecules can be used to modulate the function of PSCA. In a preferred embodiment, the PSCA amino acid sequence is linked to a peptide library. 

X.) Treatment methods and compositions

Identification of PSCAs that are normally expressed in limited tissues but are also expressed in the cancers listed in Table I will open up many approaches to the treatment of such cancers. the

Note that targeted anti-tumor therapy is effective even when the target protein is expressed in normal tissue (even normal living organ tissue). Vital organs are those necessary to maintain life, such as the heart or colon. Non-living organs are organs that can be removed but still allow the individual to survive. Examples of non-living organs are ovaries, breast and prostate. the

是一种FDA批准的药物，它是由各种称为HER2、HER2/neu和erb-b-2的蛋白质发生免疫反应的抗体组成的。它由Genentech销售并且是一种获得商业成功的抗肿瘤药。2002年Herceptin 

的销售额达到约4亿美元。Herceptin 

用于治疗HER2阳性转移性乳腺癌。然而，HER2的表达不限于这种肿瘤。这种蛋白质在许多正常组织内表达。具体地说，已知HER2/neu存在于正常的肾脏和心脏内，而这些组织存在于所有接受Herceptin 

的人当中。Latif，Z.等也证实正常肾脏内存在HER2/neu(B.J.U.International(2002)89：5-9)。如该文献所示(其评价了肾细胞癌是否应成为抗-HER2抗体如Herceptin的优选适应证)，良性肾组织制造这种蛋白和mRNA。特别地，HER2/neu蛋白在良性肾组织内强烈过度表达。  For example, Herceptin

is an FDA-approved drug that consists of antibodies that immunoreact with various proteins called HER2, HER2/neu, and erb-b-2. It is marketed by Genentech and is a commercially successful antineoplastic drug. 2002 Herceptin

Sales reached approximately $400 million. Herceptin

For the treatment of HER2-positive metastatic breast cancer. However, expression of HER2 is not limited to this tumor. This protein is expressed in many normal tissues. Specifically, HER2/neu is known to be present in normal kidneys and hearts, and these tissues were present in all patients receiving Herceptin

of people. Latif, Z. et al. also confirmed the existence of HER2/neu in normal kidney (BJU International (2002) 89: 5-9). As shown in this document, which evaluated whether renal cell carcinoma should be the preferred indication for anti-HER2 antibodies such as Herceptin, benign kidney tissue makes this protein and mRNA. In particular, HER2/neu protein is strongly overexpressed in benign kidney tissue.

Although HER2/neu is expressed in living tissues such as the heart and kidney, Herceptin is a very potent, FDA-approved and commercially successful drug. Herceptin's effect on heart tissue, "cardiotoxicity" is only one side effect of treatment. When patients were treated with Herceptin alone, significant cardiotoxicity occurred in only a few patients. In order to minimize cardiotoxicity, there are more stringent entry requirements for treatment with HER2/neu. Factors such as the influence of genetic defects on heart conditions need to be assessed before treatment can be instituted. the

Of particular note is the absence of any evaluable side effects of Herceptin in the kidney, although renal tissue showed normal expression, possibly even higher than that in cardiac tissue. In addition, side effects occurred only rarely in various normal tissues expressing HER2. Only cardiac tissue experienced evaluable side effects. In tissues with particularly significant expression of HER2/neu, such as kidney tissue, no side effects occurred. the

In addition, it was found that the anti-tumor therapy targeting epidermal growth factor receptor (EGFR); Erbitux (ImClone) has a good therapeutic effect. EGFR is also expressed in many normal tissues. Side effects in normal tissue following anti-EGFR therapy are very limited. A common side effect of EGFR therapy is a severe rash observed in 100% of patients treated. the

Therefore, the expression of the target protein in normal tissue or even normal living tissue will not destroy the effect of a targeted agent targeting this protein as a therapeutic agent in some tumors that also overexpress this protein. For example, expression in living organs is not inherently harmful. In addition, organs considered druggable (such as the prostate and ovaries) can be removed without causing death. Finally, certain vital organs are not affected by normal organ expression due to their immuno-privilege. Immunologically privileged organs are those that are shielded from blood by the blood-organ barrier and thus less susceptible to immunotherapy. Examples of immune privileged organs are the brain and testes. the

Accordingly, therapeutic methods that inhibit the activity of PSCA proteins are useful in patients with PSCA-expressing cancers. These treatments generally fall into 3 categories. The first category is because PSCA is related to the growth of tumor cells, by adjusting the function of PSCA, the growth of tumor cells is inhibited or slowed down, or the killing thereof is induced. The second category includes various methods of inhibiting the binding or association of PSCA proteins with their binding partners or other proteins. The third category includes various methods of inhibiting transcription of the PSCA gene or translation of PSCA mRNA. the

X.A.) Anticancer vaccines

The present invention provides cancer vaccines comprising PSCA-related proteins or PSCA-related nucleic acids. With regard to PSCA expression, cancer vaccines prevent and/or treat PSCA-expressing cancers with little or no effect on non-target tissues. The use of tumor antigens in vaccines that generate a cell-mediated humoral immune response for anticancer therapy is well known in the art and has been used in the treatment of prostate cancer with 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). the

This approach is readily performed using PSCA-associated proteins or nucleic acid molecules encoding PSCA and recombinant vectors (typically containing a number of T cell epitopes or antibodies) capable of expressing and presenting PSCA immunogens. Those skilled in the art are aware that many vaccine systems for delivering immunoreactive epitopes are known in the art (see for example Heryln et al., Ann Med 1999-2 31(1):66-78; Maruyama et al., Cancer Immunol Immunother 2000-649(3 ): 123-32). Briefly, this method of generating an immune response (e.g., a cell-mediated and/or humoral immune response) in a mammal comprises the steps of contacting the immune system of the mammal with an immunoreactive epitope (e.g., FIG. 1 The epitope on the indicated PSCA protein or its analogue or homologue) so that the mammal produces the specific immune response of this epitope (for example, produces the antibody that specifically recognizes this epitope). the

The entire PSCA protein, its immunogenic regions or epitopes can be delivered by various methods in combination. Such vaccine compositions may include, for example, lipopeptides (e.g., Vitiello, A. et al., J. Clin. Invest. 95:341, 1995), encapsulated in poly(DL-lactide-co-glycolide) ( "PLG") peptide composition in 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 composition contained in the immunostimulatory complex (ISCOM) (see for example Takahashi et al., Nature 344:873-875, 1990; Hu et al., Clin Exp Immunol.113:235-243, 1998), multiple antigens Peptide System (MAP) (see for example Tam, J.P., Proc. Natl. Acad. Sci. U.S.A. 85:5409-5413, 1988; Tam, J.P., J. Immunol. Methods 196: 17-32, 1996), formulated into multiple Peptides of valent peptides; peptides (usually crystallized) for ballistic delivery systems and peptides for viral delivery vehicles (Perkus, M.E. et al., cited in: Coneepts in vaccine development, eds. Kaufmann, S.H.E. , 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 (for example, Kofler, N. et al., J.Immunol. Methods.192:25,1996; Eldridge, J.H. et al., Sem.Hematol.30:16,1993; Jr. Falo, L.D. et al., Nature Med.7:649,1995), adjuvants (Warren, H.S., Vogel, F.R. and Chedid, L.A.Annu.Rev.Immunol.4:369,1986; Gupta, R.K. et al., Vaccine 11:293,1993), liposomes (Reddy, R. et al., J.Immunol.148:1585,1992; Rock, K.L., Immunol.To day 17:131, 1996), or naked cDNA or cDNA absorbed by particles (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. Cited in: Kaufmann, S.H.E., eds. Concepts in vaccine development, 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) can also be used. the

In patients suffering from PSCA-related cancers, the vaccine composition of the present invention can also be combined with other cancer treatment methods such as surgery, chemotherapy, drug therapy, radiotherapy, etc., including IL-2, IL-12, GM-CSF, etc. Combined use of immune adjuvants. the

Cellular Vaccine:

Peptides within the PSCA protein that bind the corresponding HLA alleles can be identified using specific algorithms to determine CTL epitopes (see e.g. Table IV; Epimer ^™ and Epimatrix ^™ , Brown University (URLbrown.edu/Research/TB-HIV_Lab/epimatrix/epimatrix. html); and BIMAS (URL bimas.dcrt.nih.gov/; SYFPEITHI, URLsyfpeithi.bmi-heidelberg.com/). In a preferred embodiment, the PSCA immunogen contains one or more Identified amino acid sequences, such as those shown in Tables V-XVIII and XXII-LI, or have 8, 9, 10 or 11 HLA class I motifs/supermotifs (e.g. Table IV(A), Table IV(D ) or Table IV(E)) specific amino acid peptides and/or peptides comprising HLA class II motifs/supermotifs (eg Table IV(B) or Table IV(C)) with at least 9 amino acids. In the art It is known that the end of the HLA class I binding groove is basically closed, so only peptides of a specific size range can fit into the groove and bind to it, and HLA class I epitopes are usually 8, 9, 10 or 11 amino acids in length. In contrast, HLA The end of the class II binding groove is essentially open; thus peptides of about 9 or more amino acids can be bound by HLA class II molecules. Since the binding grooves of HLA class I and II differ, the HLA class I motif is length-specific, i.e. Position 2 of a class I motif is the second amino acid in the amino to carboxyl direction of the peptide. Amino acid positions for class II motifs are relative to each other only, not to the entire peptide, i.e. the amino and amino acids of the sequence with the motif / or the carboxy terminus can be combined with other amino acids. HLA class II epitopes are usually 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids, or more than 25 amino acids.

A number of methods are known in the art to generate an immune response in mammals (eg, the first step in generating hybridomas). The method of generating an immune response in a mammal comprises exposing the immune system of the mammal to an immunogenic epitope of a protein (eg, PSCA protein) to generate an immune response. A specific embodiment includes a method of generating 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 thereafter at least The host is re-exposed to PSCA B-cell or cytotoxic T-cell epitopes or analogs thereof at practical intervals. A specific embodiment includes a method for generating an immune response against a PSCA-related protein or an artificial polyepitopic peptide: administering to a human or other mammal a PSCA immunogen (such as a PSCA protein or a peptide fragment thereof, a PSCA fusion protein) contained in a vaccine preparation or similar, etc.). Typically, such vaccine preparations also contain a suitable adjuvant (see, e.g., U.S. Patent 6,146,635) or a universal helper epitope, such as the PADRE™ peptide (Epimmune Inc., San Diego, CA; see, e. 3); 164(3):1625-1633; Alexander et al., Immunity 1994 1(9):751-761, and Alexander et al., Immunol. Res. 1998 18(2):79-92). Another method involves generating an immune response against a PSCA immunogen in an individual by in vivo administering in the muscle or skin of a mammalian organism a DNA molecule comprising a DNA sequence encoding a PSCA immunogen operably linked to Regulatory sequences that control the expression of a DNA sequence; wherein the DNA molecule is taken up by a cell, the DNA sequence is expressed within the cell, and an immune response is generated against the immunogen (see eg, US Pat. No. 5,962,428). Genetic vaccine facilitators, such as anionic lipids; saponins; lectins; estrogenic compounds; hydroxylated lower alkyls; dimethylsulfoxide; and urea, may also optionally be administered. In addition, anti-idiotypic antibodies that mimic PSCA can be administered to generate a response to the target antigen. the

Nucleic acid vaccine:

Vaccine compositions of the invention include nucleic acid-mediated forms. DNA or RNA encoding a protein of the invention can be administered to a patient. Genetic immunization can be used to generate prophylactic or therapeutic humoral and cellular immune responses against PSCA-expressing cancer cells. A construct containing 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 muscle or skin cells take up the construct and express the encoded PSCA protein/immunogen. Or the vaccine contains PSCA-related proteins. Expression of PSCA-associated protein immunogens results in prophylactic or therapeutic humoral and cellular immunity against T cells bearing PSCA proteins. Various prophylactic and therapeutic genetic immunization techniques known in the art can be used (see, eg, information and references published at Internet address genweb.com). Nucleic acid-based delivery is described, for example, in Wolff et al., Science 247:1465 (1990) and U.S. Patents 5,580,859; 5,589,466; 5,804,566; 5,739,118; Examples of DNA-based delivery technologies include "naked DNA", agonist (bupivacaine, polymer, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated delivery ("gene gun") or pressure-mediated delivery (see, eg, US Pat. No. 5,922,687). the

For the purpose of therapeutic or prophylactic immunization, the proteins of the invention may be expressed by viral or bacterial vectors. Various viral gene delivery systems that can be used in the practice of the present invention include, but are not limited to, vaccinia, fowlpox, canarypox, adenovirus, influenza virus, poliovirus, adeno-associated virus, lentivirus, and Sindbis virus (see For example 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, in muscle or in the dermis) to induce an anti-tumor response. the

For example, vaccinia virus can be used as a vector to express the nucleotide sequence encoding the peptide of the present invention. After introduction into the host, the recombinant vaccinia virus expresses protein immunogenic peptides, thereby eliciting an immune response in the host. Vaccinia vectors and methods for immunization are described, eg, in US Patent 4,722,848. Another carrier is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al., Nature 351:456-460 (1991). From the description herein, those skilled in the art will appreciate that many other vectors can be used to therapeutically administer or immunize the peptides of the invention, such as adenoviral and adeno-associated viral vectors, retroviral vectors, Salmonella typhi vectors, detoxified Anthrax toxin carrier, etc. the

Therefore, gene delivery systems are used to deliver PSCA-related nucleic acid molecules. In one embodiment the 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. the

In vitro vaccine

Various ex vivo methods can also be used to generate an immune response. One approach involves the use of antigen-presenting cells (APCs), such as dendritic cells (DCs), to present PSCA antigens to the patient's immune system. Dendritic cells express MHC class I and II molecules, B7 co-stimulator, and IL-12 and are therefore highly specific antigen-presenting cells. In prostate cancer, autologous dendritic cells pulsed with prostate-specific membrane antigen (PSMA) were used in a phase I clinical trial to stimulate the immune system in prostate cancer patients (Tjoa et al., 1996, Prostate 28:65-69; Murphy et al., 1996, Prostate 29: 371-380). Thus, dendritic cells can be used to present PSCA peptides to MHC class I or II molecules of T cells. 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 intact PSCA protein. Yet another embodiment includes overexpressing the PSCA gene in dendritic cells using various execution vectors known in the art, such as adenovirus (Arthur et al., 1997, Cancer GeneTher. 4: 17-25), inversion Recording virus (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. the

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

PSCA is an attractive target in antibody-based therapeutic strategies. A variety of antibody strategies for targeting extracellular and intracellular molecules are known in the art (see, e.g., complement and ADCC mediated killing as well as the use of intrabodies). Since PSCA is expressed by various cancer cell lines compared to corresponding normal cells, a systemically administered PSCA-immunoreactive composition was prepared that has excellent sensitivity and is not affected by the immunoreactive composition. Toxic, non-specific and/or non-targeted effects due to binding to non-target organs and tissues. Antibodies that specifically react with the PSCA domain are useful for systemic treatment of PSCA-expressing cancers, in combination with toxins or therapeutic agents, or as naked antibodies that inhibit cell proliferation or function. the

用于转移性乳腺癌的Herceptin 

以及用于结肠直肠癌的Erbitux 

Antibodies to PSCA can be introduced into a patient such that the antibody binds to PSCA and modulates functions such as interaction with the binding partner and subsequent mediated destruction of tumor cells and/or inhibition of tumor cell growth. The mechanism by which such antibodies have therapeutic effects may include: complement-mediated cell lysis, antibody-dependent cytotoxicity, regulation of PSCA physiological functions, inhibition of ligand binding or signal transduction pathways, regulation of tumor cell differentiation, Changes in the distribution of tumor angiogenic factors, 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 will appreciate that antibodies can be used to specifically target and bind immunogenic molecules, such as the immunogenic region of the PSCA sequence shown in FIG. 1 . Furthermore, the skilled artisan will appreciate that it is routine to conjugate antibodies to cytotoxic agents (see, eg, Slevers et al., Blood 93:113678-3684 (June 1, 1999)). When cytotoxic and/or therapeutic agents are delivered directly to cells by, for example, coupling them to antibodies that specifically bind to molecules expressed by the cells (e.g., PSCA), the cytotoxic agents will exert their known effects on those cells. Biological effects (ie, cytotoxicity). the

Many compositions and methods are known in the art for antibody-cytotoxic agent conjugates to kill cells. In cancer, a common approach involves administering to a tumor-bearing animal a biologically effective amount of a conjugate comprising a selected cytotoxic and/or therapeutic agent conjugated to a targeting agent (e.g., an anti-PSCA antibody) , easy to bind or localize to the cell surface. An exemplary embodiment is a method of delivering a cytotoxic and/or therapeutic agent to cells expressing PSCA, the method comprising conjugating the cytotoxic agent to an antibody that immunospecifically binds to an epitope of PSCA, and then allowing the cell to interact with The antibody-drug conjugate is contacted. Another exemplary embodiment is a method of treating an individual suspected of having metastatic cancer, the method comprising the step of: parenterally administering to the individual a therapeutically effective amount of Pharmaceutical compositions of antibodies. the

Cancer immunotherapy using anti-PSCA can be carried out according to various approaches that have been successfully applied 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 Carcinoma (Moun et al., 1994, Cancer Res.54:6160-6166; Velders et al., 1995, Cancer Res.55:4398-4403) and breast cancer (Shepard et al., 1991, J.Clin.Immunol.11:117-127 ). Some therapeutic approaches involve conjugation of naked antibodies to toxins or radioactive isotopes, such as Y ⁹¹ or I ¹³¹ , respectively, to anti-CD20 antibodies (e.g., Zevalin™, IDEC Pharmaceuticals Corp. or Bexxar ^™ , Coulter Pharmaceuticals), while others involve conjugation together Antibodies are administered with other therapeutic agents, eg, Herceptin(TM) (trastuzumab) and paclitaxel (Genentech, Inc.) co-administered. The antibody can be conjugated to a therapeutic agent. For the treatment of prostate cancer, for example, PSCA antibody can be given simultaneously with radiotherapy, chemotherapy and hormone ablation. Antibodies can also be conjugated to toxins (e.g., Mylotarg ^™ , Wyeth-Ayerst, Madison, NJ, a recombinant humanized IgG4κ antibody conjugated to the antitumor antibiotic calicheamicin) or maytansinoid , such as taxane-based tumor active prodrugs, TAP, platform, ImmunoGen, Cambridge, MA, see also, for example, US Patent 5,416,064) or AuristatinE (Nat Biotechnol. 2003 Jul; 21(7):778-84 .(Seattle Genetics)).

While PSCA antibody therapy can be used in all stages of cancer, antibody therapy is particularly appropriate for advanced or metastatic cancer. Antibody therapy of the invention may be prescribed to patients who have received one or more rounds of chemotherapy. Alternatively, for patients not receiving chemotherapy, antibody therapy of the invention may be administered in conjunction with a chemotherapy or radiation regimen. In addition, antibody therapy can enable the use of reduced doses of combined chemotherapeutic agents, especially in patients whose toxicity from chemotherapeutic agents is not well tolerated. Described in 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) Coadministration of multiple antibodies with chemotherapeutic agents. the

While PSCA antibody therapy can be used in all stages of cancer, antibody therapy is particularly appropriate for advanced or metastatic cancer. Antibody therapy of the invention may be prescribed to patients who have received one or more rounds of chemotherapy. Alternatively, for patients not receiving chemotherapy, antibody therapy of the invention may be administered in conjunction with a chemotherapy or radiation regimen. In addition, antibody therapy can enable the use of reduced doses of combined chemotherapeutic agents, especially in patients whose toxicity from chemotherapeutic agents is not well tolerated. the

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

Anti-PSCA monoclonal antibodies for the treatment of prostate and other cancers include those that induce potent immune responses against tumors or that directly cause cytotoxicity. In this regard, anti-PSCA monoclonal antibodies (MAbs) can cause tumor cell lysis through complement-mediated or antibody-dependent cellular cytotoxicity (ADCC) mechanisms, both of which require the intact Fc of the immunoglobulin molecule Partially interacts with effector cell Fc receptor sites on complement proteins. In addition, anti-PSCA MAbs that exert direct biological effects on tumor growth can be used to treat PSCA-expressing cancers. The direct mechanisms of action of cytotoxic MAbs include: inhibition of cell growth, regulation of cell differentiation, modulation of tumor angiogenic factor properties, and induction of apoptosis. The mechanism by which a particular anti-PSCA MAb exerts its antitumor effect is assessed using a variety of in vitro assays for assessing cell death, such as ADCC, ADMMC, complement-mediated lysis, and the like, generally known in the art. the

Moderate to strong immune responses against nonhuman antibodies can be induced in some patients with murine or other nonhuman monoclonal antibodies or human/mouse chimeric MAbs. This can lead to clearance of antibody from circulation and reduced efficacy. At its most severe, this immune response can lead to massive formation of immune complexes that can lead to kidney failure. Thus, preferred monoclonal antibodies for use in the therapeutic methods of the invention are fully human or humanized and specifically bind the target PSCA antigen with high affinity, but have low or no antigenicity in the patient. the

The therapeutic methods of the invention include anti-PSCA MAbs administered alone as well as compositions or mixtures of other MAbs. Such MAb mixtures have certain advantages as they may contain MAbs targeting different epitopes, employing different effector mechanisms or directly combining cytotoxic MAbs with MAbs that depend on immune effector functions. Such combined MAbs may exert synergistic therapeutic effects. In addition, anti-PSCA MAbs can be administered with other treatment modalities including, but not limited to, various chemotherapeutic agents, androgen blocking agents, immunomodulators (e.g., IL-2, GM-CSF), surgery, or radiation therapy. Anti-PSCA MAbs are administered in their "naked" or unconjugated form, or may be conjugated to a therapeutic agent. the

Anti-PSCA antibody formulations can be administered by any route capable of delivering the antibody to tumor cells. Routes of administration include, but are not limited to, intravenous, intraperitoneal, intramuscular, intratumoral, intradermal, and the like. Treatment generally involves repeated administration of the anti-PSCA antibody preparation via an acceptable route of administration, such as intravenous (IV), at doses generally in the range of about 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. Typically, doses of 10-1000 mg weekly MAbs are effective and well tolerated. the

Based on clinical trials of Herceptin ^TM in the treatment of metastatic breast cancer, an acceptable dosage regimen is an initial loading dose of approximately 4 mg/kg of patient body weight intravenously followed by weekly intravenous injections of anti-inflammatory drugs at a dose of approximately 2 mg/kg. - PSCA MAb preparations. Preferably, the initial loading dose is given as an infusion over a period of 90 minutes or longer. Regular maintenance doses are given as infusions of 30 minutes or longer, as long as the starting dose is well tolerated. Those skilled in the art are aware that many factors affect the ideal dosage regimen in a particular pathology. These factors include, for example, the binding affinity and half-life of the Ab or MAb used, the extent of expression of PSCA in the patient, the extent of circulating PSCA antigen depletion, the desired steady-state antibody concentration level, frequency of treatment, combination with the therapeutic methods of the invention, The effects of chemotherapeutic or other agents used and the health status of the particular patient.

Optionally, the level of PSCA (eg, the level of circulating PSCA antigen and/or PSCA-expressing cells) within a given sample of a patient can be assessed to help determine the most effective dosage regimen, among other things. This evaluation can also be used for monitoring purposes throughout treatment and can be combined with assessment of other parameters (eg urine cytology and/or immunocytochemistry levels in bladder cancer treatment or, by analogy, serum PSA levels in prostate cancer treatment) to measure the treatment effect. the

Anti-idiotypic anti-PSCA antibodies can also be used as vaccins in anticancer therapy to induce immune responses against cells expressing PSCA-related proteins. In particular, the production of anti-idiotypic antibodies is well known in the art; this method is suitable for producing anti-idiotypic anti-PSCA antibodies that mimic epitopes on PSCA-related proteins (see, e.g., 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 antibodies are useful in cancer vaccine approaches. the

It is an object of the present invention to provide antibodies to PSCA that inhibit or delay the growth of tumor cells expressing PSCA. Another object of the present invention is to provide methods of inhibiting angiogenesis and other biological functions in mammals, preferably humans, thereby reducing tumor growth, using PSCA antibodies, especially combining these PSCA antibodies with radiotherapy or chemotherapeutic agents or a combination of both. the

In one embodiment, there is a synergistic effect when the PSCA antibody is used in combination with a chemotherapeutic or radiotherapeutic agent or a combination thereof to treat tumors, including human tumors. In other words, the inhibitory effect of the PSCA antibody on tumor growth was more enhanced than expected when combined with chemotherapeutics or radiotherapy or a combination thereof. Synergy can be shown, for example, by greater than expected inhibition of tumor growth by the combination treatment compared to treatment with the PSCA antibody alone or the additive effect of the PSCA antibody and a chemotherapeutic agent or radiation. Preferably, synergy is demonstrated by a reduction in cancer that is unexpected relative to treatment with naked PSCA antibodies or adjunct treatment with PSCA antibodies and chemotherapeutic agents or radiation. the

Methods for inhibiting tumor cell growth using a combination of PSCA antibodies and chemotherapy or radiotherapy or both include: administering PSCA antibodies before, during, or after starting chemotherapy or radiotherapy, and any combination thereof (i.e., after starting chemotherapy and/or radiotherapy) or before and during, before and after, or before, during and after radiation therapy). For example, the PSCA antibody is usually administered between 1-60 days, preferably 3-40 days, more preferably 5-12 days before starting radiotherapy and/or chemotherapy. However, depending on the treatment record and the needs of a particular patient, the method can be practiced in a manner that will provide the most effective treatment and ultimately prolong the patient's life. the

Administration of chemotherapeutic agents can be by a variety of means, including systemic administration by parenteral and routes. In one embodiment, the PSCA antibody and chemotherapeutic agent are administered as separate molecules. In another embodiment, the PSCA antibody is attached (eg, by conjugation) to a chemotherapeutic agent. (See Example entitled "Human Clinical Trials for Treatment and Diagnosis of Human Cancer by In Vivo Use of Human Anti-PSCA Antibodies") and (See the section entitled "PSCA as a Target for Antibody-Based Therapies"). Specific examples of chemotherapeutic agents or chemotherapy include: cisplatin, dacarbazine (DTIC), actinomycin D, dichloromethylenediethylamine (nitrogen mustard), streptozocin, cyclophosphamide, carmustine ( BCNU), lomustine (CCNU), doxorubicin (doxorubicin), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5 -Fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere), adesleukin, asparaginase, busulfan, carboplatin, cladribine , dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, interferon-alpha, leuprolide, megestrol, melphalan, mercaptopurine, mitotane, mitotane , pegaspargase, pentostatin, pipobromide, plicamycin, streptozocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil nitrogen mustard, vinorelbine, chlorambucil, paclitaxel, or combinations thereof. the

The radiation source used in combination with PSCA antibodies can be located externally or internally to the patient being treated. When the source of radiation is located outside the patient, the treatment is called external beam radiation therapy (EBRT). When the radiation source is inside the patient, this treatment is called brachytherapy (BT). the

The radiation is administered according to well-known standard techniques using standard equipment manufactured for this purpose, such as AECL radiotherapy machines and Varian medical linear accelerators. Radiation dose depends on a variety of factors known in the art. These factors include the organ being treated, healthy organs that may be adversely affected in the radiation path, the patient's tolerance to radiation therapy, and the area of the body that needs to be treated. The dose is usually 1-100Gy, more specifically 2-80Gy. Some reported doses include: 35 Gy for the spinal cord, 15 Gy for the kidneys, 20 Gy for the liver, and 65-80 Gy for the prostate. It should be emphasized, however, that the invention is not limited to any particular dosage. Dosages can be determined by the treating physician according to factors specific to a given situation, including those described above. the

The distance between the external radiation source and the point of entry into the patient can be any distance that achieves an appropriate balance of killing target T cells and minimizing side effects. Typically the distance between the external radiation source and the point of entry into the patient is 70-100 cm. the

Brachytherapy is usually delivered by placing a radiation source in the patient. Typically, the radiation source is placed about 0-3 cm from the organ to be treated. Known techniques include interstitial, intracavitary, and superficial brachytherapy. The radioactive seed source 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 other radioactive atoms that have been used in brachytherapy include americium-241 and gold-198. The radiation dose used for brachytherapy can be the same as that described above for external beam radiation therapy. In addition to the factors described above for determining doses for external beam radiation therapy, the nature of the radioactive atoms used is also taken into account when determining doses for brachytherapy. the

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

Vaccines and methods of making vaccines comprising an immunologically effective amount of one or more of the HLA-binding peptides described herein are another embodiment of the invention. Furthermore, the vaccines of the invention comprise one or more combinations of said peptides. Peptides can be present alone in the vaccine. Alternatively, the peptides may exist as homopolymers containing multiple copies of the same peptide or as heteropolymers of different peptides. The advantage of the polymer is that there is an enhanced immune response, and due to the use of different epitopes, the polymer has additional antibodies and/or antibodies that induce a reaction with a different epitope of the pathogen or tumor-associated peptide targeted by the immune response. or CTL for extra capabilities. The composition may be a naturally occurring antigenic region or this may be prepared, for example, by recombinant or chemical synthesis methods. the

Carriers that can be used with the vaccine of the present invention are well known in the art and include, for example: thyroglobulin, albumin such as human serum albumin, tetanus toxoid, polyamino acids such as poly-L-lysine, poly-L -Glutamic acid, influenza virus, hepatitis B virus nucleoprotein, etc. Vaccines may contain physiologically tolerable (ie acceptable) diluents such as water or saline, preferably phosphate buffered saline. Vaccines often also contain adjuvants. Adjuvants such as incomplete Freund's adjuvant, aluminum sulfate, aluminum hydroxide or alum are examples of materials well known in the art. Furthermore, as described herein, conjugation of the peptides of the invention to lipids such as tripalmitoyl-S-glycerylcysteinylserine-serine (P3CSS) can elicit a CTL response. Furthermore, adjuvants such as synthetic oligonucleotides containing synthetic cytosine-phosphorothioate-guanine (CpG) were found to increase CTL responses 10-100 fold (see e.g. Davila and Celis, J. Immunol. 165: 539-547 (2000)). the

After immunization with the peptide composition of the present invention by injection, aerosol, oral, transdermal, transmucosal, intrapleural, intrathecal or other suitable routes, the immune system of the host can generate a large number of desired antigen-specific CTL and/or or HTL responds to the vaccine. Thereafter, the host becomes at least partially immune to subsequent generation of PSCA-expressing or overexpressing cells, or at least somewhat therapeutically effective in tumors associated with that antigen. the

(Epimmune，San Diego，CA) 分子(描述在例如美国专利5,736,142中)。  In some embodiments, it is advantageous to combine the class I peptide component with a component that induces or promotes the production of neutralizing antibody and or helper T cell responses to the target antigen. A preferred embodiment of this composition comprises the class I and class II epitopes of the invention. Another embodiment of this composition comprises a class I and/or class II epitope of the invention and a cross-reactive HTL epitope, such as

(Epimmune, San Diego, CA) molecule (described, eg, in US Patent 5,736,142).

The vaccines of the present invention may also comprise antigen presenting cells (APCs), such as dendritic cells (DCs), as vehicles for presenting the peptides of the present invention. The dendritic cells can be loaded in vitro by generating the vaccine composition in vitro, mobilizing and harvesting the dendritic cells. For example, dendritic cells can be transfected with, for example, a minigene of the invention or pulsed with a peptide. The dendritic cells are then administered to the patient and elicit an immune response in vivo. DNA- or peptide-based vaccine compositions can also be administered in vivo while mobilizing dendritic cells, thereby loading dendritic cells in vivo. the

The following principles are preferably used when selecting epitope arrays for multi-epitopic compositions to be used in vaccines or to select non-specific epitopes contained in vaccines and/or encoded by nucleic acids (such as minigenes). continuous epitope. The following principles should be balanced in making such a choice. The multiple epitopes incorporated into a given vaccine composition may, but need not be, contiguous epitopes in the native antigenic sequence from which the epitopes arise. the

1.) Select epitopes that mimic the immune response associated with tumor clearance after administration. For HLA class I, 3-4 epitopes from at least one tumor-associated antigen (TAA) are included. A similar principle was employed for HLA class II; in turn 3-4 epitopes were selected from at least one TAA (see eg Rosenberg et al., Science 278:1447-1450). Epitopes from one TAA can be combined with epitopes from one or more other TAAs to create vaccines that target tumors and alter common TAA expression patterns. the

2.) Select an epitope with the necessary binding affinity related to immunogenicity: for HLA class I molecules, IC50 is 500 nM or lower, usually 200 nM or lower; for class II molecules, IC50 is 1000 nM or lower. the

3.) Select peptides with sufficient supermotifs, or peptide arrays with sufficient allele-specific motifs, to obtain broad population coverage. For example preferably there is at least 80% population coverage. The breadth or redundancy of population coverage can be assessed using a statistical calculation known in the art - Monte Carlo analysis. the

4.) Analogs are often chosen when choosing cancer-associated antigenic epitopes, since patients may have established tolerance to natural epitopes. the

5.) Particularly suitable epitopes are referred to as "nested epitopes". Nested epitopes are found where at least two epitopes overlap for a given peptide sequence. Nested peptide sequences may include B cell, HLA class I and/or HLA class II epitopes. When providing nested epitopes, the general aim is to provide the maximum number of epitopes per sequence. Thus, one aspect is to avoid providing peptides that are longer than the amino terminus of a peptide amino terminal epitope or longer than the carboxy terminus of a peptide carboxy terminal epitope. When providing polyepitopic sequences, eg, sequences containing nested epitopes, it is often important to screen the sequences to ensure that they do not have pathogenic or other deleterious biological properties. the

6.) If a multi-epitope protein is produced or when producing a minigene, one goal is to produce the smallest peptide that includes the epitope of interest. This principle is the same or similar to that for selecting peptides comprising nested epitopes. However, in the case of artificial polyepitopic peptides, the purpose of length minimization is to balance the spacer sequences between the epitopes integrated in the polyepitopic protein. For example, spacer amino acid residues may be introduced to avoid linking of linked epitopes (epitopes recognized by the immune system but not present in the target antigen and which can only be produced by artificially juxtaposing epitopes), or to facilitate cleavage between epitopes Thereby enhancing epitope presentation. Since the receptor may mount an immune response against non-native epitopes, it is often desirable to avoid epitope linking. Special care needs to be taken when the linked epitope is a "dominant epitope". A dominant epitope can result in a null response to other attenuated or suppressed epitopes. the

7.) When there are sequences of multiple variants of the same target protein, possible peptide epitopes can also be selected according to their secrecy. For example, a criterion of confidentiality may specify that the entire sequence of an HLA class I binding peptide or the entire 9-peptide core of a class II binding peptide is conservative in percentage to a given sequence evaluation for a particular protein antigen. the

X.C.1. Small gene vaccine

There are many different methods that can deliver multiple epitopes simultaneously. A nucleic acid encoding a peptide of the invention is a particularly useful example of the invention. Epitopes contained in minigenes are preferred according to the guidelines set in the above sections. A preferred method of administering a nucleic acid encoding a peptide of the invention uses a minigene construct encoding a peptide containing one or more epitopes of the invention. the

The use of polyepitopic minigenes is described below and in: Ishioka et al., J. Immunol. 162:3915-3925, 1999; An, L. and Whitton, JL, J. Virol. 71:2292, 1997; Thomson, SA et al., J. Immunol. 157:822, 1996; Whitton, JL, et al., J. Virol. 67:348, 1993; Hanke, R. et al., Vaccine 16:426, 1998. For example, a polyepitopic DNA plasmid can be engineered to encode PSCA-derived epitopes with supermotifs and/or motifs, Universal helper T cell epitope or multiple HTL epitopes of PSCA (see eg Tables V-XVIII and XXII-LI) and endoplasmic reticulum-transport signal sequence. Vaccines may also contain epitopes derived from other TAAs.

The immunogenicity of the polyepitopic minigene can be verified in transgenic mice to assess the magnitude of the CTL response induced against the test epitope. Furthermore, the in vivo immunogenicity of a DNA-encoded epitope can be correlated with the in vitro response of a particular CTL line against target cells transfected with that DNA plasmid. Thus, these experiments could show two roles of this minigene: 1.) generate a CTL response, and 2.) induce expression of the encoded epitope in cells recognized by the CTL. the

For example, to generate a DNA sequence encoding a selected epitope (minigene) for expression in human cells, the amino acid sequence of the epitope can be back-translated. Codons for individual amino acids can be selected using the human codon table. These epitope-encoding DNA sequences may be directly contiguous, thus resulting in a contiguous polypeptide sequence when translated. Additional elements may be incorporated in the minigene design for optimal expression and/or immunogenicity. Examples of amino acid sequences within minigene sequences that can be back-translated include: HLA class I epitopes, HLA class II epitopes, antibody epitopes, ubiquitination signal sequences, and/or endoplasmic reticulum targeting signals. In addition, HLA presentation of CTL and HTL epitopes can be improved by adding synthetic (e.g. polyalanine) or naturally occurring flanking sequences adjacent to the CTL or HTL epitopes; epitopes included in these larger peptides are part of the invention scope. the

The minigene sequence can be converted to DNA by assembling oligonucleotides encoding the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases in length) can be synthesized, phosphorylated, purified and annealed under appropriate conditions using well-known techniques. The ends of the oligonucleotides can be ligated with, for example, T4 DNA ligase. This synthetic epitope polypeptide-encoding minigene is then cloned into the desired expression vector. the

Standard regulatory sequences well known to those skilled in the art are preferably included in the vector to ensure expression in target cells. Several vector elements are required: a promoter with a downstream cloning site for insertion of the minigene; a polyadenylation signal to efficiently terminate transcription; an E. coli origin for replication; and an E. coli selectable marker (such as ampicillin or kana Mycin resistance). Many promoters can be used for this purpose, such as the human cytomegalovirus (hCMV) promoter. See, eg, US Patent Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences. the

Additional vector modifications can be added to optimize minigene expression and immunogenicity. In cases where introns are required for efficient gene expression, one or more synthetic or naturally occurring introns may be incorporated into the transcribed region of the minigene. The addition of mRNA stabilizing sequences as well as replicating sequences in mammalian cells can also enhance minigene expression. the

Once the expression vector is selected, the minigene can be 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 of the minigene as well as the DNA sequence and all other elements within the vector were verified by restriction mapping and DNA sequence analysis. Bacterial cells containing the correct plasmids can be maintained as master and working cell banks. the

Furthermore, immunostimulatory sequences (ISS or CpG) appear to play a role in the immunogenicity of DNA vaccines. If enhanced immunogenicity is desired, these sequences can be included in the vector outside of the minigene coding sequence. the

Epimmune，San Diego，CA)。辅助(HTL)表位可结合到胞内寻靶信号并与CTL表位独立表达；这可将HTL表位导向不同于CTL表位的细胞区室。需要的话，这可使HTL表位更有效地进入HLA II类途径，从而促进HTL诱导。与HTL 或CTL诱导相反，通过共表达免疫抑制分子(例如TGF-b)特异性降低免疫应答对于某些疾病有益。  In some embodiments, bicistronic expression vectors capable of producing minigene-encoded epitopes and a second protein that may enhance or reduce immunogenicity may be used. Examples of proteins or polypeptides that may advantageously enhance an immune response when co-expressed include cytokines (e.g., IL-2, IL-12, GM-CSF), molecules that induce cytokine production (e.g., LeIF), co-stimulatory molecules, or for HTL responds with pan-DR binding protein (

Epiimmune, San Diego, CA). Helper (HTL) epitopes can bind to intracellular targeting signals and be expressed independently of CTL epitopes; this can direct HTL epitopes to cellular compartments distinct from CTL epitopes. This allows for more efficient entry of HTL epitopes into the HLA class II pathway, thereby facilitating HTL induction, if desired. In contrast to HTL or CTL induction, specific reduction of the immune response by co-expression of immunosuppressive molecules such as TGF-b is beneficial in certain diseases.

Therapeutic amounts of plasmid DNA can be produced, for example, by fermentation in E. coli followed by purification. An equal amount of the working cell bank is inoculated into the growth medium and grown to saturation in shake flasks or bioreactors according to well-known techniques. Plasmid DNA can be purified using standard bioseparation techniques, such as solid-phase anion exchange resins supplied by QIAGEN, Inc. (Valencia, California). The DNA in the open circular and linear forms can be separated from the supercoiled DNA by gel electrophoresis or other methods if desired. the

Purified plasmid DNA can be prepared for injection in a variety of formulations. The easiest method is to reconstitute lyophilized DNA with sterile phosphate-buffered saline (PBS). This approach, called "naked DNA," is currently being used in clinical trials for intramuscular (IM) drug delivery. In order to improve the immunotherapeutic effect of gene DNA vaccine as much as possible, other methods of preparing purified plasmid DNA can be used. A number of methods have been described, and new techniques may also be used. Cationic lipids, glycolipids, and fusogenic liposomes can be used as reagents (see, e.g., WO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7):682 (1988); U.S. Patent 5,279,833; WO 91/06309; and Felgner et al., Proc.Nat'l Acad.Sci.USA 84:7413 (1987).In addition, peptides and collectively referred to as protective, interactive, non-condensing compounds (PINC, protective, interactive, non -condensing compound) can also form complexes with purified plasmid DNA to alter 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 the expression and HLA class I presentation of minigene-encoded CTL epitopes. For example, plasmid DNA is introduced into mammalian cell lines suitable as targets for standard CTL chromium release assays. The transfection method used will be the final preparation of clairvoyance. Electroporation can be used for "naked" DNA, while cationic lipids can be used for direct in vitro transfection. Plasmids expressing green fluorescent protein (GFP) can be co-transfected to enrich transfected cells by fluorescence activated cell sorting (FACS). These cells were then labeled with chromium-51 (51Cr) and used as target cells for epitope-specific CTL lines; cytolysis was detected by 51Cr release, which indicates production of minigene-encoded CTL epitopes and HLA presentation. Expression of HLA epitopes can be assessed using similar assays to assess HLA activity. the

In vivo immunogenicity is a second method for testing the function of minigene DNA preparations. Transgenic mice expressing the appropriate human HLA protein are immunized with the DNA product. Dosage and route of administration depend on the formulation used (eg, IM for DNA in PBS, intraperitoneal (i.p.) for lipoplexed DNA). Splenocytes were harvested 21 days after immunization and restimulated for one week in the presence of peptides encoding the respective epitopes tested. 51Cr-labeled target cell assays with peptides can then be detected in CTL effector cell lysis using standard techniques. Lysis of target cells sensitized with HLA bearing a peptide epitope corresponding to the minigene-encoded epitope demonstrated that the DNA vaccine formulation induces a CTL response in vivo. The immunogenicity of HTL epitopes in transgenic mice was similarly confirmed. the

Alternatively, nucleic acids can be administered using ballistic delivery methods such as described in US Pat. No. 5,204,253. With this technique, particles containing only DNA are administered. In yet another embodiment, DNA can be attached to particles such as gold particles. the

Bacterial or viral delivery systems known in the art can also be used to deliver the minigene, for example, the expression construct encoding the epitope of the present invention can be incorporated into a viral vector such as vaccinia. the

X.C.2. Combinations of CTL peptides and helper peptides

Vaccine compositions containing the peptides of the invention may be modified, eg, analogized, to provide desired properties, such as improved serum half-life, broadened population coverage, or enhanced immunogenicity. the

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 the CTL peptide can be linked directly to the T helper peptide, the CTL epitope/HTL epitope is usually bound via a spacer molecule. Such spacer molecules usually contain relatively small neutral molecules such as amino acids or amino acid mimics that are substantially uncharged under physiological conditions. The spacer molecule may generally be selected from, for example, Ala, Gly or other neutral spacer molecules of non-polar amino acids or neutral polar amino acids. It will be appreciated that the optional spacer molecules do not have to contain identical residues and thus can be hetero- or homo-oligomers. When present, the spacer molecule will usually have at least one or two residues, more usually 3-6 residues, and sometimes even 10 or more residues. The CTL peptide epitope can be linked to the T helper peptide epitope either directly or through a spacer at the amino or carboxy terminus of the CTL peptide. The amino terminus of the immunogenic peptide or T helper peptide can be acylated. the

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

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

In some embodiments, at least one component capable of priming B lymphocytes or T lymphocytes may be included in the pharmaceutical composition of the present invention. Lipids have been identified that elicit CTLs in vivo. For example, palmitic acid residues can be attached to the e- and a-amino groups of lysine residues and then connected to the immunoassay via one or more linkers (such as Gly, Gly-Gly-, Ser, Ser-Ser, etc.) original peptide. The lipidated peptides can then be administered directly as micelles or particles incorporated into liposomes, or after emulsification with an emulsion such as incomplete Freund's adjuvant. In a preferred embodiment, a particularly effective immunogenic composition comprises palmitic acid appended to the e- and a-amino groups of Lys, which is then bound to the immunogenic peptide via a linker such as Ser-Ser the amino terminus. the

Another example of a lipid that elicits a CTL response is an E. coli lipoprotein, such as tripalmitoyl-S-glycerylcysteinylseryl-serine (P3CSS), which can be used when covalently bound to a suitable peptide. Virus-specific CTLs are elicited (see, eg, Deres et al., Nature 342:561, 1989). The peptides of the invention can be conjugated to, for example, P3CSS, and the lipopeptide administered to an individual to elicit a specific immune response against the target antigen. Furthermore, since neutralizing antibodies can also be induced by priming with P3CSS-conjugated epitopes, such two compositions can be combined to more effectively elicit humoral and cell-mediated responses. the

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

One embodiment of the vaccine composition of the invention comprises the ex vivo administration of a mixture of epitope-bearing peptides from PBMCs or DCs isolated from the blood of a patient. Drugs that facilitate DC harvesting can be used, such as Progenipoietin ^™ (Pharmacia-Monsanto, St. Louis, MO) or GM-CSF/IL-4. DCs are pulsed with peptide and washed to remove unbound peptide before reinfusion into the patient. In this embodiment, the vaccine comprises peptide-pulsed DCs on the surface of which complexes of pulsed peptide epitopes and HIA molecules are present.

DCs can be pulsed ex vivo with a mixture of peptides, some of which stimulate a CTL response to PSCA. Helper T cell (HTL) peptides, such as natural or artificial loosely restricted HLA class II peptides, may optionally be included to promote a CTL response. Accordingly, the vaccines of the present invention are used to treat cancers that express or overexpress PSCA. the

X.D.) Adoptive immunotherapy

Antigenic PSCA-related peptides are also used to induce CTL and/or HTL responses ex vivo. The resulting CTL or HTL cells can be used to treat tumors in patients who have not responded to other conventional treatment modalities or will not respond to the therapeutic vaccine peptides or nucleic acids of the invention. Induction of CTL or HTL to a specific antigen ex vivo by culturing patient or genetically compatible CTL or HTL precursor cells together with antigen-presenting cells (APCs) such as dendritic cells and appropriate immunogenic peptides in tissue culture answer. After an appropriate period of culture (usually about 7-28 days) during which precursor cells are activated and expanded into effector cells, they are infused back into the patient where they will either destroy (CTL) or help destroy (HTL) their Specific target cells (eg tumor cells). Transfected dendritic cells can also be used as antigen presenting cells. the

X.E.) Administration of vaccines for therapeutic or prophylactic purposes

The pharmaceutical and vaccine compositions of the present invention can generally be used to treat and/or prevent cancers that express or overexpress PSCA. In therapeutic applications, the peptide and/or nucleic acid composition may be administered to a patient in an amount sufficient to elicit an effective B cell, CTL and/or HTL response to the antigen, or to cure or at least partially arrest or alleviate symptoms and / or complications. An amount suitable for this purpose is referred to as a "therapeutically effective dose". Amounts effective for this use will depend, for example, on the particular composition administered, the mode of administration, the stage and severity of the disease being treated, the weight and health of the patient, and the judgment of the attending physician. the

The pharmaceutical compositions, immunogenic peptides or DNA encoding them of the present invention are administered to individuals already bearing tumors expressing PSCA. The peptide or its encoding DNA can be administered alone or as a fusion sequence of one or more peptide sequences. Patients can be treated with the immunogenic peptides alone or, if appropriate, in combination with other treatments such as surgery. the

In therapeutic applications, dosing typically begins when a PSCA-associated cancer is first diagnosed. The dose is then increased until symptoms are at least largely relieved and persist for some time. The vaccine composition (ie, including but not limited to, for example, peptide mixtures, multi-epitopic polypeptides, minigene or TAA-specific CTLs or pulsed dendritic cells) delivered to a patient may vary depending on the stage of the disease or the health status of the patient. For example, in patients with PSCA-expressing tumors, vaccines containing PSCA-specific CTLs were more effective at killing tumor cells in patients with advanced cancer than other examples. the

It is generally important to deliver, by one mode of administration, an amount of the peptide epitope sufficient to effectively stimulate a cytotoxic T cell response; compositions that stimulate a helper T cell response may also be administered in accordance with embodiments of the present invention. the

Dosages for initial therapeutic immunization are usually presented in unit doses ranging from about 1, 5, 50, 500 or 1,000 μg at the lower end to about 10,000, 20,000, 30,000 or 50,000 μg at the upper end. Dosage values for humans generally range from about 500 [mu]g to about 50,000 [mu]g per 70 kg patient body weight. The dose may be increased over subsequent weeks to months, between about 1.0 mg and about 50,000 mg of peptide, 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 can be continued until clinical symptoms or laboratory tests show that tumors have been ruled out or reduced, and continued for a period of time. Dosage, route of administration, and dosing regimen will be judged according to methods known in the art. the

In certain embodiments, the peptides and compositions of the invention are useful in serious disease states, ie, life-threatening or potentially life-threatening conditions. At this point, it may be, and necessary, to treat the patient with an excess of the peptide composition relative to the dose, based on the minimal amount of foreign matter and the preferred relatively non-toxic properties of the peptides in the compositions of the invention. the

The vaccine composition of the present invention may also be used only as a prophylactic agent. The initial dose of prophylactic immunization will usually be presented as a unit dose ranging from a lower limit of about 1, 5, 50, 500 or 1000 μg to an upper limit of about 10,000, 20,000, 30,000 or 50,000 μg. Dosage values for humans generally range from about 500 [mu]g to about 50,000 [mu]g per 70 kg patient. Booster doses of between about 1.0 mg and about 50,000 mg of peptide are given at predetermined intervals of about 4 weeks to 6 months after the primary vaccination. The immunogenicity of the vaccine can be assessed by measuring the specific activity of CTL and HTL obtained from patient blood samples. the

Therapeutic pharmaceutical compositions can be administered parenterally, topically, orally, nasally, intrathecally or topically (e.g., as a cream or topical ointment). The pharmaceutical composition is preferably administered parenterally, eg intravenously, subcutaneously, intradermally or intramuscularly. Accordingly, the present invention provides compositions for parenteral administration comprising a solution of the immunogenic peptide dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. the

Various aqueous carriers can be used, such as water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid, and the like. These compositions may be sterilized by well known conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solution can be packaged as a preparation for immediate use, or lyophilized, the lyophilized product requiring reconstitution with a sterile solution prior to administration. the

Said composition may contain pharmaceutically acceptable auxiliary substances to meet the needs of physiological conditions, such as pH-regulators and buffers, tonicity regulators, wetting agents, preservatives, etc., specifically, sodium acetate, sodium lactate, chlorine Sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. the

The concentration of the peptides of the present invention in the pharmaceutical formulation is variable, i.e. its proportion by weight can range from less than about 0.1% (usually or at least about 2%) to 20%-50% or higher, and will vary depending on the specific dose selected. The drug mode is mainly selected by liquid volume, viscosity, etc. the

The pharmaceutical compositions generally contain human unit dosage units of the composition comprising a human unit dosage of an acceptable carrier, in one embodiment an aqueous carrier, in a manner known to those skilled in the art. The volume/mass ratio for administering this composition to humans (see for example "Remington's Pharmaceutical Sciences" (Remington's Pharmaceutical Sciences) 17th edition, edited by A. Gennaro, Mack Publishing Co., Easton, Pennsylvania, 1985 ). For example, the initial immunizing peptide dose for a 70 kg patient may range from about 1 to about 50,000 mg, typically 100-5,000 mg. For example, for nucleic acids, primary immunization can be performed with expression vectors in the form of naked nucleic acids administered IM (or SC or ID) at multiple sites in an amount of 0.5-5 mg. Nucleic acids (0.1-1000 mg) can also be administered using a gene gun. A booster dose is given 3-4 weeks later. The booster may be recombinant fowlpox virus administered at a dose of 5-107 to 5 x 109 pfu. the

For antibodies, treatment typically involves repeated administration of the anti-PSCA antibody preparation by an acceptable route of administration, such as intravenous (IV), at a dose typically of about 0.1-10 mg/kg body weight. Typically, weekly doses of 10-500 mg MAb are effective and well tolerated. In addition, an acceptable dosing regimen for anti-PSCAMAb preparations is an initial loading dose of about 4 mg IV per kilogram of body weight of the patient, followed by weekly IV doses of about 2 mg/kg. Those skilled in the art know that there are many factors that will affect the ideal dosage in a particular case. These factors include, for example, the half-life of the composition, the binding affinity of the Ab, the immunogenicity of the substance, the degree of expression of PSCA in the patient, the degree of circulatory elimination of PSCA antigen, the desired steady-state concentration level, frequency of treatment, and The effects of chemotherapeutic or other agents used in conjunction with the methods of treatment of the invention and the health status of a particular patient. Non-limiting preferred human unit doses are, for example, 500 μg-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 dosage range is 2-5 mg per kilogram body weight, for example, followed by 1-3 mg/kg weekly; for example, followed by 0.5 mg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/kg body weight given weekly for 2, 3 or 4 weeks; eg, followed by 0.5-10 mg/kg body weight given weekly for 2, 3 or 4 weeks; 225, 250, 275, 300, 325, 350, 375, 400 mg/m2 body surface area; 1-600 mg/m2 body surface area per week; 225-400 mg/m2 body surface area per week; these doses can be given weekly for periods of 2, 3, 4, 5, 6, 7, 8, 9, 19, 11, 12 or more weeks. the

In one embodiment, human unit dosage forms of polynucleotides include a suitable dosage range or effective amount to provide any therapeutic effect. As is understood by those of ordinary skill in the art, the therapeutic effect depends on many factors, including the sequence of the polynucleotide, the molecular weight of the polynucleotide, and the route of administration. Dosages are typically selected by a physician or other health care professional based on various parameters known in the art (eg, severity of symptoms, medical history, etc.). Typically, for a polynucleotide of about 20 bases, the dosage range may be selected, for example, from an independently selected lower limit, such as about 0.1, 0.25, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 . , 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 mg/kg. For example, the dose can be any of the following: 0.1-100 mg/kg, 0.1-50 mg/kg, 0.1-25 mg/kg, 0.1-10 mg/kg, 1-500 mg/kg, 100-400 mg/kg, 200-300 mg/kg kg, 1-100 mg/kg, 100-200 mg/kg, 300-400 mg/kg, 400-500 mg/kg, 500-1000 mg/kg, 500-5000 mg/kg, or 500-10,000 mg/kg. Generally, the parenteral route of administration requires higher doses of polynucleotides than more direct application of the nucleotides to diseased tissue, and doses need to be increased as the length of the polynucleotide increases. the

In one embodiment, human unit dosage forms of T cells include a suitable dosage range or effective amount to provide any therapeutic effect. As is understood by those of ordinary skill in the art, the effect of treatment depends on many factors. Dosages are typically selected by a physician or other health care professional based on various parameters known in the art (eg, severity of symptoms, medical history, etc.). The dose can be about 104-106 cells, about 106-108 cells, about 108-1011 cells, or about 108-5 x 1010 cells. The dose can also be from about 106 cells/m2 to about 1010 cells/m2, or from about 106 cells/m2 to about 108 cells/m2. the

The protein and/or protein-encoding nucleic acid of the present invention can also be administered via liposomes, which have the following effects: 1) targeting the protein to specific tissues such as lymphoid tissue; 2) selectively targeting diseased cells; or 3) Extend the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, multilayers, and the like. In these preparations, the peptides to be delivered are incorporated as part of liposomes alone or with molecules that bind to lymphocyte ubiquitous receptors (such as monoclonal antibodies that bind the CD45 antigen) or other therapeutic or immunogenic components Incorporate. Thus, liposomes loaded with the desired peptides of the invention or liposomes decorated with such peptides can be directed to the lymphocyte site where the liposomes will then deliver the peptide composition. Liposomes for use in the invention can be formed from standard carriers to form lipids, which generally include neutrally and negatively charged phospholipids and a steroid (eg, cholesterol). Factors such as liposome size, acid instability, and stability of liposomes in blood are generally considered when choosing a lipid. There are many methods for preparing liposomes, such as those described in Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980) and US Patents 4,235,871, 4,501,728, 4,837,028 and 5,019,369. the

To target cells to the immune system, ligands incorporated into liposomes may include, for example, antibodies or fragments thereof specific for cell surface determinants of the desired immune system. The liposome suspension containing the peptide can be administered intravenously, locally, etc., and the dosage can be changed according to factors such as the mode of administration, the peptide to be delivered, and the stage of the disease to be treated. the

For solid compositions conventional nontoxic solid carriers can be used including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talc. Cellulose, glucose, sucrose, magnesium carbonate, etc. For oral administration, a pharmaceutically acceptable non-toxic composition, usually containing 10-95% active ingredient, can be formed by incorporating any of the commonly used excipients such as those carriers listed above. , ie one or more peptides of the present invention, and a more preferred concentration is 25%-75%. the

For aerosol administration, the immunogenic peptide is preferably provided in finely divided form together with a surfactant and propellant. The proportion by weight of the peptide is usually about 0.01%-20%, preferably about 1%-10%. Of course, the surfactant must be non-toxic and is preferably soluble in the propellant. Representative of such agents are fatty acids containing 6-22 carbon atoms (e.g. caproic acid, caprylic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, olesteric acid and oleic acid) with aliphatic polyhydric alcohols or their cyclic anhydrides or partial esters. Mixed esters, such as mixed or natural glycerides, can be used. Surfactants comprise from about 0.1% to 20%, preferably from about 0.25% to about 5%, by weight of the composition. The composition is equilibrated with conventional propellants. A carrier may also be included if desired, for example lecithin for intranasal delivery. the

XI.) Diagnosis and prognosis 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 for Detection of diseases associated with dysregulation of cell growth, such as cancers, especially those listed in Table I (see, e.g., their tissue-specific expression patterns and their overexpression in certain cancers, under the heading "Normal Tissues and Patient Specimens Expression analysis of PSCA" described in the Examples). the

Thus, PSCA is a kind of prostate-associated antigen PSA, and medical workers have used this prototype marker to identify and monitor the occurrence of prostate cancer for many years (see, for example, Merrill et al., J.Urol.163 (2): 503-5120 (2000); Polascik et al., J. Urol. Aug;162(2):293-306 (1999) and Fortier et al., J. Nat. Cancer Inst. 91(19):1635-1640 (1999)). Many other diagnostic markers can also be used, including p53 and K-ras (see for example Tulchinsky et al., Int J Mol Med 1999-7-4(1): 99-102 and Minimoto et al., Cancer Detect Prev 2000; 24(1) : 1-12). Thus, the disclosure of PSCA polynucleotides and polypeptides (as well as the PSCA polynucleotide probes and anti-PSCA antibodies used to identify the presence of these molecules) and their characteristics enables the skilled artisan to use these molecules in similar methods, Examples include many diagnostic tests involving cancer-related symptoms. the

Typical embodiments of diagnostic methods using PSCA polynucleotides, polypeptides, reactive T cells, and antibodies are analogous to well-established diagnostic assays using, for example, PSA polynucleotides, polypeptides, reactive T cells, and antibodies. For example, as in methods of monitoring PSA overexpression or prostate cancer metastasis using PSA polynucleotides as probes (e.g. Northern analysis, see e.g. Sharief et al., Biochem. Mol. Biol. Int. 33(3): 567-74 (1994)) and primers (e.g. 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, the same method can be used here The PSCA polynucleotide is used to monitor the overexpression of PSCA or the metastasis of prostate cancer and other cancers expressing this gene. Or, as in monitoring PSA protein overexpression (see e.g. Stephan et al., Urology 55(4):560-3 (2000)) or prostate cell metastasis (see e.g. Alanen et al., Pathol. Res. Pract. 192(3): 233-7 (1996)) in the method, use PSA polypeptide to produce PSA-specific antibody and be used for observing the presence and/or level of PSA albumen, can use the PSCA polypeptide described here to produce and be used for monitoring the overexpression of PSCA and and/or metastasis of prostate cells and other cancer cells expressing this gene. the

In particular, since metastasis involves the movement of cancer cells from one organ of origin (such as the lung or prostate, etc.) to other regions of the body (such as lymph nodes), detection of biological samples for the presence of PSCA polynucleotides and/or polypeptides can be used. Cell tests to provide evidence of metastasis. For example, when PSCA-expressing cells are found in a biological sample from a tissue (lymph node) that does not normally contain PSCA-expressing cells, such as PSCA expression observed in xenografts LAPC4 and LAPC9 isolated from lymph nodes and bone metastases, the Findings predict metastases. the

Alternatively, PSCA polynucleotides and/or polypeptides may be used to provide evidence of cancer, for example, when cells in a biological sample that do not normally express PSCA or that express PSCA at varying levels are found to express PSCA or have elevated expression of PSCA (see, e.g., Table 1). PSCA expression in the cancers listed and in the patient samples etc. shown in the corresponding figures). In such an assay, the skilled person would also wish to generate complementary evidence on metastasis by detecting the presence in the biological sample of a second tissue-restricted marker other than PSCA, such as PSA, PSCA, etc. (see e.g. Alanen et al., Pathol. Res. Pract. 192(3):233-237(1996)). the

Identification of PSCA polypeptides in tissue sections using immunohistochemistry may indicate an altered state of certain cells within the tissue. As is well known in the art, the ability of antibodies to localize to polypeptides expressed by cancer cells is a means of diagnosing the presence, stage, progression, and/or aggressiveness of a disease. The antibody also detects altered distribution of the polypeptide in cancer cells compared to corresponding non-malignant tissue. the

PSCA polypeptides and immunogenic compositions can also be used to understand changes in subcellular protein localization in disease states. Changes in cells from a normal state to a diseased state result in changes in cellular morphology and are often associated with changes in the localization/distribution of subcellular units. For example, membrane proteins that are expressed in a polarized manner in normal cells are altered in disease, which causes the proteins to be distributed in a nonpolar manner across the cell surface. the

The phenomenon of altered subcellular protein localization in disease states has been demonstrated by immunohistochemistry using MUCl and Her2 protein expression. Normal epithelial cells have a typical apical distribution of MUC1, in addition to nuclear localization of glycoproteins, whereas malignant tumor lesions usually exhibit a nonpolar staining pattern (Diaz et al., The Breast Journal, 7; 40- 45 (2001); Zhang et al., Clinical Cancer Research, 4; 2669-2676 (1998): Cao et al., The Journal of Histochemistry and Cytochemistry, 45: 1547-1557 (1997)). Furthermore, normal mammary epithelium is negative for Her2 protein or shows only a basolateral distribution, whereas malignant T cells can express the protein throughout the cell surface (DePotter et al., International Journal of Cancer, 44; 969-974 (1989): McCormick et al. 117; 935-943 (2002)). Alternatively, in a disease state, the distribution of the protein may change from localized only on the surface to dispersed into the cytoplasm. An example of this can be observed for MUCl (Diaz et al., The Breast Journal, 7:40-45 (2001)). the

Changes in intracellular protein localization/distribution detected by immunohistochemistry also provide important information on the feasibility of certain therapeutic approaches. This last point is illustrated by the situation for proteins that are intracellular in normal tissues but appear on the cell surface in malignant cells; cell surface localization makes the cells favorable for use in antibody-based diagnostic and therapeutic approaches. The PSCA protein and the immune response associated with it are very useful when PSCA undergoes altered protein localization. Thus, the ability to determine whether the subcellular protein localization of 24P4C12 is altered makes PSCA proteins and immune responses associated with them very useful. The use of PSCA compositions enables those skilled in the art to make important diagnostic and therapeutic decisions. the

Immunohistochemical reagents specific for PSCA are also useful for detecting metastases from PSCA-expressing tumors when the PSCA polypeptide is present in tissues that do not normally produce PSCA. the

Accordingly, PSCA polypeptides and antibodies raised in response to them can be used in many important ways, such as diagnostic, prognostic, prophylactic and/or therapeutic purposes known to those skilled in the art. the

Just as the skilled artisan uses PSA polynucleotide fragments and polynucleotide variants in methods for monitoring PSA, PSCA polynucleotide fragments and polynucleotide variants can be used in a similar manner. Specifically, typical PSA polynucleotides used in methods of monitoring PSA are probes or primers composed of fragments of the PSA cDNA sequence. For example, primers used to PCR amplify PSA polynucleotides must contain incomplete PSA sequences to function in the polymerase chain reaction. In such PCR reactions, the skilled artisan typically generates many different polynucleotide fragments that can be used as primers to amplify different portions of the polynucleotide of interest or to optimize the amplification reaction (see e.g. Caetano-Anolles, G. Biotechniques 25(3):472-476, 478-480 (1998); Robertson et al., Methods Mol. Biol. 98:121-154 (1998)). Another example of the use of such fragments is provided in the Example entitled "Expression Analysis of PSCA in Normal Tissue and Patient Specimens", where PSCA polynucleotide fragments are used as probes to show expression of PSCA RNA in cancer cells . Furthermore, variant polynucleotide sequences are commonly used as primers and probes for the corresponding mRNA in PCR and Northern analysis (see for example Sawai et al., Fetal Diagn. Ther. 1996 11-12 11(6): 407-13 and Frederick "Recent Methods in Molecular Biology", edited by M. Ausubel et al., 1995)). Polynucleotide fragments and variants are useful herein that are capable of binding a target polynucleotide sequence (eg, the PSCA polynucleotide shown in Figure 1 or variants thereof) under highly stringent conditions. the

In addition, PSA polypeptides containing epitopes recognized by antibodies or T cells that specifically bind the epitopes are used in methods of monitoring PSA. PSCA polypeptide fragments and polypeptide analogs or variants can also be used in a similar manner. Such use of polypeptide fragments or polypeptide variants to generate antibodies (such as anti-PSA antibodies or T cells) is common in many systems in the art, such as fusion protein systems used by skilled artisans (see, e.g., Frederick M. Ausubel et al. eds. "Recent Methods in Molecular Biology", 1995, Volume 2, Unit 16). Herein, the function of each epitope is to provide a reactive structure for antibodies or T cells. Typically, the skilled artisan creates many different polypeptide fragments that can be used to generate immune responses specific for different portions of the polypeptide of interest (see, eg, US Patent 5,840,501 and US Patent 5,939,533). For example, polypeptides containing one of the PSCA biological motifs described herein or subsequences with the motif that are readily identifiable by those skilled in the art based on sequences known in the art may be preferably used. Polypeptide fragments, variants or analogs are generally useful herein as long as they contain epitopes capable of generating antibodies or T cells specific to a target polypeptide sequence (eg, the PSCA polypeptide shown in Figure 3). the

As shown herein, PSCA polynucleotides and polypeptides (as well as PSCA polynucleotide probes and anti-PSCA antibodies or T cells used to identify the presence of these molecules) have specific properties that make them useful for diagnosing cancer, Cancers as listed in Table I. Diagnostic methods that measure the presence of the PSCA gene product to assess the presence or occurrence of the disease conditions described herein, such as prostate cancer, are used to identify patients for preventive testing or further monitoring, as has been successful with PSA. Furthermore, these substances fulfill a need in the art for small molecules with similar or complementary properties to PSA in situations where, for example, metastases of prostate origin cannot be definitively diagnosed on the basis of PSA alone (see, for example, Alanen et al., Pathol .Res.Pract.192 (3): 233-237 (1996)), therefore need to use substances such as PSCA polynucleotides and polypeptides (and the PSCA polynucleotide probes and anti-PSCA antibodies that are used to identify the presence of these molecules) To confirm metastases of prostate origin. the

Finally, in addition to their use in diagnostic assays, the PSCA polynucleotides described herein have many other uses, such as their use to identify chromosomal abnormalities associated with carcinogenesis within the chromosomal region of the PSCA gene (see below entitled "Chromosomal Mapping of PSCA") "Example). Furthermore, the PSCA-related proteins and polynucleotides described herein have many other uses besides their use in diagnostic assays, for example they can be used in forensic analysis of tissues of unknown origin (see e.g. Takahama K Forensic Sci Int 1996-6-28; 80(1-2):63-9). the

In addition, the PSCA-related proteins or polynucleotides of the invention can be used to treat pathological conditions characterized by overexpression of PSCA. For example, the amino acid or nucleic acid sequence of Figure 1, or a fragment thereof, can be used to generate an immune response against a PSCA antigen. Antibodies or other molecules reactive with PSCA can be used to modulate the function of this molecule and thus provide therapeutic benefit. the

XII.) Inhibition of PSCA protein function

The present invention includes various methods and compositions for inhibiting PSCA from binding to its binding partners or interacting with other proteins, as well as methods of inhibiting PSCA function. the

XII.A.) Inhibition of PSCA with Intrabodies

In one approach, a recombinant vector encoding a single-chain antibody that specifically binds PSCA is introduced into PSCA-expressing cells by gene delivery techniques. Thus, the encoded single chain anti-PSCA antibody is expressed intracellularly, binds the PSCA protein, and thereby inhibits its function. Methods for engineering such intracellular single chain antibodies are well known. Such intrabodies, also known as "intrabodies," can specifically target a specific compartment within the cell, making the inhibitory effect of the therapy concentrated in that compartment. This technique has been successfully used in the art (see eg Richardson and Marasco, 1995, TIBTECH Vol. 13). Intrabodies can in fact abolish the expression of highly abundant cell surface receptors (see, e.g., Richardson et al., 1995, Proc. Natl. Acad. Sci. USA 92:3137-3141; Beerli et al., 1994, J. Biol. Chem. 289:23931-23936; Deshane et al., 1994, Gene Ther. 1:332-337). the

Single-chain antibodies comprise the variable regions of the heavy and light chains connected by a flexible linker polypeptide and are expressed as a single polypeptide. Optionally, the single chain antibody is expressed as a single chain variable region fragment combined with a light chain constant region. Well-known intracellular trafficking signals are engineered into recombinant polynucleotide vectors encoding such single chain antibodies to precisely target the intrabodies to desired intracellular compartments. For example, intrabodies targeted to the endoplasmic reticulum (ER) are engineered to incorporate a leader peptide and an optional C-terminal ER retention signal peptide (such as the KDEL amino acid motif). Engineered to contain a nuclear localization signal so that intrabodies are active in the nucleus. The lipid moiety is bound to the intrabody to confine the intracellular receptor to the cytoplasmic side of the plasma membrane. Intrabodies are also targeted to function in the cytosol. For example, intrabodies in the cytoplasm sequester factors in the cytosol, which prevents their access to their natural cellular target sites. the

In one embodiment, intrabodies are used to capture PSCA in the nucleus, thereby preventing its activity in the nucleus. A nuclear targeting signal was added to this PSCA intrabody to achieve the desired targeting. Such PSCA intrabodies can be designed to specifically bind a particular PSCA domain. In another embodiment, cytoplasmic intrabodies that specifically bind PSCA proteins are used to prevent PSCA from contacting the nucleus, thereby preventing them from exerting any biological activity in the nucleus (eg, preventing PSCA from forming transcriptional complexes with other factors). the

To specifically direct the expression of such intracellular receptors in specific cells, the intracellular receptors are transcribed under the control of appropriate tumor-specific promoters and/or enhancers. For prostate-specific expression of intracellular receptors, for example, the PSA promoter and/or promoter/enhancer can be used (see, eg, US Pat. No. 5,919,652 issued Jul. 6, 1999). the

XII.B.) Inhibition of PSCA with recombinant proteins

In another approach, recombinant molecules bind PSCA and thereby inhibit PSCA function. For example, these recombinant molecules can prevent or inhibit PSCA from contacting/binding to its binding partners or association with other proteins. For example, such recombinant molecules may contain reactive portions of PSCA-specific antibody molecules. In a specific embodiment, the PSCA binding domain of the PSCA binding partner is engineered into a dimeric fusion protein such that the fusion protein contains two PSCA ligand binding domains that bind to the Fc portion of a human IgG (eg, human IgGl). Such an IgG portion may contain, for example, the CH2 and CH3 regions and the hinge region, but not the CH1 region. This dimeric fusion protein can be administered in soluble form to patients with cancers associated with PSCA expression, whereby the dimeric fusion protein specifically binds PSCA and blocks the interaction of PSCA with the binding partner. Such dimeric fusion proteins can also be assembled into multimeric proteins using known antibody conjugation techniques. the

XII.C.) Inhibition of PSCA Transcription or Translation

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

In one method, the method of inhibiting transcription of the PSCA gene comprises contacting the PSCA gene with a PSCA antisense polynucleotide. In another approach, the method of inhibiting translation of PSCA mRNA comprises contacting PSCA mRNA with an antisense polynucleotide. In another approach, translation is inhibited by cleaving the PSCA message with a PSCA-specific ribozyme. This antisense and ribozyme-based approach can also target regulatory regions of the PSCA gene, such as the PSCA promoter and/or enhancer elements. Similarly, proteins capable of inhibiting PSCA gene transcription factors are used to inhibit PSCA mRNA transcription. Various polynucleotides and compositions useful in the above methods have been described above. Methods for inhibiting transcription and translation using antisense and ribozyme molecules are well known in the art. the

Other factors that inhibit PSCA transcription by disturbing PSCA transcriptional activity may also be used to treat PSCA-expressing cancers. Similarly, factors that disrupt PSCA processing could also be used to treat PSCA-expressing cancers. Cancer treatment methods using such factors are also within the scope of the invention. the

XII.D.) General course of the method of treatment

Gene transfer and gene therapy techniques can be used to deliver therapeutic polynucleotide molecules to PSCA-synthesizing tumor cells (ie, antisense, ribozymes, polynucleotides encoding intrabodies, and other PSCA-inhibiting molecules). Many gene therapy approaches are known in the art. This gene therapy method can also be used to deliver recombinant vectors encoding PSCA antisense polynucleotides, ribozymes, factors capable of disrupting PSCA transcription, etc. to target tumor cells. the

The above-mentioned treatment methods can be combined with widely used surgery, chemotherapy or radiation therapy. The therapeutic methods of the present invention allow for a reduction in the dose and/or frequency of administration of chemotherapy (or other therapies), which is beneficial to patients, especially those who do not tolerate the toxicity of chemotherapeutic agents well. the

The antitumor activity of a particular composition (eg, antisense, ribozyme, intracellular receptor) or combination of these substances can be assessed using a variety of in vitro and in vivo assay systems. In vitro assays to assess therapeutic activity include cell growth assays, soft agar assays and other assays indicative of tumor growth activity, binding assays capable of determining the extent to which a therapeutic compound will inhibit binding of PSCA to binding partners and the like, and the like. the

The in vivo effects of PSCA therapeutic compositions can be evaluated in suitable animal models. For example, xenogeneic prostate cancer models can be used in which human prostate cancer explants or passaged xenograft tissue 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 WO 98/16628 and US patent 6,107,540 describe various xenograft models of human prostate cancer that are capable of illustrating primary tumor development, micrometastasis, and osteoblastic metastasis that characterize advanced disease. Efficacy can be predicted using assays for inhibition of tumor formation, tumor regression or metastasis, and the like. the

In vivo assays to evaluate apoptosis promotion can also be used to evaluate therapeutic compositions. In one embodiment, mice bearing tumor xenografts treated with a therapeutic composition can be tested for the presence of apoptotic foci compared to untreated mice bearing control xenografts. The apoptotic foci found within the tumors of the treated mice illustrate the therapeutic efficacy of the composition. the

Therapeutic compositions used in the methods described above may be formulated as pharmaceutical compositions containing a carrier suitable for the method of delivery. Suitable carriers include any material that, when mixed with a therapeutic composition, retains the antitumor function of the therapeutic composition and is generally non-reactive with the patient's immunogenicity. Examples thereof include, but are not limited to, any standard pharmaceutical carrier, such as sterile phosphate-buffered saline solution, bacteriostatic water, and the like (see generally "Remington's Pharmaceutical Sciences," 16th edition, edited by A. Osal., 1980). the

Therapeutic formulations can be dissolved and administered by any route capable of delivering the therapeutic composition to the tumor site. More effective routes of administration include, but are not limited to: intravenous, parenteral, intraperitoneal, intramuscular, intratumoral, intradermal, intraorgan, orthotopic, etc. The therapeutic composition contained in the preferred intravenous injection preparation can be formulated into a solution with preserved bacteriostatic water, sterile non-preserved water or diluted in a polyvinyl chloride or polyethylene bag containing 0.9% sterile sodium chloride for injection. Therapeutic protein preparations can be lyophilized and stored as sterile powders, preferably lyophilized under vacuum, and reconstituted with bacteriostatic water (containing a preservative such as benzyl alcohol) or sterile water prior to injection. the

Dosages and administration methods for treating cancer using the methods described above will vary depending on the method and target cancer, and will generally depend on a number of other factors known in the art. the

XIII.) Identification, Characterization and Use of PSCA Modulators

Methods of Identifying and Using Modulators

In one embodiment, a screen is performed to identify modulators that induce or inhibit a particular expression pattern, inhibit or induce a particular pathway, preferably thereby producing a relevant phenotype. In another embodiment, differentially expressed genes important for a particular situation have been identified; screens are performed to identify modulators that alter (increase or decrease) the expression of individual genes. In another embodiment, a screen is performed to identify modulators that alter the biological function of the expression products of differentially expressed genes. Furthermore, having identified the importance of a gene in a particular state, a screen is performed to identify agents that bind to and/or modulate the biological activity of the gene product. the

In addition, genes are screened for induction in response to candidate agents. Identification of modulators (agents that suppress expression patterns in cancer to produce normal expression patterns, or modulators that modulate oncogenes so that the gene is expressed in normal tissues) followed by screening to identify agents that are specifically affected in response to that agent Regulated genes. Comparing the expression patterns of normal tissue and cancer tissue treated with the agent can reveal genes that are not expressed in normal tissue or cancer tissue but are expressed in tissue treated with the agent, or vice versa. These agent-specific sequences are identified and used as described herein for cancer genes or proteins. Specifically, these sequences and the proteins they encode are used to label or identify reagent-treated cells. In addition, antibodies against the agent-inducible proteins were generated and used to target new therapeutic agents to treated cancer tissue samples. the

Identification and Screening Assays Related to Modulators:

Assays related to gene expression

The proteins, nucleic acids and antibodies of the invention are used in screening assays. Cancer-associated proteins, antibodies, nucleic acids, modified proteins, and cells containing these sequences are used in screening assays, eg, to evaluate the effect of drug candidates on "gene expression patterns," polypeptide expression patterns, or altered biological function. In one embodiment, expression patterns are used (preferably in combination with high-throughput screening techniques) to monitor gene expression patterns following treatment with candidate agents (e.g. Dayis, GF et al., J Biol Screen 7:69 (2002); Zlokarnik et al., Science 279:84-8 (1998); Heid, Genome Res 6:986-94, 1996). the

Oncoproteins, antibodies, nucleic acids, modified proteins, and cells containing native or modified oncoproteins or genes are used in screening assays. That is, the present invention includes screening methods for modulating the cancer phenotype or the physiological function of the oncoproteins of the present invention. This can be done genetically or by evaluating the "gene expression pattern" or biological function of the drug candidate. In one embodiment, expression patterns are used (preferably in combination with high throughput screening techniques) to monitor after treatment with candidate agents, see Zlokamik, supra. the

Various experiments involving the genes and proteins of the present invention were performed. Assays can be performed at the individual nucleic acid or protein level. That is, after identifying a particular gene that is upregulated in cancer, test compounds can be screened for the ability to modulate gene expression or bind an oncoprotein of the invention. "Modulation"herein includes increasing or decreasing gene expression. The preferred amount of modulation will depend on the initial change in gene expression in normal versus cancerous tissue, which is at least 10%, preferably 50%, more preferably 100-300%, and in some embodiments 300-1000% or more high. Thus, if a gene has a 4-fold increase in expression in cancer tissue compared to normal tissue, it will often require a reduction of about 4-fold; similarly, a 10-fold decrease in cancer tissue compared to normal tissue will often require a test compound to increase its expression by 10. times. Modulators that aggravate gene expression in cancer can also be employed, e.g., upregulate target gene expression in further analysis. the

The amount of gene expression and gene expression levels can be monitored with nucleic acid probes and quantitative assays, or the gene product itself can be monitored, for example, by using antibodies to oncoproteins and standard immunoassays. Expression can also be quantified using proteomics and separation techniques. the

Identify compounds that modify gene expression by monitoring expression

In one embodiment, gene expression monitoring is performed on many entities simultaneously, ie, expression profiling is monitored. Such profiles typically include one or more of the genes shown in Figure 1 . In this embodiment, for example, cancer nucleic acid probes are bound to biochips to detect and quantify cancer sequences within specific cells. Alternatively PCR can be used. Microtiter plates can therefore be used with primers dispersed in the desired wells. PCR reactions can then be performed and each well analyzed. the

Expression monitoring is performed to identify compounds that modify the expression of one or more cancer-associated sequences, such as the polynucleotide sequences listed in FIG. 1 . Test modulators are typically added to the cells prior to analysis. In addition, screens are performed to identify molecules that modulate cancer, modulate an oncoprotein of the invention, bind an oncoprotein of the invention, or disrupt the binding of an oncoprotein of the invention to an antibody or its binding partner. the

In one embodiment, the high-throughput screening method involves providing a library comprising 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 (particularly chemical species or subclasses) that possess the desired chemical activity. The identified compounds can be used as conventional "lead compounds", such as compounds for screening, or as therapeutic agents. the

In certain embodiments, combinatorial libraries of potential modulators are screened for the ability to bind or modulate activity of a cancer polypeptide. In general, compounds with useful properties can be generated by identifying compounds (termed "lead compounds") that possess some desired property or activity (e.g., inhibitory activity), generating variants of said lead compounds, and evaluating the properties and activities of these variant compounds. properties of new chemical entities. Typically, high throughput screening (HTS) methods are used for this analysis. the

As noted above, gene expression monitoring is often used to detect candidate modulators (eg, proteins, nucleic acids, or small molecules). After adding the candidate reagents and incubating the cells for a period of time, a sample containing the target sequence to be analyzed is, for example, applied to a biochip. the

Target sequences can be prepared, if desired, using known techniques. For example, the sample is treated with known lysis buffers, electroporation, etc. to lyse the cells, and purified and/or amplified, eg, by PCR. For example, in vitro transcription can be performed using labels covalently bound to nucleotides. Nucleic acids are usually labeled with biotin-FITC or PE or with cy3 or cy5. the

The target sequence can be labeled with a fluorescent signal, a chemiluminescent signal, a chemical signal, or a radioactive signal in order to detect the target sequence to which the probe specifically binds. The label can also be an enzyme, such as alkaline phosphatase or horseradish peroxidase, the products of which can be detected when provided with a suitable substrate. Alternatively, labels are labeled compounds or small molecules, such as enzyme inhibitors, that bind to but are not catalyzed or altered by the enzyme. The label may also be, for example, an epitope tag or biotin that specifically binds streptavidin. Using biotin as an example, streptavidin is labeled as described above, thereby providing a detectable signal for the bound target sequence. Unbound labeled streptavidin is usually removed prior to analysis. the

Those skilled in the art will appreciate that these assays can be direct hybridization assays or "sandwich assays" using multiple probes, such methods are generally described in U.S. Patent Nos. 5,681,702; 5,597,909; 5,545,730; 5,571,670; 5,591,584; 5,624,802; 5,635,352; 5,594,118; 5,359,100; 5,124,246 and 5,681,697. In this embodiment, the target nucleic acid is generally prepared as described above, and then applied to a biochip containing a plurality of nucleic acid probes under conditions capable of forming hybridization complexes. the

The present invention adopts a variety of hybridization conditions, including the above-mentioned high, medium and low stringency conditions. The assay is typically performed under stringent conditions such that a labeled probe hybridization complex is formed only in the presence of the target. Stringency can be controlled by varying thermodynamically variable step parameters including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration, pH, organic solvent concentration, and the like. These parameters can also be used to control non-specific binding as described, for example, in US Patent No. 5,681,697. Therefore, it is desirable to perform certain steps under relatively high stringency conditions to reduce non-specific binding. the

The reactions listed here can be achieved in a variety of ways. The reaction components can be added simultaneously or sequentially in different order, and the preferred embodiments thereof are as follows. In addition, many other reagents can be added to the reactants. These reagents include salts, buffers, neutral proteins (such as albumin), detergents, etc., which will facilitate optimal hybridization and detection, and/or reduce nonspecific or background interactions. Reagents that increase assay efficiency, such as protease inhibitors, ribozyme inhibitors, antimicrobials, etc., may also be used if desired, depending on the sample preparation method and the purity of the target. The assay data is analyzed to determine the expression levels of individual genes and changes in expression levels between states to form a gene expression profile. the

Assays related to biological activity

The present invention provides identification or screening of compounds that modulate the activity of the cancer-associated genes or proteins of the present invention. The method comprises adding a detection compound as defined above to cells containing an oncoprotein according to the invention. The cells contain a recombinant nucleic acid encoding an oncoprotein of the invention. In another embodiment, a pool of candidate agents is tested on a number of cells. the

In one aspect, the assay is performed in the presence or absence of, or prior to or subsequent exposure to, a physiological signal such as a hormone, antibody, peptide, antigen, cytokine, growth factor, action potential, pharmaceutical agent (including chemotherapeutic agents), radiation, carcinogens, or other cells (ie, cell-cell contact). In another embodiment, assays are performed at different stages of the cell cycle. Compounds that modulate the genes or proteins of the invention are identified in this way. Pharmacologically active compounds are capable of enhancing or disrupting the activity of the oncoproteins of the invention. Once identified, similar structures can be evaluated in order to identify defining structural features of the compound. the

In one embodiment, there is provided a method of modulating (eg, inhibiting) the division of cancer cells; the method comprising administering a cancer modulator. In another embodiment, there is provided a method of modulating (eg, inhibiting) cancer; the method comprising administering a cancer modulator. In still other embodiments, there is provided a method of treating a cancer cell or an individual having cancer; the method comprising administering a cancer modulator. the

In one embodiment, a method of modulating the state of a cell expressing a gene of the invention is provided. A state as described herein includes art-accepted parameters such as growth, proliferation, viability, function, apoptosis, aging, localization, enzymatic activity, signal transduction, and the like 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. As described herein, various methods for measuring cell growth, proliferation and metastasis are known to those skilled in the art. the

Identification of modulators by high-throughput screening

Assays to identify suitable modulators include high-throughput screening. Preferred assays are capable of detecting enhancement or inhibition of oncogene transcription, enhancement or inhibition of polypeptide expression, and inhibition or enhancement of polypeptide activity. the

In one embodiment, the modulator identified by the high throughput screen is a protein, typically a naturally occurring protein or a fragment of a naturally occurring protein. Thus, for example, protein-containing cell extracts or random or directed digests of proteinaceous cell extracts can be used. In this way, protein libraries for screening in the methods of the invention are prepared. Particularly preferred in this embodiment are bacterial, fungal, viral and mammalian protein libraries, preferably mammalian protein libraries, especially human protein libraries. Particularly useful test compounds will be directed to the protein class to which the target belongs, such as enzymes and substrates, or ligands and receptors. the

Identification and Characterization of Modulators Using Soft Agar Growth and Colony Formation

Normal cells require a solid substrate to attach and grow. When cells are transformed they lose this phenotype and can grow off the matrix. For example, transformed cells can be grown in stirred suspension culture or suspended in semi-solid media such as semi-solid agar or soft agar. When transformed cells are transfected with a tumor suppressor gene, they revert to a normal phenotype and again require a solid substrate for attachment and growth. Soft agar growth or colony formation is used in assays to identify modulators of cancer sequences that, when expressed in host cells, inhibit aberrant cell proliferation and transformation. Modulators reduce or eliminate the ability of host cells to grow in suspension in solid or semi-solid media such as agar. the

Soft agar growth or colony formation techniques in suspension assays are described in Freshney's Culture of Animal Cells a Manual of Basic Technique (Third Edition, 1994). See also the Methods section of Garkavtsev et al. (1996, supra). the

Assessing contact inhibition and growth density limitation to identify and characterize regulators

Normal cells typically grow in a flat and organized pattern in cell culture until they come into contact with other cells. When cells come into contact with other cells they become contact inhibited and stop growing. Transformed cells, however, were not contact inhibited and continued to grow to high densities within the disordered foci. Thus, transformed cells grow to a higher saturation density than normal cells. This is morphologically identified by the formation of a disorganized monolayer of cells in the lesion. Alternatively, the labeling index of ( ^3H )-thymidine at saturation density can be used to measure growth density limitation. Similarly, MTT or Alamar blue assays will show the proliferative capacity of cells and the ability of modulators to affect proliferation. See Freshney, (1994), supra. Transformed cells revert to a normal phenotype when transfected with a tumor suppressor gene, become contact-inhibited and grow to lower densities.

In this assay, the labeling index of ( ^3H )-thymidine at saturation density is the preferred measure of density limitation of growth. Transformed host cells are transfected with the cancer-associated sequences and grown at saturation density for 24 hours in non-restricted culture conditions. The percentage of ( ³ H)-thymidine labeled cells was determined by the number incorporated per minute.

Contact unrestricted growth is used to identify regulators of cancer sequences that lead to abnormal cell proliferation and transformation. A modulator can reduce or eliminate contact unrestrained growth and return the cell to a normal phenotype. the

Evaluate growth factor or serum dependence to identify and characterize modulators

Transformed cells are less serum-dependent than corresponding normal cells (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 from transformed cells. The degree of growth factor or serum dependence of transformed host cells can be compared to control cells. For example, growth factor or serum dependence of cells is monitored in methods to identify and characterize compounds that modulate cancer-associated sequences of the invention. the

Using levels of tumor-specific markers to identify and characterize modulators

Certain factors (hereinafter referred to as "tumor-specific markers") released by tumor cells are higher than corresponding normal cells. For example, human gliomas release plasminogen activator (PA) at higher levels than normal brain cells (see, e.g., Gullino, "Angiogenesis, Tumor Vascularization, and Potential Disorders of Tumor Growth" (Angiogenesis, Tumor Vascularization, and Potential Interference with Tumor Growth), cited in Biologial Responses in Cancer, pp. 178-184 (Mihich ed., 1985)). Similarly, tumor cells also release tumor angiogenesis factor (TAF) at higher levels than corresponding normal cells. See eg Folkman, Angiogenesis and Cancer, Sem Cancer Biol. (1992)), while endotheliomas release bFGF (Ensoli, B et al.). the

Various techniques for measuring 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: 305 312 (1980); Gullino, "Potential disturbances of angiogenesis, tumor vascularization, and tumor growth", cited in Biological Responses in Cancer, pp. 178-184 (Mihich ed., 1985); Freshney, Anticancer Res .5: 111-130 (1985). For example, the levels of tumor-specific markers can be monitored in methods to identify and characterize compounds that modulate cancer-associated sequences of the invention. the

Identification and Characterization of Modulators by Invasion on Matrigel

The extent of invasion into Matrigel or the composition of the extracellular matrix can be used in assays to identify and characterize compounds that modulate cancer-associated sequences. Tumor cells showed a positive correlation between malignant transformation and invasion of matrigel or other extracellular matrix components. Typically tumorigenic cells are used as host cells in this assay. Expression of tumor suppressor genes in these host cells will reduce the invasiveness of the host cells. Techniques described in Cancer Res. 1999;59:6010; Freshney (1994), supra may be used. Briefly, the level of invasion into host cells can be measured with filters coated with Matrigel or other extracellular matrix components. Invasion into the gel or through the distal side of the filter was scored as invasiveness and assessed histologically by cell number and distance traveled or by pre-labeling cells with 125I followed by radioactivity counts on the distal side of the filter or the bottom of the dish. See eg Freshney (1984), supra. the

Assessing tumor growth in vivo to identify and characterize modulators

Detecting the effect of cancer-associated sequences on cell growth in genetically modified or immunosuppressed organisms. Transgenic organisms are prepared using a variety of art-accepted methods. For example, knockout transgenic organisms can be produced, such as mammals such as mice, in which oncogenes are destroyed or in which oncogenes are inserted. Knockout transgenic mice are created by inserting marker genes or other heterologous genes at endogenous cancer gene loci in the mouse genome by homologous recombination. Such mice can also be produced by replacing endogenous oncogenes with mutated oncogenes, or by mutating endogenous oncogenes (eg, by art carcinogens). the

To create transgenic chimeric animals (eg, mice), the DNA construct can be introduced into the nucleus of embryonic stem cells. Cells with the newly constructed genetic defect were injected into host mouse embryos, which were reimplanted into female recipients. Some of these embryos developed into chimeric mice with some germ cells derived from the mutant cell line. Thus, new mouse strains containing the introduced genetic defect can be obtained by breeding such chimeric mice (see, eg, Capecchi et al., Science 244:1288 (1989)). Chimeric mice can be obtained by: U.S. Patent 6,365,797, issued April 2, 2002; U.S. Patent 6,107,540, issued August 22, 2000; Hogan et al., Manipulating the Mouse Embryo: A laboratory Manual), Cold Spring Harbor Laboratory (1988) and "Practical Advances in Teratoma and Embryonic Stem Cells" by Robertson, IRL Press, Washington, D.C., (1987). the

Alternatively, various immunosuppressed or immunodeficient host animals can be used. For example, genetically obtained athymic "nude" mice (see, e.g., Giovanella et al., J. Natl. Cancer Inst. 52:921 (1974)), SCID mice, thymectomized mice, or irradiated mice (see, e.g., Bradley etc., Br. J. Cancer 38:263 (1978); Selby et al., Br. J. Cancer 41:52 (1980)) can be used as the host. Transplantable tumor cells (typically about 106 cells) injected into a syngeneic host produce invasive tumors in high proportions, whereas normal cells of similar origin do not. Cells expressing tumor-associated sequences are injected subcutaneously or orthotopically in hosts that develop invasive tumors. The mice are then divided into a control group and a treatment experimental group (eg, treated with a modulator). After a suitable time, preferably 4-8 weeks, tumor growth is measured (eg by volume or by its two largest dimensions, or weight) and compared to controls. Statistically significant reductions in tumors (using eg Student's T-test) were considered growth inhibited. the

In vitro assays for identification and characterization of modulators

Assays can be performed in vitro to identify compounds with modulatory activity. For example, the cancer polypeptide is first exposed to a potential modulator and then incubated for a suitable period of time, eg, 0.5-48 hours. In one embodiment, cancer polypeptide levels are determined in vitro by measuring protein or mRNA levels. Protein levels are measured using immunoassays (eg, Western blot, ELISA, etc.) with antibodies that selectively bind the cancer polypeptide or fragments thereof. For measuring mRNA, amplification detection (for example with PCR, LCR) or hybridization detection (for example Northern hybridization, RNase protection, dot blot) is preferably used. Protein or mRNA levels are detected using directly or splice-labeled detection reagents such as fluorescently or radiolabeled nucleic acids, radiolabeled or enzyme-labeled antibodies, and the like, as described above. the

Alternatively, reporter gene systems can be designed with oncoprotein promoters operably linked to reporter genes such as luciferase, green fluorescent protein, CAT, or P-gal. Reporter constructs are typically transfected into cells. Following treatment with potential modulators, the amount of reporter gene transcription, translation or activity is measured using standard counts known to those skilled in the art (Davis GF, supra; Gonzalez, J. & Negulescu, P. Curr. Opin. Biotechnol .1998:9:624). the

Individual genes and gene products were screened in vitro as described above. That is, after identification of a particular gene differentially expressed that is important in a particular state, a screen is performed for modulators of expression of that gene or its gene product. the

In one embodiment, modulators of expression of a particular gene are screened. Usually the expression of only one or a few genes is evaluated. In another embodiment, the screen is designed to first look for compounds that bind differentially expressed proteins. These compounds were then evaluated for their ability to modulate differentially expressed activity. Furthermore, once an initial candidate compound is identified, its variants can be further screened to better evaluate structure-activity relationships. the

Binding assays for identification and characterization of modulators

Purified or isolated gene products of the invention are typically used in the binding assays of the invention. For example, antibodies to a protein of the invention are raised and immunoassays are performed to determine the amount and/or localization of the protein. Alternatively, cells containing oncoproteins can be used in this assay. the

Thus, the methods comprise binding an oncoprotein of the invention to a candidate compound (eg, a ligand), and determining the binding of the compound to the oncoprotein of the invention. A preferred embodiment uses human oncoproteins; animal models of human disease can also be created and used. Meanwhile, other similar mammalian proteins can also be used by those skilled in the art. Additionally, some embodiments employ variant or derivatized oncoproteins. the

Typically, an oncoprotein or ligand of the invention is non-diffusively bound to an insoluble support. For example, the support may contain separate sample receiving areas (microtiter plates, arrays, etc.). The insoluble support can be made of any composition, it can be combined with the composition, it can be easily separated from the soluble material, or it is compatible with the overall screening method. The surface of the support can be solid or porous and have any convenient shape. the

Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. They are usually made of glass, plastic (such as polystyrene), polysaccharides, nylon, nitrocellulose, or Teflon ^™ , among others. Microtiter plates and arrays are especially convenient because a large number of assays can be performed simultaneously with small amounts of reagents and samples. The particular method of combining the composition and support is not specified, so long as it is compatible with the reagents and methods of the invention, maintains the activity of the composition, and is non-diffusible. Preferred methods of binding include the use of antibodies that, when binding the protein to the support, do not sterically deter either the ligand binding site or the activation sequence; direct binding to "sticky" or ionic supports; chemical cross-linking; Synthetic proteins or reagents, etc. After binding of the protein or ligand/binding agent to the support, excess unbound material is removed by washing. The sample receiving area is then blocked by incubation with bovine serum albumin (BSA), casein or other unrelated proteins or other chemical molecules.

Once the oncoprotein of the present invention is bound to the support, detection compounds can be added and assayed. Alternatively, the candidate binding agent is bound to a support and the oncoprotein of the invention is added. Binding agents include specific antibodies, non-natural binding agents identified by screening chemical libraries, peptide analogs, and the like. the

Of particular interest is the identification of agents with low toxicity to human cells. A number of assays are available for this purpose, including proliferation assays, cAMP assays, in vitro assays of labeled protein-protein binding, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.), etc. wait. the

Binding of a test compound (ligand, binding agent, modulator, etc.) to an oncoprotein of the invention can be assayed in a number of ways. Detection compounds can be labeled and binding can be determined directly, e.g., adding all or part of the oncoprotein of the invention to a solid support, adding a labeled candidate compound (e.g., fluorescein), washing off excess reagent and determining the solid support Whether there is a mark on the . Different blocking and washing steps can be used if desired. the

In certain embodiments, only one component is labeled, eg, a protein or ligand of the invention. Or label multiple components with different labels, such as labeling the protein with I125 and labeling the compound with a fluorophore. Proximity reagents, such as quenchers or energy transfer agents, are also useful. the

Competitive binding to identify and characterize modulators

In one embodiment, binding of a "test compound" is determined by a competitive binding assay with a "competitor". A competitor is a binding moiety that binds a target molecule (eg, an oncoprotein of the invention). Competitors include compounds such as antibodies, peptides, binding partners, ligands, and the like. In some cases, the competitor competes for binding instead of the test compound. In one embodiment, the detection compound is labeled. A test compound or competitor or both can be added to the protein and allowed to allow sufficient time for binding to occur. Incubation is performed at a temperature that maximizes activity (usually between 4-40°C). Incubation times are usually optimized, eg, to facilitate rapid high-throughput screening; usually 0-1 hour is sufficient. Excess reagent is typically removed or washed away. The second component is then added, followed by or without the addition of a labeled component indicating binding. the

In one embodiment, the competitor is added first, followed by the detection compound. Displacement of the competitor indicates that the test compound binds the oncoprotein and is therefore capable of binding and potentially modulating the activity of the oncoprotein. In this embodiment, either component can be labeled. Thus, for example, if the competitor is labeled, the presence of the label in the wash solution of the detected compound indicates that the competitor has been displaced by the test compound. Alternatively, if the detection compound is labeled, the presence of the label on the support indicates displacement. the

In another embodiment, the test compound is added first, incubated and washed, and then the competitor is added. The absence of competitor binding indicates that the test compound binds the oncoprotein with a higher affinity than the competitor. Therefore, if the test compound is labeled, the presence of the labeled competitor on the support and the failure to bind indicates that the test compound can bind to the oncoprotein of the present invention and thus possibly modulate the oncoprotein. the

Thus, competitive binding methods include differential screening to identify agents capable of modulating the activity of the oncoproteins of the invention. In this embodiment, the method comprises binding the oncoprotein to a competitor within the first sample. The second sample contains the test compound, oncoprotein and competitor. Determining the binding of the competitor in the two samples, a change or difference in binding between the two samples indicates the presence of an agent capable of binding the oncoprotein and potentially modulating its activity. That is, the reagent is capable of binding the oncoprotein if the binding of the competitor in the second sample differs from that in the first sample. the

Alternatively, differential screening is used to identify drug candidates that bind native but not modified oncoproteins. For example, the structure of an oncoprotein is determined and used for rational drug design, leading to the synthesis of reagents that interact with the site as well as proteins that generally do not bind the site modification. In addition, drug candidates that affect the activity of native oncoproteins have also been identified by screening drugs for their ability to enhance or decrease the activity of this protein. the

Positive and negative controls can also be used in the assay. Control samples and test samples should be run at least three times to obtain statistically significant results. All samples were incubated for a time sufficient for the reagents to bind to the protein. Following incubation the samples are washed to remove non-specifically bound material and to determine the amount of bound and usually labeled reagent. For example, when radioactive labels are used, samples can be counted in a scintillation counter to determine the amount of compound bound. the

Other reagents may also be used in screening assays. These reagents include salts, neutral proteins (such as albumin), detergents, etc., and their use will facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Reagents that increase assay efficiency, such as protease inhibitors, ribozyme inhibitors, antimicrobials, and the like, may also be used. The mixture of components is added in an order to provide the desired combination. the

Use of polynucleotides to downregulate or inhibit proteins of the invention

As described in WO 91/04753, polynucleotide modulators of cancer can be introduced into cells containing the target nucleotide sequence by forming a conjugate with a ligand binding molecule. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind cell surface receptors. Binding of the ligand-binding molecule preferably does not substantially affect the ability of the ligand-binding molecule to bind its corresponding molecule or receptor, or prevent the entry of sense or antisense oligonucleotides or bound forms thereof into the cell. Alternatively, polynucleotide modulators of cancer may be introduced into cells containing the target nucleic acid sequence, for example by forming polynucleotide-lipid complexes, as described in WO 90/10448. In addition to therapeutic approaches, antisense molecules or knockout and knockin models can also be used in the screening methods described above. the

inhibitory and antisense nucleotides

In certain embodiments, through the use of antisense polynucleotides or inhibitory small nuclear RNA (snRNA), that is complementary to the mRNA encoding nucleic acid sequence (such as the oncoprotein of the present invention), mRNA or its subsequence and may be preferably Nucleic acids that specifically hybridize to them down-regulate or completely inhibit the activity of cancer-related proteins. Binding of antisense polynucleotides to mRNA reduces mRNA translation and/or stability. the

In the present invention, antisense polynucleotides may include naturally occurring nucleotides or synthetic species formed from naturally occurring subunits or close homologues thereof. Antisense polynucleotides may also contain altered sugar moieties or linkages within sugars. Examples are phosphorothioates and other sulfur-containing species, all of which are known to be useful in the art. Analogs are encompassed by the present invention as long as they efficiently hybridize to the nucleotides of the present invention. See, eg, Isis Pharmaceuticals, Carlsbad, CA; Sequitor, Inc., Natick, MA. the

Such antisense polynucleotides can be conveniently synthesized by recombinant means, or can be synthesized in vitro. Equipment for this synthesis is sold by several vendors, including Applied Biosystems. Methods of preparing other oligonucleotides such as phosphorothioate and alkylated derivatives are also well known to those skilled in the art. the

As used herein, antisense molecules include antisense or sense oligonucleotides. For example, transcription can be blocked by binding a sense oligonucleotide to the antisense strand. Antisense and sense oligonucleotides include single-stranded nucleic acid sequences (RNA or DNA) capable of binding cancer molecule target mRNA (sense) or DNA (antisense) sequences. The antisense or sense oligonucleotides of the present invention include fragments that generally contain at least about 12 nucleotides, preferably about 12-30 nucleotides. The ability to generate antisense or sense oligonucleotides from a cDNA sequence encoding a given protein is described, for example, in Stein & Cohen (Cancer Res. 48:2659 1988) and van der Krol et al. (BioTechniques 6:958 (1988)). the

ribozyme

In addition to antisense polynucleotides, ribozymes can be used to target and inhibit the transcription of cancer-associated nucleotide sequences. Ribozymes are RNA molecules that are capable of catalytically cleaving other RNA molecules. Different classes of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes, ribonuclease P, and axan ribozymes (see e.g. Castanotto et al., Adv. in Pharmacology 25: 289-317 (1994) for an overview of the properties of different ribozymes). the

The general properties of hairpin ribozymes are described, for example, in Hampel et al., Nucl. Acids Res. 18:299-304 (1990); European Patent Publication No. 0360257; US Patent 5,254,678. Its preparation method is well known to those skilled in the art (see for example WO 94/26877; Ojwang et al, Proc. 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)). the

Using Modulators in Phenotypic Screens

In one embodiment, a test compound is administered to a population of cancer cells having a cancer-associated expression signature. "Administering" or "contacting" herein means adding a modulator to a cell on which the modulator acts by uptake and acting intracellularly or acting on the cell surface. In some embodiments, nucleic acids encoding protein-like agents (i.e., peptides) are added to viral constructs, such as adenoviral or retroviral constructs, and added to cells, such that expression of the peptide agents can be achieved, e.g., PCT US97/01019. Tunable gene therapy systems are also available. Once the cell modulating agent is administered, the cells are washed if necessary and incubated for a period of time, preferably under physiological conditions. The cells are then harvested and a new gene expression profile generated. Screen the phenotypic changes of cancer tissue regulated (induced or inhibited) by the reagent. A change in at least one (and preferably a plurality) of gene expression profiles is indicative of an effect of the agent on cancer activity. Similarly, changes in biological function or signaling pathways are also indicative of modulator activity. By defining the characteristics of this cancer phenotype, new drugs that alter this phenotype can be screened. This approach does not require knowledge of the drug target, does not require display on traditional gene/protein expression platforms, and does not require alteration of the transcript level of the target protein. The inhibitory function of the modulator will be used as a surrogate marker. the

Genes or gene products are screened as described above. That is, after identifying a specific differentially expressed gene important for a particular state, a screen is performed for modulators of expression of that gene or the gene product itself. the

Use of modulators to affect the peptides of the invention

Various assays are used to measure the activity of cancer polypeptides or the cancer phenotype. The effect of a modulator on a cancer polypeptide is measured, for example, by detecting the above parameters. Physiological changes affecting activity are used to assess the effect of test compounds on polypeptides of the invention. Effects such as tumor growth, tumor metastasis, neovascularization, hormone release, known and unknown genetic Marked transcriptional changes (eg by Northern blot), changes in cellular metabolism such as changes in cell growth or pH, and changes in intracellular second messengers such as cGNIP. the

Method for identifying qualitative cancer-associated sequences

Expression of various gene sequences is associated with cancer. Diseases based on mutant or variant cancer genes can thus be determined. In one embodiment, the invention provides a method of identifying a cell containing a variant cancer gene, eg, determining the presence (in whole or in part) of the sequence of at least one endogenous cancer gene in the cell. This can be achieved by a variety of sequencing technologies. The present invention includes methods for identifying the cancer genotype of an individual, eg, determining the full or partial sequence of at least one gene of the invention in the individual. This is typically done in at least one individual tissue, such as those listed in Table I, and may involve evaluating many tissues or different samples of the same tissue. The method may include comparing the sequence of the sequenced gene to known cancer genes (ie, wild-type genes) to determine the presence of family members, homologs, mutants or variants. The sequence of all or part of the gene can then be compared to that of known cancer genes to determine if there are any differences. This can be performed by various known homology programs, such as BLAST, Bestfit, and the like. As described herein, a discrepancy between a patient's cancer gene sequence and known cancer gene sequences is associated with having or being likely to have a disease. the

In a preferred embodiment, cancer genes are used as probes to determine the copy number of cancer genes within the genome. Cancer genes were used as probes to determine the chromosomal location of cancer genes. Information such as chromosomal location can be used for diagnosis or prognosis, especially when chromosomal abnormalities such as translocations are identified within cancer gene loci. the

XIV.) RNAi and Therapy Using Small Interfering RNA (siRNA)

The present invention also relates to siRNA oligonucleotides, specifically double-stranded RNA comprising at least a fragment of the PSCA coding region or 5" UTR region, or any complementary strand or antisense oligonucleotides specific to the PSCA sequence. In one embodiment, such oligonucleotides are used to elucidate the function of PSCA, or to screen or evaluate modulators of PSCA function or expression. In another embodiment, the gene expression of PSCA is reduced by using siRNA transfection , and causes a significant reduction in the proliferative capacity of transformed cancer cells endogenously expressing this antigen; cells treated with specific PSCAsiRNA show reduced viability as measured by, for example, metabolic readouts of cell viability associated with reduced proliferative capacity. Thus , PSCA siRNA composition comprises siRNA (double-stranded RNA), and it corresponds to the nucleic acid ORF sequence of PSCA albumen or its subsequence; The length of these subsequences is usually 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, and contain complementary and non-complementary sequences with at least a part of the mRNA coding sequence.In a preferred embodiment, the length of the subsequence is 19-25 nucleotides, most preferably 21-23 nucleotides in length nucleotides.

RNA interference is a novel approach to gene silencing in vitro and in vivo, making small double-stranded RNAs (siRNAs) valuable therapeutic agents. The ability of siRNAs to silence specific gene activity has now been used in animal models of disease and has also been used in humans. For example, administration of siRNA solutions containing siRNA against a specific target by fluid infusion to mice has been shown to be therapeutically effective. the

Pioneering studies by Song et al. showed that a class of completely natural nucleic acids, small interfering RNA (siRNA), can be used as therapeutic agents even without further chemical modification (Song, E., et al., “RNAinterference targeting Fas protects mice from fulminant hepatitis" Nat. Med. 9(3): 347-51(2003)). The study provides the first in vivo evidence that infusion of siRNA into animals reduces disease. In this case, the authors injected mice with siRNA designed to silence the FAS protein, a cell death receptor that induces liver and other cell death when overactivated in response to inflammation. The next day, Fas-specific antibodies were administered to the animals. Control mice died of acute liver failure within days, whereas more than 80% of mice treated with siRNA remained free from severe disease and survived. About 80%-90% of their hepatocytes incorporated naked siRNA oligonucleotides. Furthermore, the RNA molecule continued to function for up to 10 days before losing potency after 3 weeks. the

When used in human therapy, siRNA is delivered through an efficient system that induces long-lasting RNAi activity. The main caveat in clinical application is the delivery of siRNA to the appropriate cells. Hepatocytes appear to be particularly receptive to exogenous RNA. Targets located in the liver are currently attractive due to the ease with which this organ is targeted by nucleic acid molecules and viral vectors. However, other organ and tissue targets are also preferred. the

siRNA formulations containing compounds that facilitate transport across cell membranes are used to improve siRNA delivery in therapy. Another embodiment is a chemically modified synthetic siRNA that is resistant to nucleases, has serum stability, and thereby allows for an increased duration of RNAi effect. the

Thus, siRNA technology is a therapy for the treatment of human malignancies by delivering siRNA molecules against PSCA to individuals with cancer, such as those listed in Table 1. Such administration of siRNA reduces the growth of PSCA-expressing cancer cells, provides an anti-tumor therapy, and reduces morbidity and/or mortality associated with malignancy. the

The effect of this mode of gene product knockdown is quite dramatic when assayed in vitro or in vivo. When in vitro methods are used to detect reductions in PSCA protein expression, the in vitro effect of the siRNA can be readily demonstrated by administering the siRNA to cultured cells (as previously described) or equivalent amounts of cancer patients or tissues. the

XV.) Manufacture of kits/products

The invention includes kits for the laboratory, prognostic, prophylactic, diagnostic and therapeutic uses described herein. Such a kit may comprise a carrier, pack or container divided to hold one or more contents such as vials, test tubes, etc., each container containing a separate element for use in the method alone, and instructions for use. For example, a container can contain a probe that is or can be detectably labeled. Such probes may be antibodies or polynucleotides specific for a protein or gene or message of the invention, respectively. When the method utilizes nucleic acid hybridization to detect the target nucleic acid, the container of the kit may also contain nucleotides for amplifying the target nucleic acid sequence. The container of the kit contains a receptor, such as a biotin-binding protein, such as avidin or streptavidin, which is bound to a reporter molecule such as an enzyme label, a fluorescent label, or a radioisotope label; this reporter molecule can be combined with, for example, nucleic acids or antibodies. The kit may contain all or part of the amino acid sequence in Figure 1 or an analog thereof, or a nucleic acid molecule encoding such an amino acid sequence. the

The kit of the present invention generally comprises the above-mentioned container and one or more other containers related thereto, containing materials required for commercial and user standpoints in these containers, including buffers, diluents, fillers, needles, syringes; carriers, Packaging, labels on vials and/or tubes identifying the ingredients and/or instructions for use, and instructions for use. the

A label may appear on or with the container to direct how the composition is to be used for a particular therapeutic or non-therapeutic use, such as prognostic, prophylactic, diagnostic or laboratory use, and may direct both in vivo and in vitro use, such as those described here. Instructions or other information may also appear on the insert or label provided with or on the kit. A label may appear on or be provided with the container. A label may appear on a container when the letters, numbers or other features making up the label are molded or embedded into the container itself; a label may be provided with the container when the label appears in the box or carrier in which the container is contained. The label may indicate how the composition is to be used to diagnose, treat, prevent, or predict symptoms, such as tumor formation of the tissues listed in Table I. the

The terms "kit" and "article of manufacture" are used interchangeably. the

In another embodiment of the invention, an article of manufacture comprises compositions, such as amino acid sequences, small molecules, nucleic acid sequences and/or antibodies, for example for diagnosis, prognosis, prevention and/or treatment of tissues (as listed in Table I out of those tissues) tumor-forming articles. An article of manufacture typically includes at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes and test tubes. The container can be made of various materials such as glass, metal or plastic. The container may contain amino acid sequences, small molecules, nucleic acid sequences, cell populations and/or antibodies. In one embodiment, the container contains polynucleotides for detecting the mRNA expression profile of a cell and reagents for this purpose. In another embodiment, the container contains antibodies, binding fragments thereof, or specific binding proteins to evaluate the protein expression of PSCA in cells and tissues, or for related laboratory, prognostic, diagnostic, prophylactic and therapeutic purposes; such containers Instructions and/or instructions for such use also appear on or therewith, and may contain reagents and other compositions or means for these purposes. In another embodiment, the container contains substances for eliciting a cellular or humoral immune response, along with instructions and/or instructions. In another embodiment, the container contains substances for adoptive immunotherapy, such as cytotoxic T cells (CTL) or helper T cells (HTL), as well as relevant instructions and/or instructions; Reagents and other compositions or means. the

The container may also contain a composition effective for treating, diagnosing, prognosing, or preventing a condition and may include a sterile outlet (e.g., the container may be an intravenous solution pack or vial with a hypodermic needle-piercable plug). The active agent in the composition may be an antibody capable of specifically binding PSCA and modulating PSCA function. the

The article of manufacture may also contain a second container containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and/or dextrose solution. It may also contain other materials desirable from a commercial and user standpoint, including other buffers, diluents, fillers, stirrers, needles, syringes and/or instructions for use and/or directions for use. the

Example

Various aspects of the present invention are further described and illustrated through the following several examples, and these examples are not meant to limit the scope of the present invention. the

Example 1

Expression analysis of PSCA variants in normal tissues and patient samples

PSCA is referred to as PSCA v.1 in this paper, and previous studies have confirmed that PSCA is expressed as an antigen in prostate cancer. PSCA is expressed in more than 80% of primary prostate cancers and in most prostate metastases. PSCA is also expressed in gallbladder, ovary, and pancreatic cancers; tumors of this type are listed in Table I. By immunohistochemical analysis, it was found that PSCA was overexpressed on the cell surface of most transitional cell carcinomas of the urethra and 60% of primary pancreatic adenocarcinomas. In numerous patent documents (PCT/US98/04664, PCT/US/28883, PCT/US00/19967) and peer-reviewed articles (Saffran et al., Proc Natl Acad Sci U S A. 2001-2-27; 98(5): 2658-2663; Amara et al., Cancer Res. 2001-6-15;61(12):4660-65; Reiter et al., Proc Natl Acad Sci USA.1998-2-17; etc., Cancer Res. 2001-6-1; 61 (11): 4320-24) have all reported data on PSCA expression. the

The present invention studies the specific expression of different PSCA variants in normal tissues and cancer patient samples. The designed primers can distinguish the difference between PSCA v.1/v.2/v.4, PSCA v.3 and PSCA v.5, and the length of the PCR product of PSCA v.1/v.2/v.4 is 425bp, the length of the PCR product of PSCA v.3 is 300bp, and the length of the PCR product of PSCA v.5 is 910bp (Figure 1I(a)). the

From normal gallbladder, brain, heart, kidney, liver, lung, prostate, spleen, skeletal muscle, testis, pancreas, colon, stomach tissue and a group of prostate cancer, gallbladder cancer, kidney cancer, colon cancer, lung cancer, ovarian cancer, breast cancer First-strand cDNA was prepared from cancer, metastatic cancer and pancreatic cancer tissues (Fig. 1I(b)). The product obtained by PCR amplification with actin primers was used as a control. Semi-quantitative PCR was performed using primers specific for the different variants, and amplification was performed for 30 cycles. the

The results showed that PSCA v.5 was mainly expressed in breast cancer, metastatic cancer and pancreatic cancer, and also expressed at low levels in colon cancer and lung cancer. PCR products of PSCA v.1/v.2/v.4 were detected in prostate, gallbladder, kidney, colon, lung, ovary, breast, metastatic and pancreatic cancers. In normal tissues, the PCR products of PSCA v.1/v.2/v.4 were only detected in prostate and stomach tissues, and low-level expression of PSCA v.1/v. 2/v.4, while PSCA v.5 was not detected in all normal tissues. No PCR product of PSCA v.3 was detected in any samples examined. the

Primers were designed to distinguish between PSCA v.4 and PSCA v.5 (Fig. 1J(a)). The PCR product length of PSCA v.4 is 460bp, while the PCR product length of PSCA v.5 is 945bp. the

From normal gallbladder, brain, heart, kidney, liver, lung, prostate, spleen, skeletal muscle, testis, pancreas, colon, stomach tissue and a panel of prostate cancer, gallbladder cancer and various xenograft mixtures (prostate cancer, kidney cancer and gallbladder cancer xenografts) to prepare first-strand cDNA (Fig. 1J(b)). The product obtained by PCR amplification with actin primers was used as a control. Semi-quantitative PCR was performed using primers specific for the different variants, and amplification was performed for 30 cycles. the

The results showed expression of PSCA v.4 in prostate cancer, gallbladder cancer, multiple xenograft mixtures, normal kidney and prostate tissues. The expression of PSCAv.5 was only detected in normal prostate tissue and gallbladder carcinoma tissue. the

The expression of PSCA variants in normal tissues is limited, but the expression of PSCA variants can be detected in the specimens of cancer patients, which shows that PSCA variants can be used as therapeutic, prognostic, experimental, and preventive measures for human tumors. sexual and diagnostic targets. the

Example 2

splice variant of PSCA

The term "variant" as used herein includes transcript variants and single nucleotide polymorphisms (SNPs). Transcript variants are variants of the mature mRNA of the same gene that result from alternative transcription or alternative splicing. Alternative transcripts are transcripts from the same gene but with different transcription initiation sites. Splice variants refer to mRNA variants formed by different splicing of the same transcript. In eukaryotes, when a multi-exon gene is transcribed from genomic DNA, the resulting RNA precursor needs to be spliced to form a functional mRNA, which has only one exon for translation into amino acids sequence. Correspondingly, a given gene may have zero to many different alternative transcripts, and each transcript may have zero to many splice variants. Each transcript variant is composed of its unique exons, containing different coding and/or non-coding (5' or 3' end) parts of the precursor source of the transcript. Transcript variants can encode the same, similar or different proteins, which can have the same or similar or different functions. Mutant proteins may be expressed at the same time in the same tissue, at the same time in different tissues, at different times in the same tissue, or at different times in different tissues . Transcript variants encode proteins that may have similar or different subcellular or extracellular localizations (eg, secreted versus intracellular). the

Many methods in the art can be used to identify transcript variants. For example, alternative transcripts and splice variants can be identified by full-length cloning or using full-length transcripts and EST sequences. First, all human-derived ESTs are divided into different clusters, which share direct or indirect identity with each other. Second, ESTs within the same cluster were divided into different subclusters and combined into a consensus sequence. The original sequence of the gene is compared to the consensus sequence or other full-length sequences. Each consensus sequence is a potential splice variant of the gene. Several methods of confirmation are well known in the art, such as identification of variants by Northern blotting, full-length cloning, or using probe libraries, among others. Even if the identified variant is not a full-length clone, this variant fragment can be used as a research tool, for example for antigen preparation by techniques well known in the art, or for further cloning of full-length splice variants. the

Additionally, several computer programs exist in the art that can identify transcript variants based on genomic sequences. Genome sequence-based transcript variant identification programs include FgenesH (A.Salamov and V.Solovyev, "Ab initio gene finding in Drosophila genomic DNA," GenomeResearch.2000-4; 10(4):516-22); Grail( URLcompbio.ornl.gov/Grail-bin/EmptyGrailForm) and GenScan (URLgenes.mit.edu/GENSCAN.html). For a discussion of splice variant identification tools see Southan, C., A genomic perspective on human proteases, FEBS Lett. 2001-6-8;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. 2000-11-7; 97(23): 12690-3. the

A variety of techniques are available in the art to further determine the parameters of transcript variants, such as full-length cloning, proteomic validation, PCR-based validation, and 5' RACE validation, etc. (see proteomic validation: Brennan, S.O. et al., Albumin banks peninsula: a new termination variant characterized by electrospray mass spectrometry, Biochem Biophys Acta.1999-8-17; 1433(1-2): 321-6; Ferranti P et al., Differential splicing of pre-messenger RNA produces multiple pHa forms of nepriple (s1)-casein, Eur J Biochem.1997-10-1;249(1):1-7. PCR-based verification: Wellmann S et al., Specific reverse transcription-PCR quantification of vascular endothelial growth factor (VEGF) splice variants by LightCycler technology, Clin Chem.2001-4; 47(4):654-60; Jia, H.P. et al., Discovery of new human beta-defensins using a genomics-based approach, Gene.2001-1-24; 263(1 -2): 211-8. PCR-based validation 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.1997-8-7; 1353 (2):191-8). the

As is well known in the art, there are variations in genomic regions of tumors. When the genetic mapping of a particular tumor's genomic region changes, there are corresponding changes in alternative transcripts or splice variants of that gene. Described here is a specific expression pattern of tumor-associated PSCA (see Table 1). Alternative transcripts and splice variants of PSCA are also contained within tumors, as in one or more of these tissues, as well as in certain other tissue-derived tumors. These variants can therefore also be considered as tumor-associated markers/antigens. the

Four additional transcript variants were identified using the full-length PSCA gene and EST sequences, designated PSCA v.2, v.3, v.4, and v.5, respectively. The exon boundaries of the original transcript PSCA v.1 are shown in Table VI. The sequences of PSCA and PSCA variants are shown in FIG. 1 . the

Example 3

Single Nucleotide Polymorphisms of PSCA

A single nucleotide polymorphism (SNP) refers to a single base pair change at a specific position in a nucleotide sequence. At any given location in the genome, there are four possible nucleotide base pairs: A/T, C/G, G/C, and T/A. As described herein, an allele refers to one of a series of different forms of a given gene that differ in their DNA sequence and can affect their products (RNA and/or protein). the

SNPs that occur on cDNA are called cSNPs. This cSNP can lead to changes in the amino acid sequence of the protein encoded by the gene, so the function of the protein may also change. Certain SNPs can cause genetic diseases; others are associated with quantitative changes in phenotypes and responses of different individuals to environmental factors such as food and drugs. Therefore, the presence of SNPs and/or combinations of alleles (known as haplotypes) has many uses, such as diagnosis of genetic diseases, determination of drug response and dosage, identification of disease-associated genes, and identification of genetic relatedness between individuals. Analysis (P. Nowotny, J.M. Kwon and A.M. Goate, "SNPanalysis to dissect human traits," Curr. Opin. Neurobiol. 2001-10; 11(5): 637-641; M.Pirmohamed and B.K.Park, "Genetic susceptibility to adverse drug reactions," Trends Pharmacol. Sci. 2001-6; 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.2000-2;1(1):39-47; R. Judson, J.C.Stephens and A.Windemuth, "The predictive power of haplotypes in clinical response," Pharmacogenomics.2000-2;1(1):15 -26). the

SNPs can be identified by various methods available in the art (P. Bean, "The promising voyage of SNP target discovery," Am. Clin. Lab. 2001 October-November; 20(9): 18-20; K.M.Weiss , "In search of human variation," Genome Res.1998-7; 8(7):691-697; M.M.She, "Enabling large-scale pharmacogeneticstudies by high-throughput mutation detection and genotyping technologies," Clin.Chem.2001- 2;47(2):164-172). For example, polymorphic DNA fragments detected by gel methods such as restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE) can be sequenced to identify their SNPs. Alternatively, SNPs can be revealed by directly sequencing DNA samples from different individuals or by comparing the sequences of different DNA samples. Using the rapidly accumulating sequence data in public and private databases, one can also use computer programs to discover SNPs through sequence comparison (Z. Gu, L. Hillier, and P. Y. Kwok, “Single nucleotide polymorphism hunting in cyberspace,” Hum. Mutat. 1998;12(4):221-225). Many methods are available for validating SNPs and determining genotypes and haplotypes in different individuals, including direct sequencing and high-throughput microarray methods (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. 2000-12;5(4):329-340). the

Thirteen SNPs were identified in the PSCA v.2 transcript using the method described above. We used variant 2 instead of variant 1 because variant 2 has fewer ambiguous bases than variant 1. Correspondingly, the SNPs of PSCA v.2 were identified at loci 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) on. Transcripts or proteins of alternative alleles were designated as variants PSCAv.6 to v.18, as shown in Figure 1B and Figure 1G. the

The nucleotide change on v.6 changes the start codon of v.1 so that its translation does not start until it encounters the next ATG (AUG on the mRNA), resulting in a protein that is 9 amino acids shorter than the v.1 protein . Nucleotide changes on v.7 and v.8 are reflected as silent at the protein level. the

Twelve of these 13 SNPs were also present on variant 4. The 12 SNPs associated with PSCA v.4 were named PSCA v.19 to v.30, respectively. Variants 19 to 27 encode different amino acids as shown in Figure 1H. the

Example 4

Production of recombinant PSCA in a prokaryotic system

To express recombinant PSCA and PSCA variants in prokaryotic cells, the full-length PSCA and PSCA variant cDNA sequences or fragments thereof are cloned into any expression vector well known in the art. Expression of one or more of the following fragments of PSCA variants: the full-length sequence shown in Figure 1, or any of 8, 9, 10, 11, 12, 13, 14, 15, A fragment of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids. the

A. In vitro transcription and translation constructs:

pCRII : To generate PSCA sense and antisense RNA probes for in situ detection of RNA, pCRII constructs (Invitrogen, Carlsbad CA) encoding full-length PSCA cDNA or fragments thereof were prepared. The pCRII vector contains Sp6 and T7 promoters, which are respectively located on both sides of the insert fragment. These two promoters drive the transcription of PSCARNA, and the transcribed RNA can be used as a probe for RNA in situ hybridization experiments. These probes can be used to analyze the expression of PSCA in cells and tissues at the RNA level. PSCA RNA transcripts representing the amino acid coding region of the cDNA of the PSCA gene can be used in an in vitro translation system such as the TnT™ Coupled Reticulolysate System (Promega, Corp., Madison, WI) to synthesize PSCA protein.

B. Bacterial Constructs:

pGEX construct : To prepare recombinant PSCA protein fused with glutathione S-transferase (GST) protein in bacteria, the full-length or partial PSCA cDNA protein coding sequence was cloned into the pGEX plasmid family of GST fusion vector (Amersham Pharmacia Biotech , Piscataway, NJ). These constructs can controllably express the recombinant PSCA protein with GST fused at the N-terminus and 6×His epitope (6×His) at the C-terminus. The presence of GST and 6×His tags is mainly for the purification of recombinant fusion proteins from induced bacteria with appropriate affinity matrices, which are recognized by anti-GST and anti-His antibodies. A 6×His tag can be added to the protein by adding 6 histidine codons to the 3′ end of the open reading frame (ORF) cloning primer. By introducing a protease cleavage site, such as the PreScission™ recognition site on pGEX-6P-1, the GST tag can be cleaved from the PSCA-related protein. Ampicillin resistance gene and pBR322 origin of replication are used for selection and replication of pGEX plasmid in Escherichia coli.

pMAL construct : In order to produce recombinant PSCA protein fused with mannan-binding protein (MBP) in bacteria, the full-length or partial PSCA cDNA protein coding sequence was cloned into pMAL-c2X and pMAL-p2X vectors (New England Biolabs, Beverly , MA) to make it fused with the MBP gene. These constructs can controllably express the recombinant PSCA protein fused with MBP at the N-terminus and tagged with 6×histidine epitope at the C-terminus. The MBP and 6×His tags are present mainly for the purification of recombinant fusion proteins from induced bacteria with appropriate affinity matrices, which are recognized by anti-MBP and anti-His antibodies. A 6×His tag can be added to the protein by adding 6 histidine codons to the 3'-end cloning primer. The Factor Xa recognition site was added to cleave the pMAL tag from PSCA. The pMAL-c2X and pMAL-p2X vectors are used for cytoplasmic and periplasmic expression of recombinant proteins, respectively. Periplasmic expression facilitates protein folding using disulfide bonds.

pET constructs : For expression of PSCA in bacterial cells, the full-length or partial PSCA cDNA protein coding sequence was cloned into the pET family of vectors (Novagen, Madison, WI). These vectors can controllably express recombinant PSCA proteins in bacteria that are fused with proteins that increase solubility and epitope tags or without fusion proteins. Proteins that increase solubility include NusA and thioredoxin (Trx), and epitope tags include 6 ×His and S-Tag ^TM , these tags can be used for the purification and detection of recombinant proteins. For example, using the pET NusA fusion system 43.1 to construct an expression vector can make the expressed PSCA protein fused with NusA at the amino terminus.

C. Yeast constructs:

pESC constructs : In order to express PSCA in Saccharomyces cerevisiae to prepare recombinant proteins and conduct functional studies, the full-length or partial PSCA cDNA protein coding sequence is cloned into pESC family vectors, which contain the following four selection markers One: HIS3, TRP1, LEU2 and URA3 (Stratagene, La Jolla, CA). These vectors allow us to controllably express different proteins in the same yeast cell from the same plasmid with 2 different genes or cloned sequences containing FlagTM and Myc epitope tags respectively. This system can be used to determine protein-protein interactions of PSCA. In addition, expression in yeast can produce similar post-translational modifications as eukaryotic expression, such as glycosylation and phosphorylation.

pESP Construct : For expression of PSCA in Saccharomyces pombe, the full-length or partial PSCA cDNA protein coding sequence was cloned into the pESP family of vectors. These vectors can controllably and high-level express the PSCA protein sequence fused with GST at the amino-terminus or carboxy-terminus, and GST is helpful for the purification of recombinant proteins. The presence of the FlagTM epitope allowed us to detect the recombinant protein with an anti-FlagTM antibody.

Example 5

Production of recombinant PSCA in higher eukaryotes

A. Mammalian constructs:

To express recombinant PSCA in eukaryotic cells, the full-length or partial-length PSCA cDNA sequence 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 1-123 in PSCA v.1, PSCA variants or their analogs or any 8, 9, 10, 11, 12, 13 thereof , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids. the

The construct can be transfected into any of a variety of mammalian cells such as 293T cells. Lysates of transfected 293T cells can be probed with anti-PSCA polyclonal sera as described herein. the

pcDNA4/HisMax Construct : For expression of PSCA in mammalian cells, the PSCA ORF of PSCA, or a portion thereof, was cloned into pcDNA4/HisMax Version A (Invitrogen, Carlsbad, CA). Protein expression was driven by the cytomegalovirus (CMV) promoter and SP16 transcriptional enhancer. The recombinant protein has 6 histidine (6×His) epitopes and XPressTM fused to the amino terminus. The pcDNA4/HisMax vector also contains a bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to increase mRNA stability, as well as an SV40 origin for episomal replication and in cell lines expressing the large T antigen. A simple vehicle to get rescued. Mammalian cells expressing the protein were selected by virtue of the Zeocin resistance gene, while plasmids in E. coli were selected and retained by the ampicillin resistance gene and the ColE1 origin.

pcDNA3.1/MycHis Construct : For expression of PSCA in mammalian cells, the PSCA ORF of PSCA, or a portion thereof, was cloned into pcDNA3.1/MycHis Version A (Invitrogen, Carlsbad, CA) together with a consensus Kozak translation initiation site. Protein expression was driven by a cytomegalovirus (CMV) promoter. The recombinant protein has 6 histidine (6×His) epitopes and myc epitopes fused to the carboxy terminus. The pcDNA3.1/MycHis vector also contains a bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to increase mRNA stability, as well as an SV40 origin for episomal replication and in cell lines expressing the large T antigen A single vector that can be rescued in . Mammalian cells expressing the protein can be selected using the neomycin resistance gene, while plasmids in E. coli are selected and maintained by virtue of the ampicillin resistance gene and the ColE1 origin.

pcDNA3.1/CT-GFP-TOPO construct : To express PSCA in mammalian cells and allow fluorescence to detect the recombinant protein, the PSCA ORF or part thereof was cloned into pcDNA3.1/ CT-GFP-TOPO (Invitrogen, CA). Protein expression was driven by a cytomegalovirus (CMV) promoter. The recombinant protein has green fluorescent protein (GFP) fused to the carboxy terminus, which makes non-invasive, in vivo detection and cell biology research easier. The pcDNA3.1CT-GFP-TOPO vector also contains bovine growth hormone (BGH) polyadenylation signal and transcription termination sequence to improve the stability of mRNA, as well as the SV40 origin for episomal replication and expression of large T antigen. A single vector for rescue in cell lines. Mammalian cells expressing the protein were selected by virtue of the neomycin resistance gene, while plasmids in E. coli were selected and maintained by the ampicillin resistance gene and the ColE1 origin. Additional constructs with a GFP fusion at the amino terminus were made in pcDNA3.1/NT-GFP-TOPO to span the full length of the PSCA protein.

PAPtag: The PSCA ORF or a portion thereof was cloned into pAPtag-5 (GenHunter Corp. Nashville, TN). This construct produces an alkaline phosphatase fusion at the carboxy-terminus of the PSCA protein and an IgGK signal sequence at the amino-terminus. A construct in which the amino terminus of alkaline phosphatase and IgGκ signal sequence was fused to the amino terminus of PSCA protein was also constructed. The resulting recombinant PSCA protein is optimized for secretion into the medium of transfected mammalian cells and can be used to identify proteins (eg, ligands or receptors) that interact with the PSCA protein. The expression of the protein is driven by a CMV promoter, and the recombinant protein also contains a 6×His epitope and myc fused at the carboxy terminus to facilitate detection and purification. Mammalian cells expressing the protein were selected for the Zewortsin resistance gene in the vector, while the plasmid in E. coli was selected for the ampicillin resistance gene. the

ptag5 : clone the PSCA ORF or part thereof into pTag-5. This vector is similar to pAPtag, but without the alkaline phosphatase fusion. The PSCA protein produced by this construct has the IgGκ signal sequence at the amino terminal and the 6×His epitope tag and myc at the carboxy terminal, which makes detection and affinity purification easier. The resulting recombinant PSCA protein is optimized for secretion into the medium of transfected mammalian cells and used as an immunogen or ligand to identify proteins (eg, ligands or receptors) that interact with the PSCA protein. Expression of the protein was driven by the CMV promoter. Mammalian cells expressing the protein were selected for the Zewortsin resistance gene in the vector, while the plasmid in E. coli was selected for the ampicillin resistance gene.

PsecFc : The PSCA ORF or part thereof was cloned into psecFc. The psecFc vector was assembled by cloning human immunoglobulin G1 (IgG) Fc (hinge region, CH ₂ region, CH ₃ region) into pSecTag2 (Invitrogen, California). This construct produces an IgG1 Fc fusion at the carboxy-terminus of the PSCA protein and an IgGK signal sequence at the N-terminus. A PSCA fusion employing the mouse IgG1 Fc region was also used. The resulting recombinant PSCA protein is optimized for secretion into the medium of transfected mammalian cells and can be used as an immunogen or to identify proteins (eg, ligands or receptors) that interact with the PSCA protein. Expression of the protein was driven by the CMV promoter. Mammalian cells expressing the protein were selected for the hygromycin resistance gene in the vector, while the plasmid in E. coli was selected for the ampicillin resistance gene.

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

pSRα Construct : To generate mammalian cell lines that constitutively express PSCA, the PSCAORF of PSCA, or a portion thereof, was cloned into the pSRα construct. Amphitropic and homophilic retroviruses were generated by transfection of the pSRα construct into the 293T-10A1 packaging line or by co-transfection of pSRα and a helper plasmid (containing the deleted packaging sequence) into 293 cells, respectively. Infection of various mammalian cell lines with this retrovirus resulted in the integration of the cloned gene PSCA into the host cell line. Expression of proteins is driven by long terminal gene repeats (LTRs). Mammalian cells expressing the protein were selected by virtue of the neomycin resistance gene in the vector, whereas the plasmid in E. coli was initially selected and maintained by virtue of the ampicillin resistance gene and ColE1. This retroviral vector can then be used to infect and generate various cell lines using, for example, PC3, NIH 3T3, TsuPrl, 293 or rat-1 cells.

Figure 6 shows PSCA expression in recombinant mouse, rat and human cell lines using the PSCA.pSRα construct. Infection of indicated mouse, rat and human cell lines with retroviral vectors carrying human PSCA cDNA and the neomycin resistance gene or only the neomycin resistance gene. A stable recombinant cell line was obtained by G418 drug screening. PSCA expression was determined by FACS stained with 1G8 anti-PSCA MAb (5 μg/ml). Shown are FACS profiles of the respective cell lines, showing that the fluorescence shift occurs only in PSCA-transfected cell lines, indicative of PSCA expression on the cell surface. These cell lines can be used as immunogens in MAb development, MAb screening reagents, and for functional assays. the

Additional pSRα constructs were prepared by fusing an epitope tag (eg, FLAG ^™ tag) to the carboxy-terminus of the PSCA sequence for detection using an anti-Flag antibody. For example, the FLAG ^™ sequence 5'gat tac aag gat gac gac gat aag 3' (SEQ ID NO: 76) was added to the cloning primer at the 3' end of the ORF. Additional pSRα constructs were made to generate amino- and carboxy-terminal GFP and myc/6X His fusion proteins of the full-length PSCA protein.

Other Viral Vectors : Other constructs were prepared for virus-mediated delivery and expression of PSCA. High viral titers leading to high-level expression of PSCA were obtained in viral delivery systems such as adenovirus and herpes amplicon vectors. The PSCA coding sequence or a fragment thereof was amplified by PCR and subcloned into the AdEasy shuttle vector (Stratagene). Recombination and viral packaging were performed according to manufacturer's instructions to generate adenoviral vectors. Alternatively, the PSCA coding sequence or fragments thereof were cloned into HSV-1 vectors (Imgenex) to generate herpesvirus vectors. This viral vector is then used to infect various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells.

Regulated expression system: To control the expression of PSCA in mammalian cells, clone the PSCA coding sequence or part thereof into a mammalian regulated expression system, such as the T-Rex system (Invitrogen), the GeneSwitch system (Invitrogen), and the tightly regulated ecdysone System (tightly-regulated Ecdysone System, Stratagene). These systems were used to study the time- and concentration-dependent effects of recombinant PSCA. These vectors were then used to control expression of PSCA in various cell lines such as PC3, NIH 3T3, 293 or rat-1 cells. the

B. Baculovirus expression system

To produce recombinant PSCA protein in the baculovirus expression system, the PSCA ORF or part thereof was cloned into the baculovirus transformation vector pBlueBac 4.5 (Invitrogen), which provides a His-tag at the N-terminus. Specifically, pBlueBac-PSCA and helper plasmid pBac-N-Blue (Invitrogen) were co-transfected into SF9 (Spodoptera frugiperda) insecT cells to produce recombinant baculovirus (see Invitrogen instructions for details). Baculoviruses were then harvested from the cell supernatant and purified by plaque analysis. the

Then, HighFive insecT cells (Invitrogen) were infected with purified baculovirus to produce recombinant PSCA protein. Anti-PSCA or anti-His-tag antibodies can be used to detect recombinant PSCA protein. PSCA protein can be purified and used in various cell-based assays or as an immunogen to generate PSCA-specific polyclonal and monoclonal antibodies. the

C. Expression vectors for PSCA orthologs

The mouse and monkey orthologs of PSCA were cloned into pcDNA3.1/MycHis VersionA (Invitrogen, Carlsbad, CA). Protein expression was driven by a cytomegalovirus (CMV) promoter. The recombinant protein has a 6X His epitope and a myc epitope fused at the carboxyl terminus. These vectors allow the expression of PSCA orthologs to test the cross-reactivity of anti-human PSCA monoclonal antibodies. the

The mouse and monkey orthologs of PSCA were also cloned into the pSRα construct. This pSRα construct generates a mammalian cell line that constitutively expresses the PSCA ortholog. Protein expression was driven by a cytomegalovirus (CMV) promoter. The recombinant protein has a 6X His epitope and a myc epitope fused at the carboxyl terminus. These vectors enable the expression of PSCA orthologs for testing the cross-reactivity of anti-human PSCA monoclonal antibodies and for studying the functional activity of PSCA orthologs. Amphitropic and homophilic retroviruses were generated by transfection of the pSRα construct into the 293T-10A1 packaging line or co-transfection of pSRα and a helper plasmid (containing the deleted packaging sequence) into 293 cells, respectively. Infection of various mammalian cell lines with the retrovirus allows integration of the cloned gene, the PSCA ortholog, into the host cell line. the

Figure 7 shows the expression of mouse and monkey PSCA.pcDNA3.1/MycHis after transfection into 293T cells. 293T cells were transfected with mouse PSCA.pcDNA3.1/MycHis or simian PSCA.pcDNA3.1/MycHis or pcDNA3.1/MycHis vector control. After 40 hours, cells were harvested and analyzed by flow cytometry using an anti-PSCA monoclonal antibody. the

Example 6

Antigenicity Map and Secondary Structure

Amino acid maps of PSCA variants 1, 3 and 4 can be found by accessing the ProtScale website on the ExPasy molecular biology server on the World Wide Web (expasy.ch/cgi-bin/protscale.pl). the

These maps: hydrophilicity (hydrophilicity, Hopp T.P., Woods K.R., 1981.Proc.Natl.Acad.Sci.U.S.A.78:3824-3828); hydropathicity (hydropathicity, Kyte J., Doolittle R.F., 1982.J.Mol .Biol.157:105-132); Percentage of accessible residues (Janin J., 1979Nature 277:491-492: Average Flexibility (Bhaskaran R. and PonnuswamyP.K., 1988.Int.J.Pept.Protein Res .32:242-255); β-turn (Deleage, G., Roux B. 1987 Protein Engineering 1:289-294); and other maps well known in the art, as listed on the ProtScale website, can be used to identify each Antigenic regions on each PSCA variant protein. Each of the above-mentioned amino acid profiles of PSCA variants can be described by the following ProtScale parameter: 1) the window size is 9; 2) compared with the window germline, the window edge accounts for 100% weight; And 3) the amino acid map value is between 0 and 1 after normalization. the

Hydrophilicity, Hydrophilicity, and Percentage of Accessible Residues were used to determine the distribution area of hydrophilic amino acids (ie, amino acids with a percentage of Hydrophilic and Accessible Amino Acids greater than 0.5 and a Hydrophilicity less than 0.5). Such regions, which may be exposed to aqueous environments, are located on the surface of proteins and thus serve as immune recognition sites, such as those of antibodies. the

The average buckling and β-turn determine the distribution area of amino acids not contained in secondary structures such as β-sheets and α-helices (ie amino acids with β-turn and average buckling greater than 0.5). Such regions may also be exposed on the surface of the protein and thus also be used for immune recognition, such as that of antibodies. the

The antigenic sequences of the PSCA variant proteins indicated in the above map can be used to prepare immunogens (whether peptides or nucleic acids encoding them) to generate therapeutic and diagnostic anti-PSCA antibodies. The immunogen can comprise 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 contiguous amino acids, or the corresponding nucleic acid encoding these amino acids, its amino acid map can also be deduced, because the sequence contained in the variant is similar to that in Figure 1 The variants shown are the same. In particular, it should be noted that the peptide immunogen of the present invention may comprise a peptide region containing at least 5 amino acids as shown in Figure 1, the number of amino acids contained therein may be increased by any integer, and the amino acid positions contained therein are within the range of hydrophilicity in Figure 5 The median value of the map is greater than 0.5; it contains at least the peptide region of 5 amino acids in Figure 1, the number of amino acids contained in it can be increased by any integer, and the value of the amino acid position contained in it is less than 0.5 in the hydrophilicity map; it contains at least Figure 1 The peptide region of 5 amino acids, the number of amino acids contained in it can be increased by any integer, and the value of the amino acid position contained in it is greater than 0.5 in the accessible residue percentage map; the peptide region containing at least 5 amino acids in Figure 1, The number of amino acids contained therein can be increased by any integer, and the value of the amino acid position contained in it in the flexibility map is greater than 0.5; and a peptide region containing at least 5 amino acids in Figure 1, and the number of amino acids contained therein can be increased by any integer , which contains amino acid positions with a value greater than 0.5 in the β-turn map. The peptide immunogen of the present invention also includes nucleic acid encoding the above-mentioned peptide segment. the

All immunogens of the invention, whether peptides or nucleic acids, may be presented in unit dosage form for human use, or contained in compositions comprising pharmaceutically acceptable excipients which are compatible with the physiological environment of the human body. the

The secondary structures of PSCA variant proteins 1, 3, 4, and 6, that is, the predicted presence and location of α-helixes, extended chains, and irregular helices, can be determined based on the primary amino acid sequence of the protein using the HNN-Hierarchical Neural Network method. Speculation (NPS: Network Protein Sequence Analysis TIBS March 2000, Vol. 25, No 3[291]: 147-150; Combet C., Blanchet C., Geourjon C. and Deléage G., http://pbil.ibcp. fr/cgi-bin/npsa_automat.pl?page=npsa_nn.html), the method can be found by accessing the ExPasy Molecular Biology Server (expasy.ch/tools/ on the World Wide Web). Through analysis, it was found that PSCA variant 1 was composed of 30.89% α helix, 21.95% extended chain and 47.15% irregular helix. PSCA variant 3 consists of 14.89% alpha helices, 28.51% extended strands and 76.60% random helices. PSCA variant 4 consists of 9.52% alpha helices, 8.99% extended strands and 81.48% random helices. PSCA variant 6 consists of 24.56% alpha helices, 21.93% extended strands and 53.51% random helices. the

Whether there is a transmembrane region on the PSCA variant protein can be analyzed by various transmembrane prediction algorithms on the ExPasy molecular biology server on the World Wide Web (.expasy.ch/tools/). the

Example 7

Generation of PSCA polyclonal antibody

Polyclonal antibodies can be prepared by injecting mammals with one or more immunization preparations, if necessary, adding adjuvants to the preparations. Generally, the immunizing agent and/or adjuvant is administered to a mammal by subcutaneous or intraperitoneal injection at multiple sites. In addition to immunizing with full-length PSCA protein variants, computer programs can also be used to design immunogens, such as finding antigenic sequences and sequences that can be recognized by the immune system of the immunized host according to the results of amino acid sequence analysis (see the heading " Antigenic Characteristics and Secondary Structure" example). It is predicted that this fragment has a hydrophilic, flexible, β-turn configuration, and is located on the surface of the protein. the

血蓝蛋白(KLH)、血清白蛋白、牛甲状腺球蛋白和大豆胰蛋白酶抑制因子。在一个实施方式中，编码PSCA变体4的氨基酸103-133的肽连接上一个KLH用于免疫家兔。另外，免疫制剂还可以包含PSCA变体蛋白的全长或部分序列、其模拟物或融合蛋白。例如，可以利用重组DNA技术将PSCA变体的氨基酸序列与本领域熟知的任意一种融合蛋白伴侣相融合，如含谷胱甘肽S转移酶(GST)和HIS标记的融合蛋白。在一个实施方式中，利用重组技术将PSCA变体1的氨基酸18-98与GST融合，在pGEX表达载体内表达，纯化后用于免疫家兔和小鼠，可以分别制备出多克隆抗体和单克隆抗体。这种融合蛋白可用适当的亲和基质从被诱导的细菌内纯化出来。  For example, recombinant bacterial fusion proteins or polypeptides containing the hydrophilic, flexible, β-turn regions of PSCA protein variants can be used as antigens for the production of polyclonal antibodies in New Zealand white rabbits or titled "Generation of Monoclonal Antibodies to PSCA" The monoclonal antibodies described in the Examples. For example, in PSCA variant 1, such regions include, without limitation, amino acids 28-56 and amino acids 66-94. In variant 3, such regions include, without limitation, amino acids 6-18, amino acids 27-39, amino acids 103-133, and amino acids 177-189. In variant 6, such regions include, without limitation, amino acids 19-35 and amino acids 57-85. It may be advantageous to attach to the immune factor a protein known to be immunogenic in the mammal being immunized. Such immunogenic proteins include, but are not limited to, keyhole

Hemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. In one embodiment, a peptide encoding amino acids 103-133 of PSCA variant 4 linked to a KLH is used to immunize rabbits. In addition, the immune preparation can also contain the full-length or partial sequence of PSCA variant protein, its mimic or fusion protein. For example, the amino acid sequence of the PSCA variant can be fused with any fusion protein partner known in the art, such as a fusion protein containing glutathione S-transferase (GST) and a HIS tag, using recombinant DNA technology. In one embodiment, amino acids 18-98 of PSCA variant 1 are fused with GST by recombinant technology, expressed in the pGEX expression vector, purified and used to immunize rabbits and mice, and polyclonal antibodies and monoclonal antibodies can be prepared respectively. Cloned antibodies. Such fusion proteins can be purified from induced bacteria using appropriate affinity matrices.

Other recombinant bacterial fusion proteins that can be used in the present invention include maltose-binding protein, LacZ, thioredoxin, NusA, or constant regions of immunoglobulins (see "PSCA Production in Prokaryotic Systems" section and Frederick M. Ausubu et al. eds. Current Methods in Molecular Biology, Volume 2, Unit 16, 1995; Linsley, P.S., Brady, W., Urnes, M., Grosmaire, L., Damle, N., and Ledbetter, L. (1991) J. Exp. Med. 174, 561-566). the

In addition to fusion proteins of bacterial origin, mammalian expressed protein antigens can also be used. These antigens can be expressed using mammalian expression vectors such as Tag5 and Fc-fusion vectors (see section "Preparation of Recombinant PSCA in Eukaryotic Systems"), which preserve post-translational modifications, such as glycosyl groups of native proteins change. In one embodiment, the cDNA, N-terminal leader peptide and C-terminal GPI-anchored protein of PSCA variant 1 were cloned into a Tag5 mammalian secretion vector and expressed in 293T cells. The recombinant protein was purified from the tissue culture supernatant of 293T cells stably expressing the recombinant vector by metal chelation chromatography. The purified Tag5 PSCA protein can be used as an immunogen. the

During immunization, it is beneficial to mix or emulsify the antigen with an adjuvant to enhance the immune response of the host animal. Examples of adjuvants include, but are not limited to, complete Freund's adjuvant (CFA) and MPL-TDM adjuvant (monophosphoryl lipid A, a synthetic trehalose dicorynomycolate.

In a typical immunization method, rabbits are first immunized by subcutaneously injecting up to 200 mg, generally 100-200 mg, of a fusion protein or peptide linked to KLH and mixed with complete Freund's adjuvant (CFA). Then 200 mg, usually 100-200 mg, of the immunogen mixed with incomplete Freund's adjuvant (IFA) is injected subcutaneously every two weeks. Blood samples were collected 7-10 days after each immunization for detection, and the titer of antiserum was determined by ELISA. the

To determine the reactivity and specificity of immune sera, such as rabbit sera immunized with GST fusion proteins of PSCA variant 3 or 4 proteins, the respective full-length PSCA variant cDNAs were cloned into the pCDNA 3.1myc-his expression vector (Invitrogen , see Example "Preparation of recombinant PSCA in eukaryotic systems"). After transfection of the constructs into 293T cells, specific reactivity with denatured mutant proteins was determined by western blotting hybridization of cell lysates with anti-variant sera and anti-His antibody (Santa Cruz Biotechnologies, Santa Cruz, CA) . Additionally, immune sera were analyzed using fluorescence microscopy, flow cytometry, and immunoprecipitation of cells expressing cells against 293T and other recombinant PSCA variants to determine the specific recognition activity of the native protein. Protein reactivity and specificity can also be determined by Western blot, immunoprecipitation, fluorescence microscopy, and flow cytometry analysis using cells endogenously expressing PSCA. the

Purification of rabbit antisera immunized with PSCA variant fusion proteins such as GST and MBP fusion proteins to remove antibodies reactive with fusion partner sequences, using an affinity layer containing only the fusion partner or an irrelevant fusion protein in addition to the fusion partner Antiserum can be purified by column separation. For example, antisera derived from the GST-PSCA variant 1 fusion protein were first purified by passage through a column containing an AffiGel matrix (BioRad, Hercules, Calif.) to which the GST protein was covalently bound. It is then purified by passage through a column containing an Affigel matrix to which the MBP-PSCA fusion protein is covalently bound. The antiserum was then purified by protein G affinity chromatography to separate the IgG component therein. Sera from rabbits immunized with other His-tagged antigens and peptides, as well as fusion partner-depleted sera, were affinity-purified by chromatographic columns containing native protein immunogens or free peptides. the

Example 8

Generation of PSCA Monoclonal Antibody (MAb)

In one embodiment, therapeutic polyclonal antibodies ("Mabs") to PSCA and PSCA variants include those antibodies that react with an epitope specific for each protein or with an epitope specific for a consensus sequence, the antibodies Those capable of binding, internalizing, preventing or modulating the biological function of PSCA or PSCA variants, such as those that abolish interactions with ligands and binding partners. Immunogens useful in the preparation of such MAbs include those encoding or comprising extracellular domains or the complete PSCA protein sequence, regions predicted to comprise functional motifs, or comprising PSCA proteins predicted to be antigenic by computer analysis of the amino acid sequence. An immunogen for a region of the variant. Immunogens include peptides, recombinant bacterial proteins (such as GST-PSCA fusion protein (Figure 8) and His-tagged PSCA pET carrier protein (Figure 6)), purified His-tagged protein expressed from mammalian cells (Figure 7), and human and Mouse IgG FC fusion protein. Alternatively, cells genetically engineered to express high levels of PSCA variant 1 by retroviral transduction, such as RAT1-PSCA, 293T-PSCA, 3T3-PSCA, or 300.19-PSCA, can be used to immunize mice. the

In order to prepare monoclonal antibodies to PSCA, usually 5-50 μg of protein immunogen or 10 ⁶ -10 ⁷ PSCA-expressing cells are firstly immunized in the mouse footpad (FP) mixed with a suitable immune adjuvant. An example of a suitable immunization method for FP is an initial FP injection with TiterMax (Sigma) followed by an alum gel containing (Qiagen). After the initial injection, mice were immunized twice a week until sacrifice, and B cells were obtained from lymph nodes for fusion.

During immunization, blood samples are collected to monitor the titer and specificity of the immune response. In most cases, once sufficient reactivity and specificity have been achieved by ELISA, western blot, immunoprecipitation, fluorescence microscopy or flow cytometry, cell fusion can be performed using electrocytofusion (BTX, ECM2000) and hybridoma preparation. the

In one embodiment, the invention provides monoclonal antibodies designated Ha1-1.16, Ha1-5.99, Ha1-4.117, Ha1-4.120, Ha1-4.121 and Ha1-4.37. The antibodies were identified and shown to react and bind to cell surface or immobilized PSCA. the

产生针对PSCA的Mab，在该技术中小鼠重链和κ轻链上的基因座已被灭活且主要的人重链和κ轻链免疫球蛋白座位已插入：Ha1-1.16，它是用PSCA-GST免疫可产生人γ1的异源小鼠13次后产生的；Ha1-5.99，它用Rat1-PSCA细胞免疫可产生人γ2的异源小鼠6次后再用PSCA-tag5注射2次产生的；Ha1-4.117、HA1-4.37、Ha1-4.120和Ha1-4.121，它们是用Rat1-PSCA细胞免疫可产生人γ1的异源小鼠6次后再用PSCA-tag5注射4次产生的。所述抗PSCA Mab，Ha1-1.16、Ha1-5.99、Ha1-4.117、Ha1-4.120和Ha1-4.121结合在前列腺癌异种移植物细胞中表达的内源性细胞表面PSCA。  Using xenogeneic mouse (Xenomice) technology

Generate Mabs against PSCA in which the loci on the mouse heavy and kappa light chains have been inactivated and the major human heavy and kappa light chain immunoglobulin loci have been inserted: Ha1-1.16, which was developed with PSCA -GST immunization with human γ1-producing heterogeneous mice 13 times; Ha1-5.99, which was immunized with Rat1-PSCA cells and human γ2-producing heterogeneous mice 6 times and then injected with PSCA-tag5 2 times ; Ha1-4.117, HA1-4.37, Ha1-4.120, and Ha1-4.121, which were generated by immunizing human γ1-producing heterologous mice 6 times with Rat1-PSCA cells followed by 4 injections with PSCA-tag5. The anti-PSCA Mabs, Ha1-1.16, Ha1-5.99, Ha1-4.117, Ha1-4.120 and Ha1-4.121 bind endogenous cell surface PSCA expressed in prostate cancer xenograft cells.

Antibodies designated Ha1-5.99, Ha1-4.117, Ha1-4.120, Ha1-4.37, Ha1-1.16, and Ha1-4.121 were sent (via FedEx) to the American Type Culture Collection (ATCC) on May 4, 2004 ), P.O. Box 1549, Manassas, VA 20108, and were given deposit numbers PTA-6703, PTA-6699, PTA-6700, PTA-6702, PTA-6698, PTA-6701, respectively. the

After mRNA was isolated from each hybridoma cell, the DNA of anti-PSCA MAb Ha1-1.16, Ha1-5.99, Ha1-4.117, Ha1-4.120, Ha1-4.121 and Ha1-4.37 was assayed with Trizol reagent (Life Technologies, Gibco BRL) coding sequence. Total RNA was purified and quantified. First-strand cDNA was generated from the oligo(dT)1218 primer using the Gibco BRL Superscript Preamplification system. First-strand cDNA was amplified with human immunoglobulin variable heavy chain primers and human immunoglobulin variable light chain primers. The PCR product was cloned into pCRScript vector (Stratagene, La Jolla). Multiple clones were sequenced to determine the heavy and light chain variable regions. The nucleic acid and amino acid sequences of the heavy and light chain variable regions are listed in Figures 2 and 3 . Alignments of PSCA antibodies to germline V-D-J sequences are shown in Figures 4A-4M. the

Example 9

Screening and identification of PSCA antibodies

Antibodies generated using the procedure described in the Example entitled "Generation of PSCA Monoclonal Antibodies (MAbs)" were tested using a combination of ELISA, FACS, epitope grouping, and avidity testing for PSCA expressed on the cell surface. screening and identification. the

A. Screening of human MAbs by FACS. the

Primary hybridomas were screened for MAbs against PSCA by FACS. The experimental design is as follows: 50 μl/well hybridoma supernatant (pure) or purified antibody (serial dilution) was added to a 96-well FACS plate, and cells expressing PSCA (endogenous or recombinant, 50,000 cells/well) mix. The mixture was incubated at 4°C for 2 hours. After incubation, cells were washed with FACS buffer and incubated with 100 μl of detection antibody (anti-hIgG-PE) at 4° C. for 45 minutes. After incubation, cells were washed with FACS buffer, fixed with formaldehyde, and analyzed with FACScan. Data analysis was performed using CellQuest Pro software. Filled histograms represent data obtained from control cells, and open histograms represent data obtained from PSCA-positive cells (Figure 9). the

Positive hybridomas identified from the original screen were transferred into 24-well plates, and supernatants were collected for confirmatory screening. Confirmatory screening includes: 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) Line) for FACs analysis, and using Tag5-PSCA, GST-PSCA, GST-PSCA N-terminal, Med.C-terminal and pET-PSCA for ELISA analysis. the

B. Relative affinity analysis of PSCA human MAb

Hybridoma supernatants were tested to determine their relative binding affinities for cell surface PSCA. Hybridoma supernatants were serially diluted from μg/ml to sub-ng/ml in FACS buffer (FB); assessed in FACS binding assays using LAPC9AI cells. High avidity antibodies give high MFI values. The MFI values for each point were obtained using CellQuestPro software and used for affinity calculations using Graphpad Prism software (Table VII and Table VIII): Sigmoid dose-response (variable slope) formula. The results of the relative affinity analysis are shown in FIG. 10 . the

C. Epitope Grouping

PSCA antibodies were grouped according to epitopes by assessing their binding pattern to LAPC9AI cells. Briefly, a small amount of each antibody was biotinylated; each biotinylated antibody was then incubated for 1 hour at 4°C in the presence of an excess (100x) of non-biotinylated antibody. Often, competition will occur between excess antibody and biotinylated antibody if they bind to the same epitope. At the end of the incubation, the cells were washed and incubated with streptavidin-PE for 45 min at 4°C. After washing away unbound streptavidin-PE, the cells were analyzed by FACS. Data analysis was performed using the MFI assay (Table VII). As shown in Table XI, cells highlighted in yellow indicate self-competition (100% competition), and the MFI in these cells is a background control for each biotinylated antibody. Uncolored cells indicate that the two antibodies compete with each other (low MFI), high MFI (highlighted in blue) indicate that the two antibodies bind two different epitopes. Antibodies with the same binding mode bind to the same epitope in the antibody. Six sets of epitopes were tested in the antibodies. Table XI shows that PSCA 4.121 binds to its unique epitope. the

Example 10

Characterization and expression of PSCA antibodies

A. Cross-reactivity with monkey PSCA and mouse PSCA

Screen MAbs and characterize their reactivity with mouse and simian sources. This property was used to understand the binding of MAbs to PSCA on cells and in tissues using mouse and simian animal models. Cynomolgus and mouse PSCA genes were cloned, expressed in retrovirus and transiently infected 293-T cells. _____ was incubated with various antibodies using the following experimental design. The test antibody was incubated with 293-T cells expressing macaque or mouse PSCA, or with 293T cells expressing the neo gene as a negative control. Specific recognition was determined using anti-hIgG-PE detection secondary antibody. A typical histogram depicting specific cross-reactivity is shown in FIG. 11 . The summary presented in Table X shows that all but one of the anti-human PSCA antibodies cross-reacted with monkey PSCA and only one anti-human PSCA antibody cross-reacted with mouse PSCA. the

B. Avidity Determination by FACS

A set of seven (7) anti-human PSCA antibodies were tested for their binding affinities to PSCA on SW780 cells, a human bladder cancer cell line expressing high levels of PSCA. Twenty-three serial 1 :2 dilutions of purified antibody were incubated with SW780 cells (50,000 cells per well) at final concentrations ranging from 167 nM to 0.01 pM overnight at 4°C. After incubation, cells were washed and incubated with anti-hIgG-PE detection antibody for 45 min at 4°C. After washing away the unbound detection antibody, the cells were analyzed by FACS: the MFI value of each point was obtained by CellQuest Pro software, and it was used for the affinity calculation by Graphpad Prism software: Sigmoid dose-response (variable slope) formula (Table VII and Table VIII). The results of the relative affinity analysis for seven (7) antibodies are shown in Table IX. the

Example 11

Internalization of PSCA Antibody

Internalization studies of Ha1-4.121 were performed using PC3-PSCA cells. Briefly, Hal-4.121 was incubated with cells at 4°C to 90°C to allow the antibody to bind to the cell surface. Cells were then divided into two groups and incubated either at 37°C for antibody internalization or at 4°C as a control (no internalization). After incubation at 37°C/4°C, acid wash was performed to remove PSCA 4.121 bound to the cell surface. Subsequent permeabilization enables detection of antibody binding to internalized PSCA. After incubation with detection secondary antibodies, cells were analyzed by FACS or visualized under a fluorescence microscope. About 30% of Hal-4.121 was internalized after 2 hours of incubation at 37°C (Fig. 12). the

Example 12

Antibody-mediated secondary killing

Antibodies against PSCA mediate saporin-dependent killing in cells expressing PSCA. B300.19-PSCA-expressing cells (750 cells/well) were seeded into 96-well plates on day 1. On the following days, an equal volume of anti-human (Hum-Zap) or anti-human (Hum-Zap) or anti-saporin conjugated to saporin toxin (Advanced Targeting Systems, San Diego, CA) containing 2× concentration of the indicated primary antibody was added to each well in a 2-fold excess. Medium for Goat (Goat-Zap) Polyclonal Antibody. Cells were incubated at 37°C for 5 days. After the incubation period, MTS (Promega) was added to each well and incubation was continued for 4 hours. The OD value at 450 nM was determined. The results in Figure 13(A) show that PSCA antibodies HA1-4.121 and HA1-4.117 mediate saporin-dependent cytotoxicity in B300.19-PSCA cells, while a non-specific human IgGl control antibody had no effect. The results in Figure 13(B) show that the addition of a saporin-conjugated secondary antibody that does not recognize human Fc fails to mediate cytotoxicity (Figure 13(A) and Figure 13(B)). These results suggest that drugs or toxic proteins can be selectively delivered to PSCA-expressing cells by using appropriate anti-PSCA MAbs. the

Example 13

Antibody immune-mediated cytotoxicity

Antibodies to PSCA were evaluated for their ability to mediate immune-dependent cytotoxicity. PSCA antibody (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 ¹⁰⁶ cells/ml. In a typical assay, 50 μl of PSCA antibody, 50 μl of diluted rabbit complement serum (Cedarlane, Ontario, Can) and 50 μl of cell suspension were added together in a flat bottom tissue culture 96-well plate. The mixture was incubated in a 37 °C, 5% _CO2 incubator for 2 h to facilitate complement-mediated cell lysis. Then 50 μl of Alamar Blue (Biosource Intl. Camarillo, CA) was added to each well, and the incubation was continued at 37° C. for 4-5 hours. Fluorescence was read using a 96-well fluorometer with excitation at 530 nm and emission at 590 nm. The results showed that PSCA antibodies with Ig1 (HA1-4.121) or IgG2 isotype (HA1-5.99.1) but not IgG4 isotype (HA1-6.46) could mediate complement-dependent lysis (Figure 14).

ADCC (antibody-dependent cellular cytotoxicity) is an immune-mediated lytic attack on cells bound to antibodies targeting specific cell surface antigens. In this application it is PSCA. Immune cells recognize the Fc portion of the antibody and initiate a lytic attack leading to cell death through binding to Fcγ receptors on leukocytes, monocytes and NK cells. The ability of PSCA antibodies to mediate this response can be demonstrated by labeling tumor cells in vitro with ⁵¹ chromium, europium or fluorescent molecules and incubating them in the presence of human PSCA MAb and peripheral blood mononuclear cells. Specific lysis of tumor cells can be determined by measuring the percent lysis of target tumor cells. Common endpoints measured include: release of radioactive, europium or fluorescent dyes from dead cells using appropriate detection methods. Alternatively, the release of intracellular enzymes (eg, lactate dehydrogenase, LDH) can be assayed.

Example 14

F(Ab') ₂ generation of fragments

F(Ab') ₂ fragments of MAbs were generated to study the effects of MAb molecules retaining the bivalent antigen-binding site of the MAb molecule but lacking the immune effector Fc region in in vitro and in vivo therapeutic models. 20 mg of MAbHa1-4.121 in 20 mM sodium acetate buffer pH 4.5 were incubated with or without immobilized pepsin (Pierce. Rockford IL) for the indicated times. Intact MAb and digested Fc fragments were removed by protein A chromatography. Figure 15 shows PAGE Coomassie staining of intact undigested unreduced MAb, unreduced aliquots of digested material taken at indicated times, and _reduced samples of final digested F(ab') products gel. The agent can be used to treat animals bearing tumors expressing PSCA. The antitumor activity observed with this antibody fragment can distinguish between intrinsic biological activity and activity mediated by immune-dependent mechanisms.

Example 15

Human Antibody Expression Using Recombinant DNA Method

To express recombinant anti-PSCA MAbs in transfected cells, the 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 and light chain cassettes were cloned into the cloning vector downstream of the CMV promoter/enhancer. A polyadenylation site is included downstream of the MAb coding sequence. The recombinant anti-PSCA MAb expressing constructs were transfected into 293T, Cos and CHO cells. Binding of HA1-4.121 antibody secreted from recombinant 293-T cells to cell surface PSCA in Figure Pia-3A was assessed and compared to the same antibody produced by the original hybridoma (Figure 16). the

Example 16

Binding assay of HLA class I molecules and class II molecules

Binding experiments of HLA class I molecules and class II molecules were performed using purified HLA molecules using methods published in the literature (see PCT patent applications 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 (5-500 nM) were combined with various unlabeled peptide inhibitors and 1-10 nM ¹²⁵ I-labeled peptide probes, and after co-incubation, the MHC-peptide complexes were separated from the MHC-peptide complexes by gel filtration. The free peptide is separated and the fraction bound to the peptide is determined. Generally, in pilot experiments, each MHC preparation is titered in the presence of a fixed amount of radiolabeled peptide to determine the concentration of HLA molecules required to bind 10-20% of the total radioactivity. Subsequent inhibition and direct binding assays were performed with these concentrations of HLA.

Since the concentration of the marker is less than the concentration of HLA and the _IC50 is greater than or equal to the concentration of HLA under these conditions, the measured _IC50 value should be approximately equal to the true _KD value. Peptide inhibitor concentrations are generally between 120 μg/ml and 1.2 ng/ml, and 2 to 4 completely independent experiments should be performed. To compare data from different assays, the relative binding index for each peptide is calculated by dividing the _IC50 of the positive control by the _IC50 of each test peptide (typically the unlabeled version of the radiolabeled probe peptide). For database construction and intra-assay comparisons, relative binding values need to be compiled. These values can then be converted to IC ₅₀ nM values by dividing the IC ₅₀ nM value of the positive control by the relative binding value of the peptide of interest. This method of data compilation is accurate and robust when used to compare data from peptides tested at different times or from experiments with different batches of purified MHC.

The binding assays described above can also be used to analyze HLA supermotif and/or motif-containing peptides (see Table IV). the

Example 17

Construction of "Minigene" Multi-epitope DNA Plasmid

This example discusses the construction of minigene expression plasmids. Of course, minigene plasmids may contain B cell, CTL and/or HTL epitopes or epitope mimics in various combinations as described herein. the

Small gene expression plasmids generally contain multiple CTL and HTL peptide epitopes. In this example, peptide epitopes containing HLA-A2, -A3, -B7 supermotifs and peptide epitopes containing HLA-A1 and -A24 motifs are combined with epitopes containing DR supermotifs and/or DR3 epitopes linked. PSCA-derived peptide epitopes containing HLA class I molecular supermotifs or motifs were selected so that multiple supermotifs/motifs were included in the plasmid, thereby ensuring a broad population coverage. Likewise, selection of PSCA-derived HLA class II epitopes can also provide broad population coverage, i.e., both HLA DR-1, -4, -7 supermotif-containing epitopes and Epitopes of HLA DR-3 motifs. The selected CTL and HTL epitopes are then inserted into the minigene for expression in the expression vector. the

Such constructs may also contain sequences that direct HTL epitopes to the endoplasmic reticulum. For example, the Ii protein can be fused to one or more HTL epitopes described in the art, wherein the CLIP sequence of the Ii protein is deleted and replaced with an HLA class II epitope sequence, so that the HLA class II epitope can be directed To the endoplasmic reticulum, where the epitope binds to HLA class II molecules. the

This example describes the method for constructing an expression plasmid containing a minigene. Other expression vectors can also be used for minigene compositions, and such vectors are also well known to those skilled in the art. the

The minigene DNA plasmid of this example contains a consensus Kozak and a consensus murine kappa Ig light chain signal sequence, followed by CTL and/or HTL epitopes selected according to the principles described herein. These sequences can encode an open reading frame, and the Myc and His antibody epitope tags can be fused by using the pcDNA 3.1Myc-His expression vector. the

Overlapping oligonucleotides containing an average of about 70 nucleotides with 15 overlapping nucleotides can be synthesized by chemical synthesis and purified by HPLC. The oligonucleotides encode the selected peptide epitopes and appropriate linker oligonucleotides, Kozak sequences and signal sequences. The final multi-epitopic minigene can be assembled by extending the overlapping oligonucleotides by PCR through three sets of reactions. A Perkin/Elmer 9600 PCR machine was used for 30 cycles under the following conditions: 95°C for 15 seconds, annealing temperature (5°C lower than the lowest calculated Tm for each primer pair) for 30 seconds, and 72°C for 1 minute. the

For example, minigenes are prepared as follows. The first round of PCR reaction uses two oligonucleotides, 5 mg each, for renaturation and extension: in one example, 8 oligonucleotides are used, that is, 4 pairs of primers, oligonucleotides 1+2, 3+4 , 5+6 and 7+8 were mixed in a 100ml reaction solution containing Pfu polymerase buffer (1×=10mM KCL, 10mM (NH4)2SO4, 20mM Tris-Cl, pH8.75, 2mM MgSO4, 0.1% TritonX- 100, 100mg/ml BSA), dNTP each 0.25mM and 2.5U Pfu polymerase. The full-length dimerization products were purified by gel, and the two reactions containing products 1+2 and 3+4 and products 5+6 and 7+8 were mixed, renatured and extended for 10 cycles. Then half of the two reaction solutions were mixed, followed by 5 cycles of annealing and extension, and primers on both sides were added to amplify the full-length product. The full-length product was gel purified, cloned into pCR-blunt (Invitrogen), and each clone was screened by sequencing. the

Example 18

Plasmid constructs and the extent to which they induce immunogenicity

A plasmid construct, such as the plasmid constructed in the above examples, can induce immunogenicity by inducing or transfecting APC with an epitope expressing nucleic acid construct in vitro, and then measuring the ability of APC to present an epitope. Sure. This test determines "antigenicity" and human APC can be used. This assay measures the ability of an epitope to be presented by APCs. If an epitope can be recognized by T cells, this ability can be determined by quantitatively measuring the density of the epitope-HLA class I molecule complex on the cell surface. Quantification can be determined by direct measurement of the amount of peptide eluted from APC (see Sijts et al., J. Immunol. 156:683-692, 1996; Demotz et al., Nature 342:682-684, 1989), or by measuring To determine the amount of peptide-HLA class I molecule complexes, lytic release or lymphokine release is induced by diseased or transfected target cells, and then it can be determined that the same level of lytic release is achieved Or the peptide concentration required for lymphokine release (see Kageyama et al., J. Immunol. 154:567-576, 1995). the

In addition, immunogenicity can also be determined by injecting mice in vivo, and then measuring CTL and HTL activities in vitro. CTL activity and HTL activity are determined by cytotoxic activity assay and proliferation assay, such as Alexander et al., Immunity 1:751- 761, 1994 as described in detail. the

For example, to determine the ability of DNA minigene constructs containing at least one HLA-A2 supermotif peptide to induce CTLs in vivo, HLA-A2.1/Kb transgenic mice were immunized with 100 mg of naked cDNA intramuscularly. In order to compare the CTL levels induced by cDNA immunization, control animals were immunized with a peptide composition containing multiple epitopes on one polypeptide chain, like the polypeptide chain encoded by the minigene. the

Splenocytes were isolated from immunized animals, stimulated twice with the two compositions (minigene-encoded peptide epitope and multi-epitope peptide-encoded peptide epitope), and peptide-specific cytotoxic activity was determined by 51Cr release assay . The results can show the intensity of the CTL response against the A2-restricted epitope, thus illustrating the immunogenicity of minigene vaccines and multi-epitope vaccines in vivo. the

Thus, the results of this trial demonstrate that minigenes can elicit an immune response against HLA-A2 supermotif peptide epitopes, as can multiepitopic peptide vaccines. Using other HLA-A3 and HLA-B7 transgenic mouse models can also evaluate the ability of HLA-A3 and HLA-B7 motifs or supermotif epitopes to induce CTL through similar experiments. Immune responses to these epitopes. the

To evaluate the ability of minigenes encoding class II epitopes to induce HTL in vivo, DR transgenic mice or I-Ab-restricted mice were injected intramuscularly with 100 mg of plasmid DNA to assess crossover to appropriate murine MHC molecules Those epitopes that react. In order to compare the level of HTL induced by DNA immunization, a group of control animals were set up and immunized with the peptide composition emulsified in complete Freund's adjuvant. Spleen cells were isolated from the immunized animals, and the CD4+ T cells in them, ie, HTL, were purified and stimulated with two compositions (peptides encoded by minigenes) respectively. HTL responses were determined by ^the3H thymidine incorporation proliferation assay (see Alexander et al., Immunity 1:751-761, 1994). The results indicated that a strong HTL response was generated, thus demonstrating that the minigene is immunogenic in vivo.

The DNA minigene constructed according to the description of the above examples can also be evaluated as a vaccine co-boosting factor through a prime-boost strategy. Boosters include recombinant proteins (see Barnett et al., Aids Res. and Human Retroviruses 14, Suppl. 3:S299-S309, 1998) or recombinant vaccinia viruses, such as those expressing a minigene or DNA encoding the entire protein of interest (see Hanke et al., Vaccine 16 : 439-445, 1998; Sedegah et al, Proc. -34, 1999). the

For example, the efficiency of DNA minigenes used in a prime-boost strategy was first evaluated using transgenic mice. In this example, A2.1/Kb transgenic mice were immunized with IM plus 100 mg of DNA minigenes encoding immunogenic peptides, wherein the immunogenic peptides included at least one peptide containing the HLA-A2 supermotif. After an incubation period (3-9 weeks), the mice were boosted by intraperitoneal injection of recombinant vaccinia virus at a dose of 107 pfu per mouse, which expresses the same sequence encoded by the DNA minigene. Control mice were immunized with 100 mg of DNA or recombinant vaccinia virus without the minigene, or with DNA encoding the minigene but without booster immunization with vaccinia virus. After another 2-week incubation period, the splenocytes of the mice were collected and immediately analyzed for their peptide-specific cytotoxic activity by ELISPOT assay. Alternatively, in vitro stimulation with A2-restricted peptide epitopes encoded by minigenes or recombinant vaccinia viruses was followed by α, β, and/or γIFN ELISA to measure peptide-specific cytotoxic activity. the

It was found that the minigene used in the prime-boost strategy elicited a stronger immune response against the HLA-A2 supermotif peptide than DNA alone. Similar assays can be performed using HLA-A11 or HLA-B7 transgenic mouse models to assess the ability of HLA-A3 or HLA-B7 motif or supermotif epitopes to induce CTLs. Methods for implementing primary boost regimens in humans are described in the Example "Induction of CTL responses using a prime-boost strategy" below. the

Example 19

Multi-epitope vaccine composition derived from multiple antigens

The PSCA peptide epitope of the present invention can be combined with epitopes derived from other tumor-associated target antigens, and the formed vaccine composition can be used to prevent or treat tumors expressing PSCA and the tumor-associated target antigen. For example, a vaccine composition provided by the present invention exists in the form of a single-chain polypeptide, which contains PSCA and multiple epitopes derived from tumor-associated antigens, wherein the target tumor expressing PSCA usually also expresses the tumor-associated antigen, Alternatively the vaccine composition is a mixture of one or more discrete epitopes. Alternatively, vaccines can be injected as minigene constructs or as dendritic cells that have been loaded with peptide epitopes in vitro. the

Example 20

Using Peptides to Assess the Immune Response

The peptides of the present invention can be used to analyze the immune response to determine whether there are specific antibodies against PSCA, CTL or HTL. This analysis can be performed as described by Ogg et al., Science 279:2103-2106,1998. In this example, the peptides of the present invention are used as reagents rather than immunogens for diagnostic or prognostic purposes. the

In this example, the highly sensitive human leukocyte antigen tetrameric complex ("tetramer") is used as a representative example to analyze PSCA peptides with A*0201 motif in different disease stages. Probability of producing PSCA HLA-A*0201-specific CTL in HLA A*0201-positive individuals after immunization. The tetrameric complex was synthesized according to the method described in the literature (Musey et al., N. Engl. J. Med. 337:1267, 1997). Briefly, purified HLA heavy chain (A*0201 in this example) and b2-microglobulin were synthesized in a prokaryotic expression system. The heavy chain was modified by deleting the transmembrane-cytoplasmic tail and adding a sequence containing a BirA enzyme-catalyzed biotinylation site at the COOH terminus. Heavy chain, b2-microglobulin and peptide are refolded by dilution. The 45-kD refolded product was isolated by fast protein liquid chromatography and biotinylated with BirA in the presence of biotin (Sigma, St. Louis, Missouri), 5'-adenosine triphosphate, and magnesium ions. The streptavidin-phycoerythrin linker was added at a molar ratio of 1:4, and the tetrameric product was concentrated to 1 mg/ml. The resulting product is called tetramer-phycoerythrin. the

For analysis of patient blood samples, approximately 1 x ¹⁰⁶ PBMC were centrifuged at 300 g for 5 minutes and then resuspended in 50 ml of cold phosphate buffered saline. Tricolor analysis using tetramer-phycoerythrin, anti-CD8-Tricolor and anti-CD38. PBMC were incubated with tetramers and antibodies for 30 to 60 minutes on ice, washed twice and fixed with formaldehyde. The gate was set to be greater than 99.98% of the control samples. Tetramer controls included A*0201 negative individuals and A*0201 positive non-diseased donors. The percentage of tetramer-stained cells was then determined by flow cytometry. The results show the number of cells containing epitope-restricted CTLs within the PBMC sample, allowing the strength of the immune response to the PSCA epitope to be easily determined and therefore the protective response or treatment elicited after exposure to PSCA or vaccine The state of the sexual response.

Example 21

Induction of CTL responses by a prime-boost strategy

A prime-boost strategy similar to that described in the above example "Plasmid constructs and their degree of induction of immunogenicity" for determining the efficacy of DNA vaccines in transgenic mice can also be used for evaluation under the principles described below Efficacy of vaccines in humans. This vaccine immunization scheme includes primary immunization with naked DNA, and then booster immunization with recombinant virus or recombinant protein/polypeptide or peptide mixture that encodes the vaccine mixed with adjuvant. the

For example, the initial immunization can use an expression vector, such as the expression vector constructed in the embodiment "Construction of a small gene multi-epitope DNA plasmid", to inject intramuscularly (or subcutaneously or ID) to multiple sites in the form of naked nucleic acid, The dosage is 0.5-5 mg. Nucleic acid (0.1-1000 mg) can also be injected using a gene gun. After an incubation period of 3-4 weeks, another booster immunization was performed. Booster immunization can use 5×107 to 5×109 pfu of recombinant fowlpox virus. Other recombinant viruses, such as MVA, canarypox virus, adenovirus, or adeno-associated virus can also be used for booster immunization, or polyepitopic protein or peptide mixtures. To evaluate the efficacy of the vaccine, blood samples were collected from patients before immunization, after primary immunization, and after booster immunization. Peripheral blood mononuclear cells were isolated from heparinized fresh blood samples by Ficoll-Hypaque density gradient centrifugation, resuspended in cryopreservation medium and stored in cryopreservation tubes. Samples were analyzed for CTL and HTL activity. the

The test results show that the intensity of the immune response generated is sufficient to achieve the purpose of generating anti-PSCA therapeutic or protective immunity. the

Example 22

complementary polynucleotides

A sequence complementary to the PSCA coding sequence (Figure 1 or Figure 3) or any portion thereof is used to detect, reduce or inhibit the expression of native PSCA. Although the use of oligonucleotides of about 15 to 30 base pairs has been described herein, substantially similar methods can be used for smaller or larger sequence fragments. Appropriate oligonucleotides were designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of PSCA. To inhibit transcription, complementary oligonucleotides are designed based on the most unique 5' sequence to prevent binding of the promoter to the coding sequence. To inhibit translation, complementary oligonucleotides were designed to prevent ribosome binding to PSCA-encoding transcripts. the

Example 23

Purification of native or recombinant PSCA using PSCA-specific antibodies

Native or recombinant PSCA was purified by immunoaffinity chromatography using antibodies specific for PSCA. Immunoaffinity columns are prepared by covalently binding anti-PSCA antibodies to activated chromatography resins such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After bonding the resin was blocked and washed according to the manufacturer's instructions. the

The PSCA-containing medium is passed through the immunoaffinity column, and the column is washed under conditions that allow preferential adsorption of PSCA, such as a high ionic strength buffer containing a detergent. Then use conditions that break the antibody/PSCA binding (such as buffer solution from pH2 to pH3, or high concentration of chaotropic agent, such as urea or thiocyanate ion) to elute the chromatography column to collect GCR.P. the

Example 24

Identification of molecules that can interact with PSCA

PSCA or its biologically active fragments are labeled with 121 1 Bolton-Hunter reagent (see Bolton et al. (1973) Biochem. J. 133:529). Candidate molecules arrayed in the wells of the multiwell culture plate described above were incubated with labeled PSCA, and after washing, all wells containing labeled PSCA complexes were detected. The data obtained from different concentrations of PSCA were used to calculate the amount, affinity and interaction between PSCA and candidate molecules. the

Example 25

Determination of the promoting effect of PSCA on tumor growth in vivo

The effect of PSCA protein on tumor growth in vivo was determined by evaluating the development and growth of cells expressing or lacking PSCA. For example, 1×10 ⁶ 3T3 cells or prostate cancer cell lines (such as PC3 cells) containing tkNeo empty vector or PSCA are subcutaneously injected on one side of SCID mice. At least two strategies are available: (1) constitutive expression of PSCA under the regulation of a promoter such as a constitutive promoter, which can be derived from the genome of a virus, such as polyoma virus, fowlpox virus ( UK2,211,504, published 5 July 1989), adenoviruses (such as adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus and simian virus 40( SV40), or heterologous promoters from mammals, such as actin promoters or immunoglobulin promoters, as long as these promoters are compatible with the inducible vector system, and (2) in the inducible Regulatable expression under the control of a vector system, such as ecdysone, tetracycline, etc., as long as the promoter is compatible with the host cell system. If the tumor is palpable, the volume of the tumor is measured with calipers, and over time it is determined whether the PSCA-expressing cells are growing at a faster rate, and whether the aggressive characteristics of the tumors formed by PSCA-expressing cells have changed (eg, increased metastatic capacity, vascularization, and reduced sensitivity to chemotherapeutic agents).

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

This assay can also be used to analyze the inhibitory effect of PSCA on candidate therapeutic compositions, such as PSCA endosomes, PSCA antisense molecules, and ribozymes. the

Example 26

In vivo tumor inhibition mediated by PSCA monoclonal antibody

PSCA is significantly expressed on the cell surface in tumor tissues, but restricted in normal tissues, which makes PSCA an ideal target for antibody therapy. PSCA is also a target for T cell immunotherapy. Therefore, recombinant cell lines such as PC3-PSCA and 3T3-PSCA (see Kaighn, M.E. et al., Invest Urol, 1979.17 (1): 16-23) and human prostate xenograft models such as LAPC 9AD (Saffran et al., PNAS 1999, 10 : 1073-1078) can be used to evaluate the therapeutic effect of anti-PSCA monoclonal antibody in human prostate cancer xenograft mouse model and human pancreatic cancer xenograft mouse model. the

The effect of antibodies on tumor growth and metastatic carcinogenesis can be studied in murine orthotopic prostate or pancreatic cancer xenograft models. Antibodies can be unattached as discussed in this example, or attached to therapeutic carriers as is well known in the art. Anti-PSCA MAbs inhibit tumor formation in pancreatic and prostate xenograft models. Anti-PSCA MAbs also inhibited the growth of existing orthotopic tumors and prolonged the survival of tumor-bearing mice. These results suggest that anti-PSCA MAbs may be useful in the treatment of several localized and advanced prostate cancers, pancreatic cancers, and those cancers listed in Table I (see Saffran, D. et al., PNAS 10:1073-1078; or visit the site pnas.org/cgi /doi/10.1073/pnas.051624698). the

Injection of anti-PSCA MAb resulted in inhibition of the growth of existing orthotopic tumors and prevented tumor metastasis to distant sites, thus significantly prolonging the survival of tumor-bearing mice. These studies suggest that PSCA is an ideal target for immunotherapy and that PSCA MAbs are effective in both localized and metastatic prostate and pancreatic tumors. This example shows that PSCA monoclonal antibodies not linked to any substance can effectively inhibit the growth of human prostate tumor xenografts in SCID mice; correspondingly, combinations of such monoclonal antibodies are also effective. the

Tumor suppression with multiple PSCA MAbs

Materials and methods

PSCA monoclonal antibody:

Anti-PSCA monoclonal antibody can be prepared according to the method described in the example "Preparation of PSCA monoclonal antibody (MAb)". The ability of antibodies to bind PSCA was determined by ELISA, Western blot, FACS and immunoprecipitation techniques. Epitope mapping data for anti-PSCA MAb obtained by ELISA and Western blot assays indicated that the antibody recognizes epitopes on the PSCA protein. Immunohistochemical analysis of prostate tumor tissues and cells using these antibodies. the

Monoclonal antibodies were purified from ascites or hybridoma tissue culture supernatants using Protein-G or Protein-A Sepharose chromatography columns, dialyzed with PBS, and stored at -20°C after filter sterilization. Protein content was determined using the Bradford assay (Bio-Rad, Hercules, CA). Preparation of therapeutic monoclonal antibodies or mixtures comprising different monoclonal antibodies for treatment of mice injected subcutaneously or orthotopically with LAPC9 AD and HPAC tumor xenografts. the

Cell Lines and Xenografts

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

PC3-PSCA and 3T3-PSCA cell populations were generated by retroviral gene transformation as described in Hubert et al., Proc Natl Acad Sci USA, 1996, 96(25):14523. the

LAPC-9 xenografts expressing wild-type androgen receptor and producing prostate-specific antigen (PSA) were implanted via subcutaneous trocar into 6-8 week-old male ICR-severe combined immunodeficiency (SCID) pups. In vivo in mice (Taconic Farms) (Craft, N. et al., 1999, 5:280). Single cell suspensions of LAPC-9 tumor cells were prepared as described by Craft et al. the

Xenograft mouse model

Subcutaneous (sc) tumors were prepared by subcutaneously injecting 1×10 ⁶ tumor cells mixed with Matrigel (Collaborative Research) at a ratio of 1:1 in the right abdomen of male SCID mice. In order to test the effect of the antibody on tumor formation, the antibody injection was started on the same day as the tumor cell injection. Control mice were injected with purified murine IgG (ICN) or PBS; or purified monoclonal antibodies that recognized unrelated antigens not expressed by human cells. In the preliminary experiment, it was found that there was no significant difference in the effect of mouse IgG and PBS on tumor growth. The size of the tumor was measured using calipers, and the volume of the tumor was equal to length×width×height. Mice were sacrificed when subcutaneous tumors exceeded 1.5 cm in diameter.

Orthotopic injections were performed after anesthesia with ketamine/xylazine. When performing prostate orthotopic transplantation experiments, the abdomen of mice was cut open to expose the prostate. Matrigel-mixed LAPC or PC3 tumor cells (2×10 ⁶ ) were injected into the prostatic capsule with an injection volume of 10 μl. To monitor tumor growth, mice were palpated and blood samples were collected weekly to measure PSA expression levels.

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

The effect of anti-PSCA MAbs on tumor formation was evaluated using HPAC and LAPC9 orthotopic models. Compared with the subcutaneous tumor model, the orthotopic tumor transplantation model requires direct injection of tumor cells into the mouse pancreas or prostate, respectively, resulting in local tumor formation, tumor metastasis to distant sites, deterioration of the health status of the mice, and small The mice subsequently died (Saffran, D. et al., PNAS, supra). These features make the orthotopic model more representative of human disease progression and allow us to judge the therapeutic effect of mAbs based on clinically relevant indicators. the

Correspondingly, tumor cells were injected into the mouse prostate, and after 2 days, the mice were divided into 2 groups and treated with a) 250-1000 μg of anti-PSCA Ab; or b) control antibody, 3 times a week for 2-5 weeks. the

A major advantage of orthotopic tumor models is the ability to study the development of tumor metastasis. The formation of metastatic tumors in mice with orthotopic tumors can be studied by IHC analysis using antibodies against tumor-specific cell surface proteins such as anti-CK20 antibodies for prostate cancer (Lin S et al., Cancer Detect Prev. 2001; 25: 202) . the

Another advantage of orthotopic tumor models is the ability to study neovascularization and angiogenesis. Tumor growth depends in part on the formation of new blood vessels. Although the capillary system and resulting vascular network are of host origin, xenograft tumors can also modulate the initiation and structure of neovascularization (Davidoff et al., Clin Cancer Res. 2001;7:2870; Solesvik O et al., Eur.J Cancer Clin Oncol. 1984, 20:1295). The effects of antibodies and small molecules on neovascularization can be studied using methods well known in the art, such as by IHC analysis of tumor tissue and its surrounding microenvironment. the

Mice with orthotopic tumors were injected with anti-PSCA MAb or control antibody within 4 weeks. Both groups of mice were loaded with a high tumor burden to ensure the formation of metastases in the lungs of the mice. Mice were then sacrificed and analyzed by IHC for the presence of tumor cells in their bladders, livers, bones and lungs. The results of these experiments demonstrate that anti-PSCA antibodies have potent activity against prostate cancer development and progression in a murine xenograft model. Anti-PSCA antibodies inhibited tumor formation, prevented the growth of existing tumors, and prolonged the survival of treated mice. Moreover, anti-PSCA MAbs had a strong inhibitory effect on metastasis of localized prostate tumors to distant sites, even in the presence of high tumor burden. Therefore, anti-PSCA MAbs help to improve the main clinical indicator (tumor growth), prolong survival, and improve the health status of patients. the

Effect of PSCA Mab on the Growth of Human Prostate Cancer in Mice

Using the method described above, LAPC-9AI tumor cells (2.0×10 ⁶ cells) were subcutaneously injected into male SCID mice. Mice were randomized into groups (n=10 in each group) and treatment was initiated on day 0 with HA1-4.120 or isotype Mab control intraperitoneally (ip) as indicated. Animals were treated twice a week for a total of 7 doses until day 28 of the study. Tumor growth was monitored by caliper measurements every 3-4 days as indicated. The results showed that human anti-PSCA monoclonal antibody Ha1-4.120 significantly inhibited the growth of human prostate cancer xenografts implanted subcutaneously in SCID mice (p<0.05) ( FIG. 18 ).

In another experiment, LAPC-9AI tumor cells (2.0 x ¹⁰⁶ cells) were injected subcutaneously into male SCID mice. When tumor volumes reached 50 ^mm3 , mice were randomized into groups (n=10 in each group) and the indicated HA1-5.99.1 or isotype Mab control initial treatment was given intraperitoneally (ip) as indicated. Animals were treated twice a week for a total of 5 doses until day 14 of the study. Tumor growth was monitored by caliper measurements every 3-4 days as indicated. The results showed that fully human anti-PSCA monoclonal antibody Ha1-5.99 significantly inhibited the growth of established androgen-dependent human prostate cancer xenografts implanted subcutaneously in SCID mice (p<0.05) (Figure 19).

In another experiment, LAPC-9AD tumor cells (2.5 x ¹⁰⁶ cells) were injected subcutaneously into male SCID mice. When tumor volumes reached 40 ^mm3 , mice were randomized (n=10 in each group) and treatment was initiated with intraperitoneal (ip) injections of increasing concentrations of HA1-4.121 or isotype MAb controls as indicated. Animals were treated twice a week for a total of 7 doses until day 21 of the study. Tumor growth was measured with calipers every 3-4 days as indicated. The results of the present study show that HA1-4.121 inhibits the growth of established human androgen-dependent prostate cancer xenografts implanted subcutaneously in SCID mice. The results were statistically significant on days 14, 17 and 21 (p<0.05, Kruskal-Wallis test, two-tailed α=0.05) of the 300 μg dose group and 10, 14, 17 and 21 days of the 700 μg dose group ( p<0.05, Kruskal-Wallis test, two-tailed α=0.05) (Figure 20).

In another experiment, patient-derived androgen-dependent LAPC-9AD tumor cells (2.0 x ¹⁰⁶ cells) were injected into the dorsal lobe of the prostate of male SCID mice. Tumors were allowed to grow for approximately 10 days and mice were randomized into groups. Treatment with 500 mg of human HA1-4.117, HA1-4.121 or isotype control was initiated 10 days after tumor implantation. Antibody was delivered intraperitoneally twice a week for a total of 7 doses. Four days after the last dose, animals were sacrificed and primary tumors were removed and weighed. The results showed that human anti-PSCA monoclonal antibodies Ha1-4.121 (p<0.01) and Ha1-4.117 (p<0.05) significantly inhibited the growth of LAPC-9AD prostate cancer xenografts transplanted into SCID mice (Figure 21) .

In another experiment, androgen-dependent LAPC-9AD tumor cells (2.0 x ¹⁰⁶ cells) obtained from a patient were injected into the dorsal flap of the prostate of male SCID mice. Tumors were allowed to grow for approximately 9 days and mice were randomized into groups. Animals randomized into the survival group included 11 mice in the isotype MAb control group and 12 mice in the HA1-4.121 treated group. Animals were treated ip with 1000 μg Ha1-4.121 or 1000 μg isotype MAb control twice weekly for a total of 9 doses. The results showed that HA1-4.121 significantly (log-graded test: p<0.01) prolonged the survival time of SCID mice bearing human androgen-dependent prostate tumors. 110 days after the last treatment, 2 mice in the HA1-4.121 treated group remained free of palpable tumors (Figure 22).

The effect of the combination of PSCA Mabs and taxotere in mice

In another experiment, LAPC-9AI tumor cells (2 x ¹⁰⁶ cells/animal) were injected subcutaneously into male SCID mice. When the tumor volume reached 65 ^mm3 , animals were randomized into 4 different groups (n=10 in each group) as indicated. Ha1-4.121 or the isotype MAb control were administered twice weekly at a dose of 500 μg starting on day 0 for a total of 6 administrations. The last dose was given on day 17. Taxotere was administered intravenously at a dose of 5 mg/kg on days 0, 3 and 7. Tumor growth was measured with calipers every 3-4 days. The results of this study showed that Ha1-4.121 as a single agent inhibited the growth of androgen-independent prostate cancer xenografts in SCID mice by 45% compared with control antibody alone at day 28 (ANOVA/Tukey Test: p<0.05). Administration of the isotype MAb control plus taxotere inhibited tumor growth by 28%, which was not statistically significant, compared to control antibody treatment alone. The combined administration of HA1-4.121 and taxotere had a potentiated effect and resulted in a 69% inhibition of tumor growth compared to the control antibody alone (ANOVA/Tukey test: p<0.01). When the combination group of HA1-4.121 and taxotere was compared with the HA1-4.121 or isotype MAb control plus taxotere group, both showed statistically significant differences (ANOVA/Tukey test: p < 0.05) (Figure 23).

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

In another experiment, human HPAC pancreatic cancer cells (2 x ¹⁰⁶ /mouse) were injected subcutaneously into immunodeficient ICR SCID mice (Taconic Farm, Germantown, NY). Mice were randomized into groups (n=10 animals/group) and treatment with indicated human PSCA monoclonal antibodies was initiated on the same day. Antibody (500 mg/mouse) was delivered intraperitoneally twice a week for a total of 8 doses. The results showed that human anti-PSCA monoclonal antibodies Ha1-4.121, Ha1-4.117 and Ha1-1.16 significantly inhibited the growth of subcutaneously implanted human pancreatic cancer xenografts in SCID mice. Statistical analysis was performed using t-test (two-tailed, a=0.05) (Figure 24).

In another experiment, HPAC cells (3.0 x ¹⁰⁶ cells) were ordinarily implanted into the pancreas of SCID mice. Mice were randomly assigned into 3 groups (n=9 for each group) as indicated. Treatment with HA1-4.121 (250 μg or 1000 μg) or isotype MAb control (1000 μg) was initiated on the day of transplantation. Antibodies were administered intraperitoneally twice a week for a total of 10 administrations. Thirteen days after the last dose, animals were sacrificed and primary tumors were removed and weighed. The results of the present study showed that both dose levels of HA1-4.121 tested significantly inhibited the orthotopic growth of human pancreatic cancer xenografts in SCID mice. 250 μg and 1000 μg AGS-PSCA inhibited tumor growth by 66% and 70%, respectively (Kruskal-Wallis/Tukey test: p<0.01 and p<0.01, respectively) ( FIG. 25 ).

At autopsy, visible metastases to lymph nodes and distant organs were observed in the control antibody-treated group. No visible metastases were observed in either HA1-4.121-treated group. Lymph nodes, lungs and livers were removed from all animals and examined histologically for the presence of transitional tumors. Sections from the lungs and lymph nodes of each animal were stained with human cytokeratin, and the number of metastases was determined microscopically. Results of histological analysis showed a significant reduction in lymph node (LN) metastases in animals treated with HA1-4.121 (p=0.0152, as measured by Fishers exact test). The incidence of metastasis and invasion was also significantly reduced in animals treated with both concentrations of HA1-4.121 (p=0.0152, as measured by Fishers exact test). The number of lung metastases was significantly reduced in mice treated with only 1.0 mg dose of HA1-4.121 (p=0.0498, as measured by Fishers exact test) (Figure 26). the

Effect of human PSCA MAb on bladder cancer growth in mice

In another experiment, human SW780 bladder cancer cells (2 x ¹⁰⁶ /mouse) were injected subcutaneously into immunodeficient ICR SCID mice (Taconic Farm, Germantown, NY). Mice were randomized into groups (n=10 animals/group) and treatment with the indicated human PSCA MAbs was initiated on the same day. Antibody (250 mg/mouse) was delivered intraperitoneally twice a week for a total of 7 doses. The results showed that HA1-4.117 (p=0.014), HA1-4.37 (p=0.0056), HA1-1.78 (p=0.001), Ha1-5.99 (p=0.0002) and HA1-4.5 (p=0.0008) significantly inhibited SCID Growth of subcutaneously implanted SW780 bladder tumors in mice. Statistical analysis was performed by t-test (two-tailed, α=0.05) (Fig. 27).

The results of these trials show that PSCA Mab can be used for therapeutic and diagnostic purposes in the treatment and management of the cancers listed in Table I. the

Example 27

Human therapy and diagnosis using anti-PSCA antibodies

Anti-PSCA monoclonal antibodies are safe and effective for diagnostic, prophylactic, prognostic and/or therapeutic purposes in humans. Analysis of cancerous tissues and tumor xenografts by Western blot and immunohistochemistry with anti-PSCA Mab showed strong widespread staining in carcinomas with significantly reduced or undetectable levels in normal tissues. Detection of PSCA in cancer and in metastatic disease suggests the use of this Mab as a diagnostic and/or prognostic indicator. Anti-PSCA antibodies are therefore useful for diagnostic purposes, such as immunohistochemical analysis of kidney biopsy samples to detect cancer in suspected patients. the

As detected by flow cytometry, it was found that the anti-PSCA Mab specifically binds to cancer cells. Anti-PSCA antibodies are therefore useful in diagnostic applications of whole-body imaging, such as radioimmunoscintigraphy and radioimmunotherapy (see, e.g., Potamianos S. et al., Anticancer Res 20(2A):925-948 (2000)), to detect expression of PSCA. localized and metastatic cancers. The extracellular domain of PSCA that is shed or released into the extracellular environment, such as basic phosphodiester B10 (Meerson, N. R., Hepatology 27:563-568 (1998)), allows the use of serum obtained from suspected patients and/or Anti-PSCA antibodies in urine samples for the diagnosis of PSCA become possible. the

Anti-PSCA antibodies that specifically bind PSCA are useful in the treatment of PSCA-expressing cancers. The anti-PSCA antibodies are used in unconjugated molecule form and in conjugated forms in which the antibody is conjugated to a variety of therapeutic or imaging molecules (e.g., prodrugs, enzymes, or radioisotopes) well known in the art. In preclinical studies, the effect of non-conjugated and conjugated anti-PSCA antibodies on tumor prevention and growth inhibition in SCID mouse cancer xenograft models (e.g. kidney cancer models AGS-K3 and AGS-K6) was tested (see e.g., title is an example of "PSCA monoclonal antibody-mediated tumor suppression in vivo"). Both conjugated and nonconjugated anti-PSCA antibodies can be used as treatment modalities in human clinical trials, either alone or in combination with other therapies as described in the Examples below. the

Example 28

Human Clinical Study Using Human Anti-PSCA Antibody In Vivo for Therapy and Diagnosis of Human Tumors

Antibodies of the invention recognize epitopes on PSCA and are useful in the treatment of certain tumors, such as those listed in Table I. Tumors such as those listed in Table I are currently the preferred indications considering a number of factors, including the expression level of PSCA. In connection with these indications, we have successfully implemented the following three clinical programs. the

I.) Adjuvant therapy: As an adjuvant therapy, anti-PSCA antibodies are used in combination with chemotherapy drugs or antineoplastic drugs and/or radiation therapy to treat patients. Primary tumor targets, such as those listed in Table I, were treated with standard therapy, in addition to standard first-line and second-line therapy, with anti-PSCA antibodies. The efficacy of the treatment regimen was judged by the reduction of tumor mass and the ability to reduce the dose of standard chemotherapy drugs used. Reductions in doses of standard chemotherapeutics lead to a reduction in dose-related toxicity of chemotherapeutics, thus enabling the introduction of additional and/or longer durations of treatment. Anti-PSCA antibodies are available in several adjuvant clinical studies in combination with chemotherapy or antineoplastic agents such as doxorubicin (advanced prostate cancer), cisplatin (advanced head and neck and lung cancer), paclitaxel (breast cancer), and doxorubicin Bixing (preclinical research). the

II.) Monotherapy: Using anti-PSCA antibody alone to treat tumors refers to giving patients only antibodies without chemotherapeutics or anti-tumor drugs. In one embodiment, a single therapeutic approach is used to treat patients with advanced tumors and severe metastases. The patient's condition appears to be somewhat stable. Experiments have shown that the antibody also has a certain effect on patients with stubborn tumors. the

III.) Contrast agents: Radiolabeled antibodies prepared by linking radionuclides such as iodine or yttrium (I ¹³¹ , y ⁹⁰ ) to anti-PSCA antibodies can be used as diagnostic reagents and/or contrast agents. Use For this purpose, the labeled antibody is localized to solid tumors and PSCA-expressing metastases. When anti-PSCA antibody is used as a contrast agent, the antibody can be used as an add-on to solid tumor surgical treatment for pre-operative scans and post-operative scans To determine whether the tumor is cleared and/or relapsed. In one embodiment, the ( ¹¹¹ In)-PSCA antibody is used as a contrast agent in Phase I human clinical trials to screen patients with tumors expressing PSCA (similar examples see Divgi et al. J.Natl.Cancer Inst.83:97-104 (1991)). The patient then undergoes standard anterior and posterior gamma-ray angiography. From the results of the angiography, the location of the primary lesion and metastases can be identified.

Dosage and route of administration

As is well known to those skilled in the art, the dosage can be determined by comparing with other similar drugs used in clinic. Therefore, the dose of anti-PSCA antibody administered ranges from 5 to 400 mg/m ² , and lower doses may be considered based on the results of safety studies. The relative affinity of an anti-PSCA compared to the affinity of a known antibody for its target is one parameter used by those skilled in the art in determining similar dosing regimens. In addition, anti-PSCA antibodies are fully human antibodies, and their clearance rate is relatively low compared with chimeric antibodies; therefore, the dose required for patients using this antibody should be correspondingly reduced, perhaps in the range of 50 to 300mg/ ^m2 between is still valid. The dosage unit is mg/ ^m2 , rather than the conventional dosage unit mg/kg, and administration is based on surface area, which facilitates the determination of the required dosage for people of all sizes from infants to adults.

Three different methods of administration are available for the administration of anti-PSCA antibodies. Conventional intravenous administration is the standard method of administration for most tumors. However, if the tumor is located in the peritoneal cavity, such as tumors in the ovary, tumors in the bile duct, and tumors in other cavities, intraperitoneal injection is considered to be better, so that a higher antibody dose can be achieved at the tumor site, and at the same time It also minimizes antibody clearance. Certain solid tumors contain blood vessels, so administration by local infusion is more appropriate. Local perfusion concentrates high doses of antibody to the tumor site while minimizing short-term clearance of the antibody. the

Clinical Development Program (CDP)

Overview: CDP utilizes and develops the clinical potential of anti-PSCA antibodies in relation to adjuvant therapy, monotherapy and as a contrast agent. Prove its safety through clinical studies first, and then prove its effectiveness with repeated dosing. The clinical trial was open-label and compared standard chemotherapy with standard treatment plus an anti-PSCA antibody. It should be noted that one of the inclusion criteria related to the screening of subjects was the level of expression of PSCA in their tumors by biopsy. the

As with any protein or antibody infusion therapy, safety considerations are primarily related to: (i) cytokine release syndrome, such as hypotension, fever, tremors, chills; (ii) immunogenicity of the drug The development of a response (eg, an antibody produced by the patient against the therapeutic antibody, or a HAHA response); and (iii) toxicity to normal cells expressing PSCA. Standard testing and follow-up should be performed to monitor these safety indicators. Tests have shown that anti-PSCA antibodies are safe when used in humans. the

Example 29

Human Clinical Trials: Monotherapy Using Human Anti-PSCA Antibodies

Anti-PSCA antibodies are safe in the adjuvant clinical trials mentioned above, and phase II human clinical trials have also confirmed the efficacy and determined the optimal dosage for monotherapy. After this clinical trial, it was found that its safety and clinical efficacy were the same as the results of the above-mentioned adjuvant therapy clinical trial, except that patients did not use chemotherapy drugs while receiving anti-PSCA antibodies. the

Example 30

Human Clinical Trials: Diagnostic Imaging Using Human Anti-PSCA Antibodies

As mentioned above, adjuvant therapy with antibodies is safe according to the safety criteria mentioned above, so we conducted another clinical trial using anti-PSCA antibody as a diagnostic contrast agent. The assay protocols were substantially similar to those described in the art, eg, Divgi et al., J. Natl. Cancer Inst. 83:97-104 (1991). The antibody was found to be safe and effective as a diagnostic reagent. the

Example 31

With human anti-PSCA antibody and chemotherapy, radiotherapy and/or hormone ablation therapy

Human clinical trials for adjuvant therapeutic measures

First, a phase I clinical trial of 6 intravenous doses of human anti-PSCA antibody was conducted to evaluate its safety related to the treatment of solid tumors, which refer to the tumors listed in Table I. In this study, a phase I clinical trial was conducted to evaluate the safety of a single dose of anti-PSCA antibody as an adjuvant therapy of antineoplastic drugs or chemotherapy drugs or hormone removal, as described but not limited to cisplatin, doxorubicin, Bixing, doxorubicin, paclitaxel, leuprolide, Norad, flutamide, casodex, nilutamide, etc. The test protocol includes about 6 single doses of anti-PSCA antibody, and 6 doses are gradually increased from about 25 mg/m ² to about 275 mg/m ² . The arrangement of the test process is as follows or can be similar to the following arrangement:

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

MAb dosage 25mg/m ² 75mg/m ² 125mg/m ² 175mg/m ² 225mg/m ² 275mg/m ²

Chemotherapy drugs

+ + + + + + + + + +

(standard dose)

Monitor patients closely for 1 week after each dose of antibody therapy and chemotherapy. Special attention should be paid to the evaluation of the above-mentioned safety indicators: (i) cytokine release syndrome, such as hypotension, fever, trembling, chills; antibody, or HAHA response); and (iii) toxicity to normal cells expressing PSCA. Standard testing and follow-up should be performed to monitor these safety indicators. Also evaluate the patient's clinical presentation, especially the reduction of the tumor mass observed by MRI or other contrast techniques. the

Experiments have proved that anti-PSCA antibody is safe and effective, and the purpose of phase II clinical trials is to further confirm its effectiveness and determine the optimal therapeutic dose. the

Example 32

RNA interference (RNAi)

RNA interference (RNAi) technology is used in various cellular analyzes related to tumors. RNAi is a post-transcriptional gene silencing mechanism triggered by double-stranded RNA (dsRNA). RNAi-induced specific mRNA degradation leads to changes in protein expression and subsequent gene function. In mammalian cells, these dsRNAs, known as short interfering RNAs (siRNAs), have the correct structure to activate RNAi pathways targeted for degradation, especially certain mRNAs. See "Duplexes of 21-nucleotide RNAs Mediate RNA interference in Cultured Mammalian Cells" by Elbashir S.M. et al., Nature 411(6836):494-8 (2001). Therefore, RNAi technology can be successfully applied to mammalian cells to silence target genes. the

Uncontrolled cell proliferation is a hallmark of cancerous cells; therefore, assessment of PSCA in cell survival/proliferation assays is relevant. Therefore, RNAi was used for the function of PSCA antigen. To generate siRNA against PSCA, an algorithm was employed that predicts oligonucleotides with key molecular parameters (G:C content, melting point stability, etc.) that are capable of significantly reducing PSCA protein expression levels when introduced into cells. According to this example, a PSCA siRNA composition comprising siRNA (double-stranded short interfering RNA) corresponding to the PSCA protein nucleic acid ORF sequence or a subsequence thereof was used. Thus, siRNA subsequences used in this manner are typically 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 contiguous RNA nucleotides. These siRNA sequences are complementary and non-complementary to at least part of the mRNA coding sequence. In a preferred embodiment, the subsequence is 19-25 nucleotides in length, most preferably 21-23 nucleotides in length. In a preferred embodiment, these siRNAs have the effect of knocking out PSCA antigen in cells expressing PSCA protein, and have the effects as described below. the

Selected siRNAs (PSCA.b oligos) were tested in a variety of cell lines in a survival/proliferation MTS assay (measures cellular metabolic activity). The colorimetric assay based on tetrazolium salts (i.e., MTS) can exclusively detect viable cells because living cells are metabolically active and thereby reduce the tetrazolium salts to colored nail compounds; dead cells You can't. In addition, according to the following experimental design, the PSCA.b oligomer has the effect of knocking out PSCA antigen in cells expressing PSCA protein and has the following efficacy. the

Mammalian siRNA transfection: The day before siRNA transfection, 2×10 ³ cells/well of different cell lines were seeded in 80 μl (96-well plate format) in medium (containing 10% FBS w/o antibiotics) RPMI1640), for survival/MTS testing. In parallel with PSCA-specific siRNA oligos, the following sequences were included as controls in each experiment: a) mock-transfected cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and annealing buffer (without siRNA); b) Luciferase-4-specific siRNA (targeted sequence: 5'-AAGGGACGAAGACGAACACUUCTT-3') (SEQ ID NO: 77); and c) Eg5-specific siRNA (targeted sequence: 5'- AACTGAAGACCTGAAGACAATAA-3') (SEQ ID NO: 78). SiRNA was used at a final concentration of 10 nM and 1 μg/ml Lipofectamine 2000.

The assay design was as follows: firstly, siRNAs were diluted to 0.1 uM μM (10-fold concentrated) in OPTIMEM (serum-free transfection medium, Invitrogen) and incubated at room temperature for 5-10 minutes. Lipofectamine2000 was diluted to 10 μg/ml (10-fold concentrated) for all transfections and incubated at room temperature (RT) for 5-10 minutes. Mix an appropriate amount of diluted 10-fold concentrated Lipofectamine 2000 with diluted 10-fold concentrated siRNA at a ratio of 1:1, and incubate at room temperature for 20-30 minutes (5-fold concentrated transfection solution). 20 μl of 5-fold concentrated transfection solution was added to each sample and assayed after incubation at 37° C. for 96 hours. the

MTS test: The MTS test is a colorimetric method for determining the number of surviving cells in a proliferation, cytotoxicity or chemosensitivity test based on the tetrazolium salt compound [3-(4,5-dimethylthiazole- 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS(b)] and electron coupler (sulfoethyl Base (ethosulfate) phenazine; PES). The test is performed as follows: add a small amount of solution reagent directly into the culture well, incubate for 1-4 hours, and then record the absorbance at 490 nm with a 96-well plate reader. The amount of colored formazan product measured by the amount of absorbance at 490 nm is directly proportional to mitochondrial activity and/or the number of viable cells in culture. the

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

Another embodiment of the present invention is a method of analyzing cell proliferation associated with PSCA by measuring DNA synthesis as a marker of proliferation. Labeled DNA precursors (ie ³ H-thymidine) were used and their incorporation into DNA was quantified. The amount of labeled precursor incorporated into the DNA is directly proportional to the amount of cell division present in the cell culture. Another way to measure cell proliferation is to perform a clonogen test. In these tests, a defined number of cells are plated on a suitable substrate and the number of colonies formed over a period of growth following siRNA treatment is counted.

Complementary cell survival/proliferation assays with apoptosis and cell cycle distribution studies are considered in PSCA cancer target validation. A biochemical hallmark of the apoptotic process is genomic DNA fragmentation, an irreversible event that leads to cell death. One way to visualize fragmented DNA in cells is by immunoassays for the detection of histone-complexed DNA fragments (i.e., cell death detection ELISA), which measure the concentration of histone-complexed DNA fragments in the cytoplasm of apoptotic cells (mononucleosomes and oligonucleosomes). The assay does not require pre-labeling of cells and enables the detection of DNA degradation in cells that do not proliferate in vitro (ie, freshly isolated tumor cells). the

The most important effector molecules that trigger apoptotic cell death are caspases. Caspases are proteases that, once activated, cleave at the carboxyl site of aspartic acid residues, which, once activated, mediate the very early stages of apoptosis. All caspases are synthesized as precursor enzymes, the activation of which involves cleavage of aspartic acid residues. Specifically, caspase 3 appears to play an important role in the initiation of apoptotic cellular events. Early events of apoptosis can be detected by measuring caspase 3 activity. Active induction of apoptosis was further supported by detection of the presence of active caspase 3 or proteolytic cleavage product (i.e., PARP) in apoptotic cells by protein blot assay after RNAi treatment. Since the cellular mechanisms leading to apoptosis are complex, each approach has its advantages and limitations. Examination of other criteria/endpoints such as cell morphology, chromatin condensation, membrane blebbing, apoptotic bodies helps further support that cell death is apoptotic. Since not all gene targets regulating cell growth are anti-apoptotic, DNA content of permeabilized cells was measured to obtain DNA content profile or cell cycle profile. The nuclei of apoptotic cells contain less DNA (sub-G1 population) due to leakage to the cytoplasm. In addition, due to the different amounts of DNA present in G0/G1, S and G2/M, DNA staining (ie, propidium iodide) can also be used to distinguish different cell cycle phases in a cell population. In these studies, subpopulations can be quantified. the

For the PSCA gene, RNAi studies have advanced understanding of the role of this gene product in cancer pathways. These active RNAi molecules can be used in identification assays to screen for MAbs with anti-tumor therapeutic activity. In addition, siRNAs can be administered to cancer patients as therapeutic agents to reduce malignant growth in various cancer types, including those listed in Table I. When PSCA plays a role in cell survival, cell proliferation, tumorigenesis or apoptosis, it can be used as a target for diagnostic, prognostic, preventive and/or therapeutic purposes. the

Example 33

Detection of PSCA protein in cancer patient samples by IHC

The expression of PSCA protein in tumor samples obtained from cancer patients was detected using antibody HA1-4.117. Formaldehyde-fixed, paraffin-embedded tissues were sectioned into 4 micron thick sections and mounted on glass slides. Sections were dewaxed, dehydrated, and treated with Antigen Retrieval Citra Solution (BioGenex, 4600 Norris Canyon Road, San Ramon, CA, 94583) at high temperature. Sections were then incubated in fluorescein-conjugated human monoclonal anti-PSCA antibody Ha1-4.117 for 16 hours at 4°C. The slides were washed 3 times in buffer, incubated with rabbit anti-fluorescein for 1 hour, and after washing in buffer, immersed in DAKO EnVision+ ^TM peroxidase-conjugated goat anti-rabbit immunoglobulin secondary antibody (DAKO Corporation, Carpenteria, CA) for 30 minutes. Sections were then washed in buffer, developed with a DAB kit (SIGMA Chemicals), counterstained with hematoxylin, and analyzed by brightfield microscopy. The results showed that PSCA was expressed in prostate cancer tumor cells (A, B), bladder metastatic cancer tumor cells (C) and pancreatic ductal carcinoma tumor cells (D). These results indicate that PSCA is expressed in human cancers and that antibodies against this antigen can be used as diagnostic reagents (Figure 17).

These results suggest that PSCA is a target for diagnostic, prognostic and therapeutic uses in cancer. the

Various web site materials, published documents, patent applications, and patents are referenced throughout this application (sites cited are identified by their resource uniform locators, or URLs, whose addresses are on the World Wide Web). The disclosures of these references are hereby incorporated by reference in their entirety. the

The present invention is not limited within the scope specified by the embodiments described herein, these embodiments are only for illustrating various aspects of the present invention, and any modified versions with functional equivalents are included in the scope of the present invention. In addition to what is described herein, those skilled in the art should be able to understand various modifications to the models and methods of the present invention based on the above descriptions and illustrations, and these modifications also fall within the scope of the present invention. Such modifications or other embodiments can be made without departing from the true scope and spirit of the invention. the

sheet

Table I: Organs expressing PSCA when malignant

Prostate

pancreas

bladder

kidney

colon

lung

ovaries

breast

Table II: Amino Acid Abbreviations

single letter three letters full name the the the F Phe Phenylalanine L Leu Leucine S Ser Serine Y Tyr Tyrosine C Cys cysteine W Trp Tryptophan P Pro Proline h His Histidine Q Gln Glutamine R Arg Arginine I Ile Isoleucine m Met Methionine T Thr threonine N Asn Asparagine K Lys Lysine V Val Valine A Ala Alanine D Asp Aspartic Acid E Glu glutamic acid G Gly Glycine

Table III : Amino Acid Substitution Matrix

Adopted from GCG software 9.0BLOSUM62 amino acid substitution matrix (closed substitution matrix). Larger numbers are more likely to be substituted in the related native protein. (see World Wide Web URLikp.unibe.ch/manual/blosum62.html)

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

4 0 -2 -1 -2 0 -2 -1 -1 -1 -1 -2 -1 -1 -1 1 0 0 -3 -2 A

9 -3 -4 -2 -3 -3 -1 -3 -1 -1 -3 -3 -3 -3 -1 -1 -1 -2 -2 C

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

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

6 -3 -1 0 -3 0 0 -3 -4 -3 -3 -2 -2 -1 1 3 F

6 -2 -4 -2 -4 -3 0 -2 -2 -2 0 -2 -3 -2 -3 G

8 -3 -1 -3 -2 1 -2 0 0 -1 -2 -3 -2 2 H

                                                                         

5 -2 -1 0 -1 1 2 0 -1 -2 -3 -2 K

                                                                        

5 -2 -2 0 -1 -1 -1 1 -1 -1 M

6 -2 0 0 1 0 -3 -4 -2 N

7 -1 -2 -1 -1 -2 -4 -3 P

5 1 0 -1 -2 -2 -1 Q

5 -1 -1 -3 -3 -2 R

4 1 -2 -3 -2 S

5 0 -2 -2 T

4 -3 -1 V

                                                         

7 Y

Table IV:

HLA class I/II motif/supermotif

Table IV(A): HLA class I supermotifs/motifs

supermotif Location Location Location the 2 (primary anchor position) 3 (primary anchor position) C-terminus (primary anchor position) A1 TILVMS the FWY A2 LIVMATQ the IVMATL A3 VSMATLI the RK A24 YFWIVLMT the FIYWLM B7 P the VILFMWYA B27 RHK the FYLWMIVA B44 ED the FWYLIMVA B58 ATS the FWYLIVMA B62 QLIVMP the FWYMIVLA the the the the Motif the the the A1 TSM the Y A1 the DEAS Y A2.1 LMVQIAT the VLIMAT A3 LMVISATFCGD the KYRHFA A11 VTMLISAGNCDF the KRYH A24 wxya the FLIW A*3101 MV TALIS the RK A*3301 MVALFIST the RK A*6801 AVTM SLI the RK B*0702 P the LMFWYAIV B*3501 P the LMFWYIVA B51 P the LIVFWYAM B*5301 P the IMFWYALV B*5401 P the ATIVLMFWY

Residues in bold are preferred, residues in italics are less preferred: A peptide is considered to have a motif if it has a major anchor at every major anchor position in the motif or supermotif shown in the table above . the

Table IV(B): HLA class II supermotifs

1 6 9 the the the W, F, Y, V, .I, L A, V, I, L, P, C, S, T A, V, I, L, C, S, T, M, Y

Table IV (C): HLA class II motifs

Residues in italics are less preferred or "marginally available" residues

Table IV(D): HLA class I supermotifs

Residues in italics are less preferred or "marginally available" residues

Table IV(E): HLA class I motifs

Table IV(F):

HLA-supertype summary

Total phenotypic frequencies of HLA-supertypes in different ethnic populations

Table IV(G):

B44 and A1

A2, A3, B7, A24,

B44, A1, B27,

B62, and B58

Each motif represents the residues that determine supertype specificity. Each motif incorporates residues identified from published data that are recognized by multiple alleles within the supertype. Residues in parentheses are also additional residues predicted to be tolerated by multiple alleles within the supertype. the

Table VI: Exon boundaries of transcript PSCA v.1

number of exons start terminate length 1 10 69 60 2 70 177 108 3 178 985 808

Table VII : MFI value for each data point of affinity calculation

MFI value

nM PSC A PSC A PDP3 T＝8 +4 PDP3 T＝8 +4 40 869.3 777.06 795.24 661.66 20 875.19 835.94 816.34 824.07 10 856.28 847.83 777.85 842.72 5 866.94 817.45 758.15 818.83 2.5 835.47 769.79 742.45 783.5 1.25 813.12 782.84 806.2 792.44 0.625 766.52 689.3 683.64 666.28 0.3125 588.3 541.25 549.03 499.22 0.15625 389.95 354.24 366.82 355.83 0.07813 234.85 230.37 225.82 211.77 0.03906 138.05 132.14 134.25 134.07 0.01953 84.35 78.49 77.62 80.14 0.00977 48.13 48.38 50.93 46.87 0.00488 33.49 30.58 30.71 30.04 0.00244 21.77 20.33 18.14 20.44 0.00122 14.45 13.59 13.82 13.11 0.00061 11.07 10.14 9.52 10.35 0.00031 8.61 8.3 8.57 9.09 0.00015 7.45 7.17 7.28 7.85 0.00008 6.91 6.73 7.32 7.86 0.00004 6.94 6.41 6.81 6.51

Table VIII : Affinities calculated using Graphpad Prism software: Sigmoid dose-response (variable slope) formula

Kd value

Table IX : FACS-based affinities of full-length human PSCA Mabs

FACS-based affinity

Sample ID Kd(nM) Ha1-4.37 0.23 Ha1-4.121 0.26 Ha1-5.99.1 0.28 Ha1-4.117 0.32 Ha1-4.120 0.41 Ha1-4.5 1.00 Ha1-1.16.1 6.91

Table X : Antibodies that cross-react with monkey PSCA and/or mouse PSCA.

Hybridoma ID Cross-reacts with monkey PSCA Cross-reacts with mouse PSCA

H1-1.10 - -

Ha1-1.16 + + -

Ha1-1.78 + + -

Ha1-1.41 + + -

Ha1-4.5 + + -

Ha1-4.37 + + -

Ha1-4.117 + +

Ha1-4.120 + + -

Ha1-4.121 + -

Ha1-5.99 + + -

Table XI : PSCA: Epitope panels analyzed by FACS

Legend:

sequence listing

<110> Akinsys Co., Ltd. (AGENSYS, INC.)

J. Gudas (Gudas, Jean)

A. Jakobovits (Aya)

X. Jia (Xiao-Chi, Jia)

R.K. Morrison (Morrison, Robert Kendall)

K.J.M. Morrison (Morrison, Karen Jane Meyrick)

H. Shao (Shao, Hui)

P.M. Challita-Eid, Pia M.

A.B. Raitano (Raitano, Arthur B.)

<120>Antibodies and related molecules that bind to PSCA protein

<130>51158-20088.41

<140> not granted

<141> Apply here

<150>60/616,381

<151>2004-10-05

<150>60/617,881

<151>2004-10-12

<150>60/621,310

<151>2004-10-21

<150>60/633,077

<151>2004-12-02

<150>10/857,484

<151>2004-05-28

<150>60/475,064

<151>2003-05-30

<160>78

<170>FastSEQ for Windows Version 4.0

<210>1

<211>990

<212>DNA

<213> Homo sapiens

<220>

<221> CDS

<222>(18)...(389)

<221>misc_feature

<222>543

<223>n=a, t, c or g

<400>1

agggaggc agtgacc atg aag gct gtg ctg ctt gcc ctg ttg atg gca 50

              Met Lys Ala Val Leu Leu Ala Leu Leu Met Ala

1 5 10

ggc ttg gcc ctg cag cca ggc act gcc ctg ctg tgc tac tcc tgc aaa 98

Gly Leu Ala Leu Gln Pro Gly Thr Ala Leu Leu Cys Tyr Ser Cys Lys

15 20 25

gcc cag gtg agc aac gag gac tgc ctg cag gtg gag aac tgc acc cag 146

Ala Gln Val Ser Asn Glu Asp Cys Leu Gln Val Glu Asn Cys Thr Gln

30 35 40

ctg ggg gag cag tgc tgg acc gcg cgc atc cgc gca gtt ggc ctc ctg 194

Leu Gly Glu Gln Cys Trp Thr Ala Arg Ile Arg Ala Val Gly Leu Leu

45 50 55

acc gtc atc agc aaa ggc tgc agc ttg aac tgc gtg gat gac tca cag 242

Thr Val Ile Ser Lys Gly Cys Ser Leu Asn Cys Val Asp Asp Ser Gln

60 65 70 75

gac tac tac gtg ggc aag aag aac atc acg tgc tgt gac acc gac ttg 290

Asp Tyr Tyr Val Gly Lys Lys Asn Ile Thr Cys Cys Asp Thr Asp Leu

80 85 90

tgc aac gcc agc ggg gcc cat gcc ctg cag ccg gct gcc gcc atc ctt 338

Cys Asn Ala Ser Gly Ala His Ala Leu Gln Pro Ala Ala Ala Ile Leu

95 100 105

gcg ctg ctc cct gca ctc ggc ctg ctg ctc tgg gga ccc ggc cag cta 386

Ala Leu Leu Pro Ala Leu Gly Leu Leu Leu Trp Gly Pro Gly Gln Leu

110 115 120

tag gctctggggg gccccgctgc agccacact gggtgtggtg ccccaggcct 439

*

ttgtgccact cctcacagaa cctggcccag tgggagcctg tcctggttcc tgaggcacat 499

cctaacgcaa gtttgaccat gtatgtttgc accccttttc cccnaaccct gaccttccca 559

tgggcctttt ccaggattcc cacccggcag atcagtttta gtgacacaga tccgcctgca 619

gatggcccct ccaacccttt ctgttgctgt ttccatggcc cagcattttc cacccttaac 679

cctgtgttca ggcacttctt cccccaggaa gccttccctg cccacccccat ttatgaattg 739

agccaggttt ggtccgtggt gtcccccgca cccagcaggg gacaggcaat caggagggcc 799

cagtaaaggc tgagatgaag tggactgagt agaactggag gacaagagtt gacgtgagtt 859

cctggggagtt tccagagatg gggcctggag gcctggagga aggggccagg cctcacattt 919

gtggggctcc cgaatggcag cctgagcaca gcgtaggccc ttaataaaca cctgttggat 979

aagccaaaaa a 990

<210>2

<211>123

<212>PRT

<213> Homo sapiens

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Met Lys Ala Val Leu Leu Ala Leu Leu Met Ala Gly Leu Ala Leu Gln

1 5 10 15

Pro Gly Thr Ala Leu Leu Cys Tyr Ser Cys Lys Ala Gln Val Ser Asn

20 25 30

Glu Asp Cys Leu Gln Val Glu Asn Cys Thr Gln Leu Gly Glu Gln Cys

35 40 45

Trp Thr Ala Arg Ile Arg Ala Val Gly Leu Leu Thr Val Ile Ser Lys

50 55 60

Gly Cys Ser Leu Asn Cys Val Asp Asp Ser Gln Asp Tyr Tyr Val Gly

65 70 75 80

Lys Lys Asn Ile Thr Cys Cys Asp Thr Asp Leu Cys Asn Ala Ser Gly

85 90 95

Ala His Ala Leu Gln Pro Ala Ala Ala Ile Leu Ala Leu Leu Pro Ala

100 105 110

Leu Gly Leu Leu Leu Trp Gly Pro Gly Gln Leu

115 120

<210>3

<211>1020

<212>DNA

<213> Homo sapiens

<220>

<221> CDS

<222>(56)...(427)

<400>3

tttgaggcca tataaagtca cctgaggccc tctccaccac agcccaccag tgacc atg 58

Met

1

aag gct gtg ctg ctt gcc ctg ttg atg gca ggc ttg gcc ctg cag cca 106

Lys Ala Val Leu Leu Ala Leu Leu Met Ala Gly Leu Ala Leu Gln Pro

5 10 15

ggc act gcc ctg ctg tgc tac tcc tgc aaa gcc cag gtg agc aac gag 154

Gly Thr Ala Leu Leu Cys Tyr Ser Cys Lys Ala Gln Val Ser Asn Glu

20 25 30

gac tgc ctg cag gtg gag aac tgc acc cag ctg ggg gag cag tgc tgg 202

Asp Cys Leu Gln Val Glu Asn Cys Thr Gln Leu Gly Glu Gln Cys Trp

35 40 45

acc gcg cgc atc cgc gca gtt ggc ctc ctg acc gtc atc agc aaa ggc 250

Thr Ala Arg Ile Arg Ala Val Gly Leu Leu Thr Val Ile Ser Lys Gly

50 55 60 65

tgc agc ttg aac tgc gtg gat gac tca cag gac tac tac gtg ggc aag 298

Cys Ser Leu Asn Cys Val Asp Asp Ser Gln Asp Tyr Tyr Val Gly Lys

70 75 80

aag aac atc acg tgc tgt gac acc gac ttg tgc aac gcc agc ggg gcc 346

Lys Asn Ile Thr Cys Cys Asp Thr Asp Leu Cys Asn Ala Ser Gly Ala

85 90 95

cat gcc ctg cag ccg gct gcc gcc atc ctt gcg ctg ctc cct gca ctc 394

His Ala Leu Gln Pro Ala Ala Ala Ile Leu Ala Leu Leu Pro Ala Leu

100 105 110

ggc ctg ctg ctc tgg gga ccc ggc cag cta tag gctctggggg gccccgctgc 447

Gly Leu Leu Leu Trp Gly Pro Gly Gln Leu *

115 120

agcccacact gggtgtggtg ccccaggcct ctgtgccact cctcacacac ccggcccagt 507

gggagcctgt cctggttcct gaggcacatc ctaacgcaag tctgaccatg tatgtctgcg 567

cccctgtccc ccaccctgac cctcccatgg ccctctccag gactcccacc cggcagatcg 627

gctctattga cacagatccg cctgcagatg gcccctccaa ccctctctgc tgctgtttcc 687

atggcccagc attctccacc cttaaccctg tgctcaggca cctcttcccc caggaagcct 747

tccctgccca ccccatctat gacttgagcc aggtctggtc cgtggtgtcc cccgcaccca 807

gcagggggaca ggcactcagg agggcccggt aaaggctgag atgaagtgga ctgagtagaa 867

ctggaggaca ggagtcgacg tgagttcctg ggagtctcca gagatggggc ctggaggcct 927

ggaggaaggg gccaggcctc acattcgtgg ggctccctga atggcagcct cagcacagcg 987

taggccctta ataaacacct gttggataag cca 1020

<210>4

<211>123

<212>PRT

<213> Homo sapiens

<400>4

Met Lys Ala Val Leu Leu Ala Leu Leu Met Ala Gly Leu Ala Leu Gln

1 5 10 15

Pro Gly Thr Ala Leu Leu Cys Tyr Ser Cys Lys Ala Gln Val Ser Asn

20 25 30

Glu Asp Cys Leu Gln Val Glu Asn Cys Thr Gln Leu Gly Glu Gln Cys

35 40 45

Trp Thr Ala Arg Ile Arg Ala Val Gly Leu Leu Thr Val Ile Ser Lys

50 55 60

Gly Cys Ser Leu Asn Cys Val Asp Asp Ser Gln Asp Tyr Tyr Val Gly

65 70 75 80

Lys Lys Asn Ile Thr Cys Cys Asp Thr Asp Leu Cys Asn Ala Ser Gly

85 90 95

Ala His Ala Leu Gln Pro Ala Ala Ala Ile Leu Ala Leu Leu Pro Ala

100 105 110

Leu Gly Leu Leu Leu Trp Gly Pro Gly Gln Leu

115 120

<210>5

<211>888

<212>DNA

<213> Homo sapiens

<220>

<221> CDS

<222>(423)...(707)

<400>5

tttgaggcca tataaagtca cctgaggccc tctccaccac agcccaccag tgaccatgaa 60

ggctgtgctg cttgccctgt tgatggcagg cttggccctg cagccaggca ctgccctgct 120

gtgctactcc tgcaaagccc aggcgcagtt ggcctcctga ccgtcatcag caaaggctgc 180

agcttgaact gcgtggatga ctcacaggac tactacgtgg gcaagaagaa catcacgtgc 240

tgtgacaccg acttgtgcac tcggcctgct gctctgggga cccggccagc tataggctct 300

ggggggcccc gctgcagccc acactgggtg tggtgcccca ggcctctgtg ccactcctca 360

cacacccggc ccagtggggag cctgtcctgg ttcctgaggc acatcctaac gcaagtctga 420

cc atg tat gtc tgc gcc cct gtc ccc cac cct gac cct ccc atg gcc 467

Met Tyr Val Cys Ala Pro Val Pro His Pro Asp Pro Pro Met Ala

1 5 10 15

ctc tcc agg act ccc acc cgg cag atc ggc tct att gac aca gat ccg 515

Leu Ser Arg Thr Pro Thr Arg Gln Ile Gly Ser Ile Asp Thr Asp Pro

20 25 30

cct gca gat ggc ccc tcc aac cct ctc tgc tgc tgt ttc cat ggc cca 563

Pro Ala Asp Gly Pro Ser Asn Pro Leu Cys Cys Cys Phe His Gly Pro

35 40 45

gca ttc tcc acc ctt aac cct gtg ctc agg cac ctc ttc ccc cag gaa 611

Ala Phe Ser Thr Leu Asn Pro Val Leu Arg His Leu Phe Pro Gln Glu

50 55 60

gcc ttc cct gcc cac ccc atc tat gac ttg agc cag gtc tgg tcc gtg 659

Ala Phe Pro Ala His Pro Ile Tyr Asp Leu Ser Gln Val Trp Ser Val

65 70 75

gtg tcc ccc gca ccc agc agg gga cag gca ctc agg agg gcc cgg taa 707

Val Ser Pro Ala Pro Ser Arg Gly Gln Ala Leu Arg Arg Ala Arg*

80 85 90

aggctgagat gaagtggact gagtagaact ggaggacagg agtcgacgtg agttcctggg 767

agtctccaga gatggggcct ggaggcctgg aggaaggggc caggcctcac attcgtgggg 827

ctccctgaat ggcagcctca gcacagcgta ggcccttaat aaacacctgt tggataagcc 887

a 888

<210>6

<211>94

<212>PRT

<213> Homo sapiens

<400>6

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

1 5 10 15

Ser Arg Thr Pro Thr Arg Gln Ile Gly Ser Ile Asp Thr Asp Pro Pro

20 25 30

Ala Asp Gly Pro Ser Asn Pro Leu Cys Cys Cys Phe His Gly Pro Ala

35 40 45

Phe Ser Thr Leu Asn Pro Val Leu Arg His Leu Phe Pro Gln Glu Ala

50 55 60

Phe Pro Ala His Pro Ile Tyr Asp Leu Ser Gln Val Trp Ser Val Val

65 70 75 80

Ser Pro Ala Pro Ser Arg Gly Gln Ala Leu Arg Arg Ala Arg

85 90

<210>7

<211>1174

<212>DNA

<213> Homo sapiens

<220>

<221> CDS

<222>(424)...(993)

<400>7

gacagtgaac cctgcgctga aggcgttggg gctcctgcag ttctggggca gccacaggcg 60

cccagggttt cgtgccgatc agcccaggac ggtcttcccg gtgcagtttc tgatgcgggg 120

agggcagtgc tgccttccgg tcaccaggac cagtgctcag cccgcctgct tgaccccctt 180

acttagctgg ggtccaatcc atacccaatt tagatgattc agacgatggg atttgaaact 240

tttgaactgg gtgcgactta agcactgccc tgctgtgcta ctcctgcaaa gcccaggtga 300

gcaacgagga ctgcctgcag gtggagaact gcacccagct gggggagcag tgctggaccg 360

cgcgcatccg cgcagttggc ctcctgaccg tcatcagcaa aggctgcagc ttgaactgcg 420

tgg atg act cac agg act act acg tgg gca aga aga aca tca cgt gct 468

Met Thr His Arg Thr Thr Thr Trp Ala Arg Arg Thr Ser Arg Ala

1 5 10 15

gtg aca ccg act tgt gca acg cca gcg ggg ccc atg ccc tgc agc cgg 516

Val Thr Pro Thr Cys Ala Thr Pro Ala Gly Pro Met Pro Cys Ser Arg

20 25 30

ctg ccg cca tcc ttg cgc tgc tcc ctg cac tcg gcc tgc tgc tct ggg 564

Leu Pro Pro Ser Leu Arg Cys Ser Leu His Ser Ala Cys Cys Ser Gly

35 40 45

gac ccg gcc agc tat agg ctc tgg ggg gcc ccg ctg cag ccc aca ctg 612

Asp Pro Ala Ser Tyr Arg Leu Trp Gly Ala Pro Leu Gln Pro Thr Leu

50 55 60

ggt gtg gtg ccc cag gcc tct gtg cca ctc ctc aca cac ccg gcc cag 660

Gly Val Val Pro Gln Ala Ser Val Pro Leu Leu Thr His Pro Ala Gln

65 70 75

tgg gag cct gtc ctg gtt cct gag gca cat cct aac gca agt ctg acc 708

Trp Glu Pro Val Leu Val Pro Glu Ala His Pro Asn Ala Ser Leu Thr

80 85 90 95

atg tat gtc tgc gcc cct gtc ccc cac cct gac cct ccc atg gcc ctc 756

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

100 105 110

tcc agg act ccc acc cgg cag atc ggc tct att gac aca gat ccg cct 804

Ser Arg Thr Pro Thr Arg Gln Ile Gly Ser Ile Asp Thr Asp Pro Pro

115 120 125

gca gat ggc ccc tcc aac cct ctc tgc tgc tgt ttc cat ggc cca gca 852

Ala Asp Gly Pro Ser Asn Pro Leu Cys Cys Cys Phe His Gly Pro Ala

130 135 140

ttc tcc acc ctt aac cct gtg ctc agg cac ctc ttc ccc cag gaa gcc 900

Phe Ser Thr Leu Asn Pro Val Leu Arg His Leu Phe Pro Gln Glu Ala

145 150 155

ttc cct gcc cac ccc atc tat gac ttg agc cag gtc tgg tcc gtg gtg 948

Phe Pro Ala His Pro Ile Tyr Asp Leu Ser Gln Val Trp Ser Val Val

160 165 170 175

tcc ccc gca ccc agg agg gga cag gca ctc agg agg gcc cgg taa 993

Ser Pro Ala Pro Ser Arg Gly Gln Ala Leu Arg Arg Ala Arg *

180 185

aggctgagat gaagtggact gagtagaact ggaggacagg agtcgacgtg agttcctggg 1053

agtctccaga gatggggcct ggaggcctgg aggaaggggc caggcctcac attcgtgggg 1113

ctccctgaat ggcagcctca gcacagcgta ggcccttaat aaacacctgt tggataagcc 1173

a 1174

<210>8

<211>189

<212>PRT

<213> Homo sapiens

<400>8

Met Thr His Arg Thr Thr Thr Trp Ala Arg Arg Thr Ser Arg Ala Val

1 5 10 15

Thr Pro Thr Cys Ala Thr Pro Ala Gly Pro Met Pro Cys Ser Arg Leu

20 25 30

Pro Pro Ser Leu Arg Cys Ser Leu His Ser Ala Cys Cys Ser Gly Asp

35 40 45

Pro Ala Ser Tyr Arg Leu Trp Gly Ala Pro Leu Gln Pro Thr Leu Gly

50 55 60

Val Val Pro Gln Ala Ser Val Pro Leu Leu Thr His Pro Ala Gln Trp

65 70 75 80

Glu Pro Val Leu Val Pro Glu Ala His Pro Asn Ala Ser Leu Thr Met

85 90 95

Tyr Val Cys Ala Pro Val Pro His Pro Asp Pro Pro Met Ala Leu Ser

100 105 110

Arg Thr Pro Thr Arg Gln Ile Gly Ser Ile Asp Thr Asp Pro Pro Ala

115 120 125

Asp Gly Pro Ser Asn Pro Leu Cys Cys Cys Phe His Gly Pro Ala Phe

130 135 140

Ser Thr Leu Asn Pro Val Leu Arg His Leu Phe Pro Gln Glu Ala Phe

145 150 155 160

Pro Ala His Pro Ile Tyr Asp Leu Ser Gln Val Trp Ser Val Val Ser

165 170 175

Pro Ala Pro Ser Arg Gly Gln Ala Leu Arg Arg Ala Arg

180 185

<210>9

<211>1660

<212>DNA

<213> Homo sapiens

<220>

<221> CDS

<222>(910)...(1479)

<400>9

gacagtgaac cctgcgctga aggcgttggg gctcctgcag ttctggggca gccacaggcg 60

cccagggttt cgtgccgatc agcccaggac ggtcttcccg gtgcagtttc tgatgcgggg 120

agggcagtgc tgccttccgg tcaccaggac cagtgctcag cccgcctgct tgaccccctt 180

acttagctgg ggtccaatcc atacccaatt tagatgattc agacgatggg atttgaaact 240

tttgaactgg gtgcgactta agcactgccc tgctgtgcta ctcctgcaaa gcccaggtga 300

gcaacgagga ctgcctgcag gtggagaact gcacccagct gggggagcag tgctggaccg 360

cgcgcatccg tgagtggggg gacgacagcc gccaggccta ggtctctgcc actgaactat 420

taatctttct ggccatctgt ccgcatctgt gtgctgtttt ccttccacct gtccccgacc 480

cgtcccgcac ctgcaccccc aacaatcacc cagcatctgt ccctccagcc atcctcctcc 540

atctgccact cctccactca tctgtccctc cccatcctcc atcttccact cctccaccca 600

tctgtccctc cccatccctg agctcactta ctcactcacc ccatttctga cgctcagcgg 660

gtggtccatc tgcctcggac atctggatag ggctgagacc agggccgaga ccaggccctc 720

gcactgcttg caatcctgag gccagcccag ggggactcta gagcattagg cagggtggga 780

caggaggagg cctggggcag gtcaggcagg tgagcacaca gggcagcccc atccccggat 840

cccgctgctc cccaggcgca gttggcctcc tgaccgtcat cagcaaaggc tgcagcttga 900

actgcgtgg atg act cac agg act act acg tgg gca aga aga aca tca cgt 951

Met Thr His Arg Thr Thr Thr Trp Ala Arg Arg Thr Ser Arg

1 5 10

gct gtg aca ccg act tgt gca acg cca gcg ggg ccc atg ccc tgc agc 999

Ala Val Thr Pro Thr Cys Ala Thr Pro Ala Gly Pro Met Pro Cys Ser

15 20 25 30

cgg ctg ccg cca tcc ttg cgc tgc tcc ctg cac tcg gcc tgc tgc tct 1047

Arg Leu Pro Pro Ser Leu Arg Cys Ser Leu His Ser Ala Cys Cys Ser

35 40 45

ggg gac ccg gcc agc tat agg ctc tgg ggg gcc ccg ctg cag ccc aca 1095

Gly Asp Pro Ala Ser Tyr Arg Leu Trp Gly Ala Pro Leu Gln Pro Thr

50 55 60

ctg ggt gtg gtg ccc cag gcc tct gtg cca ctc ctc aca cac ccg gcc 1143

Leu Gly Val Val Pro Gln Ala Ser Val Pro Leu Leu Thr His Pro Ala

65 70 75

cag tgg gag cct gtc ctg gtt cct gag gca cat cct aac gca agt ctg 1191

Gln Trp Glu Pro Val Leu Val Pro Glu Ala His Pro Asn Ala Ser Leu

80 85 90

acc atg tat gtc tgc gcc cct gtc ccc cac cct gac cct ccc atg gcc 1239

Thr Met Tyr Val Cys Ala Pro Val Pro His Pro Asp Pro Pro Met Ala

95 100 105 110

ctc tcc agg act ccc acc cgg cag atc ggc tct att gac aca gat ccg 1287

Leu Ser Arg Thr Pro Thr Arg Gln Ile Gly Ser Ile Asp Thr Asp Pro

115 120 125

cct gca gat ggc ccc tcc aac cct ctc tgc tgc tgt ttc cat ggc cca 1335

Pro Ala Asp Gly Pro Ser Asn Pro Leu Cys Cys Cys Phe His Gly Pro

130 135 140

gca ttc tcc acc ctt aac cct gtg ctc agg cac ctc ttc ccc cag gaa 1383

Ala Phe Ser Thr Leu Asn Pro Val Leu Arg His Leu Phe Pro Gln Glu

145 150 155

gcc ttc cct gcc cac ccc atc tat gac ttg agc cag gtc tgg tcc gtg 1431

Ala Phe Pro Ala His Pro Ile Tyr Asp Leu Ser Gln Val Trp Ser Val

160 165 170

gtg tcc ccc gca ccc agc agg gga cag gca ctc agg agg gcc cgg taa 1479

Val Ser Pro Ala Pro Ser Arg Gly Gln Ala Leu Arg Arg Ala Arg *

175 180 185

aggctgagat gaagtggact gagtagaact ggaggacagg agtcgacgtg agttcctggg 1539

agtctccaga gatggggcct ggaggcctgg aggaaggggc caggcctcac attcgtgggg 1599

ctccctgaat ggcagcctca gcacagcgta ggcccttaat aaacacctgt tggataagcc 1659

a 1660

<210>10

<211>189

<212>PRT

<213> Homo sapiens

<400>10

Met Thr His Arg Thr Thr Thr Trp Ala Arg Arg Thr Ser Arg Ala Val

1 5 10 15

Thr Pro Thr Cys Ala Thr Pro Ala Gly Pro Met Pro Cys Ser Arg Leu

20 25 30

Pro Pro Ser Leu Arg Cys Ser Leu His Ser Ala Cys Cys Ser Gly Asp

35 40 45

Pro Ala Ser Tyr Arg Leu Trp Gly Ala Pro Leu Gln Pro Thr Leu Gly

50 55 60

Val Val Pro Gln Ala Ser Val Pro Leu Leu Thr His Pro Ala Gln Trp

65 70 75 80

Glu Pro Val Leu Val Pro Glu Ala His Pro Asn Ala Ser Leu Thr Met

85 90 95

Tyr Val Cys Ala Pro Val Pro His Pro Asp Pro Pro Met Ala Leu Ser

100 105 110

Arg Thr Pro Thr Arg Gln Ile Gly Ser Ile Asp Thr Asp Pro Pro Ala

115 120 125

Asp Gly Pro Ser Asn Pro Leu Cys Cys Cys Phe His Gly Pro Ala Phe

130 135 140

Ser Thr Leu Asn Pro Val Leu Arg His Leu Phe Pro Gln Glu Ala Phe

145 150 155 160

Pro Ala His Pro Ile Tyr Asp Leu Ser Gln Val Trp Ser Val Val Ser

165 170 175

Pro Ala Pro Ser Arg Gly Gln Ala Leu Arg Arg Ala Arg

180 185

<210>11

<211>1020

<212>DNA

<213> Homo sapiens

<220>

<221> CDS

<222>(83)...(427)

<400>11

tttgaggcca tataaagtca cctgaggccc tctccaccac agcccaccag tgaccatgaa 60

ggctgtgctg cttgccctgt tg atg gca ggc ttg gcc ctg cag cca ggc act 112

Met Ala Gly Leu Ala Leu Gln Pro Gly ThR

1 5 10

gcc ctg ctg tgc tac tcc tgc aaa gcc cag gtg agc aac gag gac tgc 160

Ala Leu Leu Cys Tyr Ser Cys Lys Ala Gln Val Ser Asn Glu Asp Cys

15 20 25

ctg cag gtg gag aac tgc acc cag ctg ggg gag cag tgc tgg acc gcg 208

Leu Gln Val Glu Asn Cys Thr Gln Leu Gly Glu Gln Cys Trp Thr Ala

30 35 40

cgc atc cgc gca gtt ggc ctc ctg acc gtc atc agc aaa ggc tgc agc 256

Arg Ile Arg Ala Val Gly Leu Leu Thr Val Ile Ser Lys Gly Cys Ser

45 50 55

ttg aac tgc gtg gat gac tca cag gac tac tac gtg ggc aag aag aac 304

Leu Asn Cys Val Asp Asp Ser Gln Asp Tyr Tyr Val Gly Lys Lys Asn

60 65 70

atc acg tgc tgt gac acc gac ttg tgc aac gcc agc ggg gcc cat gcc 352

Ile Thr Cys Cys Asp Thr Asp Leu Cys Asn Ala Ser Gly Ala His Ala

75 80 85 90

ctg cag ccg gct gcc gcc atc ctt gcg ctg ctc cct gca ctc ggc ctg 400

Leu Gln Pro Ala Ala Ala Ile Leu Ala Leu Leu Pro Ala Leu Gly Leu

95 100 105

ctg ctc tgg gga ccc ggc cag cta tag gctctggggg gccccgctgc 447

Leu Leu Trp Gly Pro Gly Gln Leu *

110

agcccacact gggtgtggtg ccccaggcct ctgtgccact cctcacacac ccggcccagt 507

gggagcctgt cctggttcct gaggcacatc ctaacgcaag tctgaccatg tatgtctgcg 567

cccctgtccc ccaccctgac cctcccatgg ccctctccag gactcccacc cggcagatcg 627

gctctattga cacagatccg cctgcagatg gcccctccaa ccctctctgc tgctgtttcc 687

atggcccagc attctccacc cttaaccctg tgctcaggca cctcttcccc caggaagcct 747

tccctgccca ccccatctat gacttgagcc aggtctggtc cgtggtgtcc cccgcaccca 807

gcagggggaca ggcactcagg agggcccggt aaaggctgag atgaagtgga ctgagtagaa 867

ctggaggaca ggagtcgacg tgagttcctg ggagtctcca gagatggggc ctggaggcct 927

ggaggaaggg gccaggcctc acattcgtgg ggctccctga atggcagcct cagcacagcg 987

taggccctta ataaacacct gttggataag cca 1020

<210>12

<211>114

<212>PRT

<213> Homo sapiens

<400>12

Met Ala Gly Leu Ala Leu Gln Pro Gly Thr Ala Leu Leu Cys Tyr Ser

1 5 10 15

Cys Lys Ala Gln Val Ser Asn Glu Asp Cys Leu Gln Val Glu Asn Cys

20 25 30

Thr Gln Leu Gly Glu Gln Cys Trp Thr Ala Arg Ile Arg Ala Val Gly

35 40 45

Leu Leu Thr Val Ile Ser Lys Gly Cys Ser Leu Asn Cys Val Asp Asp

50 55 60

Ser Gln Asp Tyr Tyr Val Gly Lys Lys Asn Ile Thr Cys Cys Asp Thr

65 70 75 80

Asp Leu Cys Asn Ala Ser Gly Ala His Ala Leu Gln Pro Ala Ala Ala

85 90 95

Il e Leu Ala Leu Leu Pro Ala Leu Gly Leu Leu Leu Trp Gly Pro Gly

100 105 110

Gln Leu

<210>13

<211>148

<212>PRT

<213> Homo sapiens

<400>13

Val Ala Ala Pro Arg Trp Val Leu Ser Gln Val Gln Leu Gln Glu Ser

1 5 10 15

Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr

20 25 30

Val Ser Gly Gly Ser Ile Ser Ser Gly Gly Tyr Tyr Trp Ile Trp Ile

35 40 45

Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr

50 55 60

Asn Gly Asn Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Met

65 70 75 80

Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val

85 90 95

Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Gly Ile Thr

100 105 110

Met Ile Arg Gly Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr

115 120 125

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Lys Gly

130 135 140

Pro Ser Val Phe

145

<210>14

<211>155

<212>PRT

<213> Homo sapiens

<400>14

Gln Leu Thr Gln Ser Pro Ser Ser Ser Val Ser Ala Ser Val Gly Asp Arg

1 5 10 15

Val Thr Ile Thr Cys Arg Ala Ser Arg Gly Ile Ser Ser Trp Leu Ala

20 25 30

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Thr

35 40 45

Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

50 55 60

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

65 70 75 80

PheAla Thr Tyr Tyr Cys Gln Gln Ala Tyr Ser Phe Pro Arg Thr Phe

85 90 95

Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser

100 105 110

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr AIa

115 120 125

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

130 135 140

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

145 150 155

<210>15

<211>136

<212>PRT

<213> Homo sapiens

<400>15

Gln Cys Val Ala Ala Pro Arg Trp Val Leu Ser Gln Val Gln Leu Gln

1 5 10 15

Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr

20 25 30

Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly Gly Tyr Tyr Trp Ser

35 40 45

Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile

50 55 60

Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val

65 70 75 80

Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser

85 90 95

Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg

100 105 110

Ile Thr Met Val Arg Gly Gly Ile Pro Ser Gly Met Asp Val Trp Gly

115 120 125

Gln Gly Thr Thr Val Thr Val Ser

130 135

<210>16

<211>139

<212>PRT

<213> Homo sapiens

<400>16

Ser Pro Phe Thr Cys Arg Ala Ser Gln Ser Ile Thr Asn Tyr Leu Asn

1 5 10 15

Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Lys Leu Leu Ile His Val

20 25 30

Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

35 40 45

Ser Gly Arg Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

50 55 60

Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His Ser Ile Pro Arg Thr Phe

65 70 75 80

Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser

85 90 95

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

100 105 110

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

115 120 125

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

130 135

<210>17

<211>138

<212>PRT

<213> Homo sapiens

<400>17

Gln Val His Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp IleArg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Ser Tyr Lys Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Val Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Tyr Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Arg Gly Asp Tyr Gly Asp Phe Leu Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

130 135

<210>18

<211>141

<212>PRT

<213> Homo sapiens

<400>18

Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile

1 5 10 15

Gly Ser Thr Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

20 25 30

Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Glu

35 40 45

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

50 55 60

Gly Leu Glu Pro Glu Asp Phe Ala Val Phe Tyr Cys Gln Gln Cys Gly

65 70 75 80

Ser Ser Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

85 90 95

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

100 105 110

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

115 120 125

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala

130 135 140

<210>19

<211>139

<212>PRT

<213> Homo sapiens

<400>19

Gly Leu Gln Cys Val Ala Ala Pro Arg Trp Val Leu Ser Gln Val Gln

1 5 10 15

Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr Leu Ser

20 25 30

Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Asn Tyr Trp Ser

35 40 45

Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile

50 55 60

Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val

65 70 75 80

Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser

85 90 95

Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Gly

100 105 110

Ser Tyr Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

115 120 125

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Lys

130 135

<210>20

<211>236

<212>PRT

<213> Homo sapiens

<400>20

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Ser Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser

35 40 45

Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Ser Pro Gly Lys

50 55 60

Ala Pro Lys Phe Leu Ile Ser Asp Ala Ser Asn Leu Lys Thr Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Phe Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Cys Cys Gln Gln

100 105 110

Tyr Asp Ser Leu Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

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

225 230 235

<210>21

<211>239

<212>PRT

<213> Homo sapiens

<400>21

Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Ser

1 5 10 15

Gly Ser Ser Gly Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro

20 25 30

Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser

35 40 45

Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Val Trp Tyr Leu Gln Lys

50 55 60

Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Leu Gly Ser Ile Arg Ala

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr

100 105 110

Cys Met Gln Pro Leu Gln Thr Pro Ile Thr Phe Gly Gln Gly Thr Arg

115 120 125

Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

130 135 140

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

145 150 155 160

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

165 170 175

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

180 185 190

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Ser Thr Leu Thr Leu Ser Lys

195 200 205

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

210 215 220

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

225 230 235

<210>22

<211>141

<212>PRT

<213> Homo sapiens

<400>22

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Gly Tyr Ala

50 55 60

Val Ser Val Lys Ser Arg Met Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Glu Arg Leu Gly Glu Leu Tyr Gly Met Asp Val

100 105 110

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

130 135 140

<210>23

<211>161

<212>PRT

<213> Homo sapiens

<400>23

Gln Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg

1 5 10 15

Ala Thr Ile Asn Cys Lys Ser Ser Gln Asn Ile Leu Tyr Arg Ser Ser

20 25 30

Lys Lys Asn His Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Thr Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr

85 90 95

Ser Thr Pro Pro Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly

<210>24

<211>137

<212>PRT

<213> Homo sapiens

<220>

<221> VARIANT

<222>(0)...(0)

<223> Xaa = any amino acid

<400>24

Gly Pro Gly Xaa Xaa Lys Pro Ser Gln Xaa Leu Ser Leu Thr Gly Thr

1 5 10 15

Val Ser Gly Gly Ser Ile Ser Ser Gly Gly Tyr Tyr Trp Ser Trp Ile

20 25 30

Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly Asn Ile Tyr Tyr

35 40 45

Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile

50 55 60

Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ala Val

65 70 75 80

Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Asn Ile Thr

85 90 95

Met Val Arg Gly Val Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Tyr

130 135

<210>25

<211>107

<212>PRT

<213> Homo sapiens

<400>25

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

1 5 10 15

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

20 25 30

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Leu Pro Arg

35 40 45

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

50 55 60

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

65 70 75 80

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

85 90 95

Lys Val Gln Trp Lys Val Asp Asn Thr Leu Gln

100 105

<210>26

<211>144

<212>PRT

<213> Homo sapiens

<400>26

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg His

20 25 30

Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Leu Ile Ala Val Arg Pro Gly Tyr Tyr Tyr Tyr Gly

100 105 110

Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser

115 120 125

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

130 135 140

<210>27

<211>157

<212>PRT

<213> Homo sapiens

<400>27

Glu Met Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

LeuAsn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

ProSer Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

145 150 155

<210>28

<211>141

<212>PRT

<213> Homo sapiens

<400>28

Cys Pro Gly Ala Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser

1 5 10 15

Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser

20 25 30

Gly Gly Thr Tyr Tyr Trp Ile Trp Ile Arg Gln His Pro Gly Lys Gly

35 40 45

Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn

50 55 60

Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Asp Gly Ile Thr Met Val Arg Gly Ile Ser Gly

100 105 110

Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala

115 120 125

Ser Thr Lys Gly Pro Ser Val Lys Gly Pro Ser Val Phe

130 135 140

<210>29

<211>145

<212>PRT

<213> Homo sapiens

<400>29

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser

1 5 10 15

Ile Ser Ser His Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

20 25 30

Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser

35 40 45

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Ser

50 55 60

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Tyr

65 70 75 80

Ser Ile Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Thr Arg

85 90 95

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

100 105 110

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

115 120 125

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

130 135 140

Gly

145

<210>30

<211>139

<212>PRT

<213> Homo sapiens

<400>30

Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu

1 5 10 15

Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn Ser Ala

20 25 30

Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu Trp Leu

35 40 45

Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Glu Tyr Ala Val Ser

50 55 60

Val Lys Ser Arg Met Thr Ile Asn Pro Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Arg Leu Gly Glu Leu Tyr Gly Met Asp Val Trp Gly

100 105 110

Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

130 135

<210>31

<211>128

<212>PRT

<213> Homo sapiens

<400>31

Leu Gln Val Gln Pro Glu Cys Leu Tyr Thr Val Ser Asp Lys Asn Asn

1 5 10 15

Phe Leu Cys Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu

20 25 30

Met Tyr Trp Ala Ser Ile Arg Glu Ser Gly Val Pro Asp Arg Phe Ser

35 40 45

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

50 55 60

Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro

65 70 75 80

Pro Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala

85 90 95

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

100 105 110

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

115 120 125

<210>32

<211>141

<212>PRT

<213> Homo sapiens

<400>32

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Trp Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Glu Tyr Ala

50 55 60

Val Ser Val Lys Ser Arg Met Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Glu Arg Leu Gly Glu Leu Tyr Gly Met Asp Val

100 105 110

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

130 135 140

<210>33

<211>162

<212>PRT

<213> Homo sapiens

<400>33

Met Gln Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu

1 5 10 15

Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Asn Val Leu Tyr Arg Ser

20 25 30

Asn Lys Lys Asn Phe Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln Pro

35 40 45

Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg Glu Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Thr Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr

85 90 95

Tyr Ser Thr Pro Pro Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly

<210>34

<211>445

<212>DNA

<213> Homo sapiens

<400>34

gtggcagcac ccagatgggt cctgtcccag gtgcagctgc aggagtcggg cccaggactg 60

gtgaagcctt cacagaccct gtccctcacc tgcactgtct ctggtggctc catcagcagt 120

ggtggttact actggatctg gatccgccag cacccaggga agggcctgga gtggattggg 180

tacatctatt acaatgggaa cacctactac aacccgtccc tcaagagtcg agttaccatg 240

tcagtagaca cgtctaagaa ccagttctcc ctgaagctga gctctgtgac tgccgcggac 300

acggccgtgt attackgtgc gagagatggt attackatga tacgcggcta ctactacggt 360

atggacgtct ggggccaagg gaccacggtc accgtctcct cagcctccac caagggccca 420

tcggtcaagg gcccatcggt cttca 445

<210>35

<211>148

<212>PRT

<213> Homo sapiens

<400>35

Val Ala Ala Pro Arg Trp Val Leu Ser Gln Val Gln Leu Gln Glu Ser

1 5 10 15

Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr

20 25 30

Val Ser Gly Gly Ser Ile Ser Ser Gly Gly Tyr Tyr Trp Ile Trp Ile

35 40 45

Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr

50 55 60

Asn Gly Asn Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Met

65 70 75 80

Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val

85 90 95

Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Gly Ile Thr

100 105 110

Met Ile Arg Gly Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr

115 120 125

Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Lys Gly

130 135 140

Pro Ser Val Phe

145

<210>36

<211>466

<212>DNA

<213> Homo sapiens

<220>

<221>misc_feature

<222>(0)...(0)

<223>n=a, t, c or g

<400>36

cagctgacnc agtctccatc ttccgtgtct gcatctgtag gagacagagt caccatcact 60

tgtcgggcga gtcggggtat tagcagctgg ttagcctggt atcagcagaa accagggaaa 120

gcccctaagc tcctgatcta tactgcatcc agtttgcaaa gtggagtccc atcaaggttc 180

agcggcagtg gttctgggac agatttcact ctcaccatca gcagcctgca gcctgaagat 240

tttgcaactt actattgtca acaggcttac agtttccctc ggacgttcgg ccaagggacc 300

aaggtggaaa tcaaacgaac tgtggctgca ccatctgtct tcatcttccc gccatctgat 360

gagcagttga aatctggaac tgcctctgtt gtgtgcctgc tgaataactt ctatcccaga 420

gaggccaaag tacagtggaa ggtggataac gccctccaat cgggta 466

<210>37

<211>155

<212>PRT

<213> Homo sapiens

<400>37

Gln Leu Thr Gln Ser Pro Ser Ser Ser Val Ser Ala Ser Val Gly Asp Arg

1 5 10 15

Val Thr Ile Thr Cys Arg Ala Ser Arg Gly Ile Ser Ser Trp Leu Ala

20 25 30

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Thr

35 40 45

Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

50 55 60

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

65 70 75 80

Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Tyr Ser Phe Pro Arg Thr Phe

85 90 95

Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser

100 105 110

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

115 120 125

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

130 135 140

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

145 150 155

<210>38

<211>409

<212>DNA

<213> Homo sapiens

<400>38

cagtgtgtgg cagcacccag atgggtcctg tcccaggtgc agctgcagga gtcgggccca 60

ggactggtga agccttcaca gaccctgtcc ctcacctgca ctgtctctgg tggctccatc 120

agcagtggtg gttactactg gagctggatc cgccagcacc cagggaaggg cctggagtgg 180

attgggtaca tatattacag tgggagcacc tactacaacc cgtccctcaa gagtcgagtt 240

accatatcag tagacacgtc caagaaccag ttctccctga agctgagctc tgtgactgcc 300

gcggacacgg ccgtgtatta ctgtgcgaga gatcgaatta ctatggttcg gggaggtatt 360

cccagtggta tggacgtctg gggccaaggg accacggtca ccgtctcct 409

<210>39

<211>136

<212>PRT

<213> Homo sapiens

<400>39

Gln Cys Val Ala Ala Pro Arg Trp Val Leu Ser Gln Val Gln Leu Gln

1 5 10 15

Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr

20 25 30

Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly Gly Tyr Tyr Trp Ser

35 40 45

Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly Tyr Ile

50 55 60

Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val

65 70 75 80

Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser

85 90 95

Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg

100 105 110

Ile Thr Met Val Arg Gly Gly Ile Pro Ser Gly Met Asp Val Trp Gly

115 120 125

Gln Gly Thr Thr Val Thr Val Ser

130 135

<210>40

<211>417

<212>DNA

<213> Homo sapiens

<400>40

tcaccattca cttgccgggc aagtcagagc attaccaact atttaaattg gtatcagcag 60

aaaccagggg aagcccctaa gctcctgatc catgttgcat ccagtttgca aagtggggtc 120

ccatcaaggt tcagtggcag tggatctggg agagatttca ctctcaccat cagcagtctg 180

caacctgaag attttgcaac ttactactgt caacagagtc acagtatccc tcggacgttc 240

ggccaaggga ccaaggtgga aatcaaacga actgtggctg caccatctgt cttcatcttc 300

ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac 360

ttctatccca gagaggccaa aagtacagtgg aaggtggata acgccctcca atcgggt 417

<210>41

<211>139

<212>PRT

<213> Homo sapiens

<400>41

Ser Pro Phe Thr Cys Arg Ala Ser Gln Ser Ile Thr Asn Tyr Leu Asn

1 5 10 15

Trp Tyr Gln Gln Lys Pro Gly Glu Ala Pro Lys Leu Leu Ile His Val

20 25 30

Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

35 40 45

Ser Gly Arg Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

50 55 60

Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His Ser Ile Pro Arg Thr Phe

65 70 75 80

Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser

85 90 95

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

100 105 110

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

115 120 125

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

130 135

<210>42

<211>416

<212>DNA

<213> Homo sapiens

<400>42

caggtgcatc tacagcagtg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60

acctgcgctg tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc 120

ccggggaagg gactggagtg gattggggaa atcaatcata gtggaagcac cagctacaag 180

ccgtccctca agagtcgagt caccgtatca gtggacacgt ccaagaacca gttctccctg 240

aagctgagct atgtgaccgc cgcggacacg gctgtgtatt actgtgcgag agataggggt 300

gactacggtg acttcctctt tgactactgg ggccagggaa ccctggtcac cgtctcctca 360

gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag caccta 416

<210>43

<211>138

<212>PRT

<213> Homo sapiens

<400>43

Gln Val His Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Ser Tyr Lys Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Val Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Tyr Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Asp Arg Gly Asp Tyr Gly Asp Phe Leu Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

130 135

<210>44

<211>425

<212>DNA

<213> Homo sapiens

<400>44

tctccagggg aaagagccac cctctcctgc agggccagtc agagtattgg cagcacctac 60

ttagcctggt accagcagaa acctggccag gctcccaggc tcctcatcta tggtgcatcc 120

agcagggcca ctggcatccc agaaaggttc agtggcagtg ggtctgggac agacttcact 180

ctcaccatca gcggactgga gcctgaagat tttgcagtgt tttactgtca acagtgtggt 240

agctcacctc cgacgttcgg ccaagggacc aaggtggaaa tcaaacgaac tgtggctgca 300

ccatctgtct tcatcttccc gccatctgat gagcagttga aatctggaac tgcctctgtt 360

gtgtgcctgc tgaataactt ctatcccaga gaggccaaag tacagtggaa ggtggataac 420

gccct 425

<210>45

<211>141

<212>PRT

<213> Homo sapiens

<400>45

Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile

1 5 10 15

Gly Ser Thr Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

20 25 30

Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Glu

35 40 45

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

50 55 60

Gly Leu Glu Pro Glu Asp Phe Ala Val Phe Tyr Cys Gln Gln Cys Gly

65 70 75 80

Ser Ser Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

85 90 95

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

100 105 110

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

115 120 125

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala

130 135 140

<210>46

<211>417

<212>DNA

<213> Homo sapiens

<400>46

ggtctgcagt gtgtggcagc accccagatgg gtcctgtccc aggtgcagct acagcagtgg 60

ggcgcaggac tgttgaagcc ttcggagacc ctgtccctca cctgcgctgt ctatggtggg 120

tccttcagtg gtaactactg gagctggatc cgccagcccc cagggaaggg gctggagtgg 180

attggggaaa tcaatcatag tggaagcacc aactacaacc cgtccctcaa gagtcgagtc 240

accatatcag tagacacgtc caagaaccag ttctccctga agctgagctc tgtgaccgcc 300

gcggacacgg ctgtgtatta ctgtgcgaga ggggggagct acaactactt tgactactgg 360

ggccagggaa ccctggtcac cgtctcctca gcctccacca agggcccatc ggtcaag 417

<210>47

<211>139

<212>PRT

<213> Homo sapiens

<400>47

Gly Leu Gln Cys Val Ala Ala Pro Arg Trp Val Leu Ser Gln Val Gln

1 5 10 15

Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr Leu Ser

20 25 30

Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Asn Tyr Trp Ser

35 40 45

Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile

50 55 60

Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val

65 70 75 80

Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser

85 90 95

Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Gly

100 105 110

Ser Tyr Asn Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

115 120 125

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Lys

130 135

<210>48

<211>933

<212>DNA

<213> Homo sapiens

<400>48

aaaaaagtca gtcccaacca ggacacagca tggacatgag ggtccctgct cagctcctgg 60

ggctcctgct gctctggctc tcaggtgcca gatgtgacat ccagatgact cagtctccat 120

cctccctgtc tgcatctgta ggagacagag tcaccatcac ttgccaggcg agtcaggaca 180

ttagcaacta tttgaattgg tatcagcaga gtcccgggaa agcccctaag ttcctgatct 240

ccgatgcatc caatttaaaa acagggggtcc catcaaggtt cagtggaagt ggatctggga 300

cagatttttc tttcaccatc agcagcctac agcctgaaga tattgcgacc tattgctgtc 360

aacagtatga tagtctccca ttcactttcg gccctgggac caaagtggat atcaaacgaa 420

ctgtggctgc accatctgtc ttcatcttcc cgccatctga tgagcagttg aaatctggaa 480

ctgcctctgt tgtgtgcctg ctgaataact tctatcccag agaggccaaa gtacagtgga 540

aggtggataa cgccctccaa tcgggtaact cccaggagag tgtcacagag caggacagca 600

aggacagcac ctacagcctc agcagcaccc tgacgctgag caaagcagac tacgagaaac 660

acaaagtcta cgcctgcgaa gtcacccatc agggcctgag ctcgcccgtc acaaagagct 720

tcaacagggg agagtgttag agggagaagt gcccccacct gctcctcagt tccagcctga 780

ccccctccca tcctttggcc tctgaccctt tttccacagg ggacctaccc ctattgcggt 840

cctccagctc atctttcacc tcaccccccct cctcctcctt ggctttaatt atgctaatgt 900

tggaggagaa tgaataaata aagtgaatct ttg 933

<210>49

<211>236

<212>PRT

<213> Homo sapiens

<400>49

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Ser Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser

35 40 45

Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Ser Pro Gly Lys

50 55 60

Ala Pro Lys Phe Leu Ile Ser Asp Ala Ser Asn Leu Lys Thr Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Phe Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Cys Cys Gln Gln

100 105 110

Tyr Asp Ser Leu Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

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

225 230 235

<210>50

<211>968

<212>DNA

<213> Homo sapiens

<400>50

aaagatcagg actcctcagt tcaccttctc acaatgaggc tccctgctca gctcctgggg 60

ctgctaatgc tctgggtctc tggatccagt ggggatattg tgatgactca gtctccactc 120

tccctgcccg tcacccctgg agagccggcc tccatctcct gcaggtctag tcagagcctc 180

ctacatagta atggatacaa ctatttggtt tggtacctgc agaagccagg acagtctcca 240

cagctcctga tctatttggg ttctattcgg gcctccgggg tccctgacag gttcagtggc 300

agtggatcag gcacagattt tacactgaaa atcagcagag tggaggctga ggatgttggg 360

gtttaattact gcatgcaacc tctacaaact ccgatcacct tcggccaagg gacacgactg 420

gagattaaac gaactgtggc tgcaccatct gtcttcatct tcccgccatc tgatgagcag 480

ttgaaatctg gaactgcctc tgttgtgtgc ctgctgaata acttctatcc cagagaggcc 540

aaagtacagt ggaaggtgga taacgccctc caatcgggta actcccagga gagtgtcaca 600

gagcaggaca gcaaggacag cacctacagc ctcagcagca ccctgacgct gagcaaagca 660

gactacgaga aacacaaagt ctacgcctgc gaagtcaccc atcagggcct gagctcgccc 720

gtcacaaaga gcttcaacag gggagagtgt tagagggaga agtgccccca cctgctcctc 780

agttccagcc tgaccccctc ccatcctttg gcctctgacc ctttttccac agggggaccta 840

cccctattgc ggtcctccag ctcatctttc acctcacccc cctcctcctc cttggcttta 900

attatgctaa tgttggagga gaatgaataa ataaagtgaa tctttgcaaa aaaaaaaaaa 960

aaaaaaaa 968

<210>51

<211>239

<212>PRT

<213> Homo sapiens

<400>51

Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Ser

1 5 10 15

Gly Ser Ser Gly Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro

20 25 30

Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser

35 40 45

Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Val Trp Tyr Leu Gln Lys

50 55 60

Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Leu Gly Ser Ile Arg Ala

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr

100 105 110

Cys Met Gln Pro Leu Gln Thr Pro Ile Thr Phe Gly Gln Gly Thr Arg

115 120 125

Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro

130 135 140

Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu

145 150 155 160

Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp

165 170 175

Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp

180 185 190

Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Ser Thr Leu Thr Leu Ser Lys

195 200 205

Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln

210 215 220

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

225 230 235

<210>52

<211>425

<212>DNA

<213> Homo sapiens

<400>52

caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60

acctgtgcca tctccgggga cagtgtctct agcaacagtg ctgcttggaa ctggatcagg 120

cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180

aatggttatg cagtatctgt gaaaagtcga atgaccatca accccagacac atccaagaac 240

cagttctccc tgcagctgaa ctctgtgact cccgaggaca cggctgtgta ttactgtgca 300

agagagaggt taggggagtt atacggtatg gacgtctggg gccaagggac cacggtcacc 360

gtctcctcag cctccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc 420

accta 425

<210>53

<211>141

<212>PRT

<213> Homo sapiens

<400>53

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Gly Tyr Ala

50 55 60

Val Ser Val Lys Ser Arg Met Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Glu Arg Leu Gly Glu Leu Tyr Gly Met Asp Val

100 105 110

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

130 135 140

<210>54

<211>484

<212>DNA

<213> Homo sapiens

<400>54

cagctgacgc agtctccaga ctccctggct gtgtctctgg gcgagagggc caccatcaac 60

tgcaagtcca gccagaatat tttatacagg tccagcaaga agaaccactt agtttggtac 120

cagcagaaac caggacagcc tcctaagctg ctcatttact gggcatctac ccgggaatcc 180

ggggtccctg cccgattcag tggcagcggg tctggcaag atttcactct caccatcagc 240

accctgcagg ctgaagatgt ggcagtttat tactgtcagc aatattatag tactcctccc 300

accttcggcc aagggacacg actggagatt aaacgaactg tggctgcacc atctgtcttc 360

atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420

aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg 480

ggta 484

<210>55

<211>161

<212>PRT

<213> Homo sapiens

<400>55

Gln Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg

1 5 10 15

Ala Thr Ile Asn Cys Lys Ser Ser Gln Asn Ile Leu Tyr Arg Ser Ser

20 25 30

Lys Lys Asn His Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Thr Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr

85 90 95

Ser Thr Pro Pro Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly

<210>56

<211>409

<212>DNA

<213> Homo sapiens

<220>

<221>misc_feature

<222>(0)...(0)

<223>n=a, t, c or g

<400>56

ggcccaggac nggngaagcc ttcacagacc tgtccctcac cggcactgtc tctggtggcc 60

catcagcagt ggtggttat actggagctg gatccgccag cacccaggga agggcctgga 120

gtggattggg aacatctatt acagtggggag cacctactac aacccgtccc tcaagagtcg 180

agttaccata tcagtagaca cgtctaagaa ccagttctcc ctgaagctga gcgctgtgac 240

tgccgcggac acggccgtgt attackgtgc gagagataat attackatgg ttcggggagt 300

ctactacggt atggacgtct ggggccaagg gaccacggtc accgtctcct cagcctccac 360

caagggccca tcggtcttcc ccctggcacc ctcctccaag agcacctat 409

<210>57

<211>136

<212>PRT

<213> Homo sapiens

<220>

<221> VARIANT

<222>(0)...(0)

<223> Xaa = any amino acid

<400>57

Ala Gln Asp Xaa Xaa Ser Leu His Arg Pro Val Pro His Arg His Cys

1 5 10 15

Leu Trp Trp Pro Ile Ser Ser Gly Gly Tyr Tyr Trp Ser Trp Ile Arg

20 25 30

Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly Asn Ile Tyr Tyr Ser

35 40 45

Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Ile Ser

50 55 60

Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ala Val Thr

65 70 75 80

Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Asn Ile Thr Met

85 90 95

Val Arg Gly Val Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Tyr

130 135

<210>58

<211>321

<212>DNA

<213> Homo sapiens

<400>58

tatgcagcat ccagtttgca aagtggggtc ccatcaaggt tcagcggcag tggatctgga 60

acagacttca ctctcaccat cagcagcctg cagcctgaag attttgcaac ttactattgt 120

caacaggctc acagtctccc tcggacgttc ggccaaggga ccaaggtgga aatcaaacga 180

actgtggctg caccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga 240

actgcctctg ttgtgtgcct gctgaataac ttctatccca gaaaggccaa agtacagtgg 300

aaggtggata acaccctcca a 321

<210>59

<211>107

<212>PRT

<213> Homo sapiens

<400>59

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

1 5 10 15

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

20 25 30

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala His Ser Leu Pro Arg

35 40 45

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

50 55 60

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

65 70 75 80

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

85 90 95

Lys Val Gln Trp Lys Val Asp Asn Thr Leu Gln

100 105

<210>60

<211>433

<212>DNA

<213> Homo sapiens

<400>60

caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cgtccggatt caccttcagt cgccatggcg tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggctgtgt attackgtgc gagaggaggc 300

cttatagcag ttcgtccggg gtactactac tacggtatgg acgtctgggg ccaagggacc 360

acggtcaccg tctcctcagc ctccaccaag ggcccatcgg tcttccccct ggcaccctcc 420

tccaagagca cct 433

<210>61

<211>144

<212>PRT

<213> Homo sapiens

<400>61

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg His

20 25 30

Gly Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Leu Ile Ala Val Arg Pro Gly Tyr Tyr Tyr Tyr Gly

100 105 110

Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser

115 120 125

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

130 135 140

<210>62

<211>471

<212>DNA

<213> Homo sapiens

<400>62

gaaatgcagc tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta ccccgctcac tttcggcgga 300

gggaccaagg tggagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg t 471

<210>63

<211>157

<212>PRT

<213> Homo sapiens

<400>63

Glu Met Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

145 150 155

<210>64

<211>424

<212>DNA

<213> Homo sapiens

<400>64

tgtccaggtg cactgcagga gtcggggccca ggactggtga ggccttcaca gaccctgtcc 60

ctcacctgca ctgtctctgg tggctccatc agcagtggtg gtacttacta ctggatctgg 120

atccgccagc acccagggaa gggcctggag tggattgggt acatctatta cagtgggagc 180

acctactaca acccgtccct caagagtcga gttaccatat cagtagacac gtctaagaac 240

cagttctccc tgaagctgag ctctgtgact gccgcggaca cggccgtgta ttactgtgcg 300

agagatggaa ttactatggt tcggggaatt agcgggggca tggacgtctg gggccaaggg 360

accacggtca ccgtctcctc agcctccacc aagggcccat cggtcaaggg cccatcggtc 420

ttca 424

<210>65

<211>141

<212>PRT

<213> Homo sapiens

<400>65

Cys Pro Gly Ala Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser

1 5 10 15

Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser

20 25 30

Gly Gly Thr Tyr Tyr Trp Ile Trp Ile Arg Gln His Pro Gly Lys Gly

35 40 45

Leu Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn

50 55 60

Pro Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Asp Gly Ile Thr Met Val Arg Gly Ile Ser Gly

100 105 110

Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala

115 120 125

Ser Thr Lys Gly Pro Ser Val Lys Gly Pro Ser Val Phe

130 135 140

<210>66

<211>436

<212>DNA

<213> Homo sapiens

<400>66

gcatctgtag gagacagagt caccatcact tgccgggcaa gtcagagcat tagtagtcat 60

ttaaattggt atcagcagaa accagggaaa gcccctaagc tcctgatcta tgctgcttcc 120

agtttgcaaa gtggggtccc atcaaggttc agtggcagtg gatctgggac agatttcact 180

ctctccatca gcagtctgca acctgaagat tttgcaactt acttctgtca acagagttac 240

agtatccctc ggacgttcgg ccaagggacc aaggtggaaa tcacacgaac tgtggctgca 300

ccatctgtct tcatcttccc gccatctgat gagcagttga aatctggaac tgcctctgtt 360

gtgtgcctgc tgaataactt ctatcccaga gaggccaaag tacagtggaa ggtggataac 420

gccctccaat cgggta 436

<210>67

<211>145

<212>PRT

<213> Homo sapiens

<400>67

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser

1 5 10 15

Ile Ser Ser His Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

20 25 30

Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser

35 40 45

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Ser

50 55 60

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Tyr

65 70 75 80

Ser Ile Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Thr Arg

85 90 95

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

100 105 110

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Asn Phe Tyr

115 120 125

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

130 135 140

Gly

145

<210>68

<211>417

<212>DNA

<213> Homo sapiens

<400>68

cagctgcagc agtcaggtcc aggactggtg aagccctcgc agaccctctc actcacctgt 60

gccatctccg gggacagtgt ctctagcaac agtgctgctt ggaactggat caggcagtcc 120

ccatcgagag gccttgagtg gctgggaagg acatactaca ggtccaagtg gtataatgaa 180

tatgcagtat ctgtgaaaag tcgaatgacc atcaacccag acacatccaa gaaccagttc 240

tccctgcagc tgaactctgt gactcccgag gacacggctg tgtattactg tgcaagagag 300

aggttagggg agttatacgg tatggacgtc tggggccaag ggaccatggt caccgtctcc 360

tcagcctcca ccaagggccc atcggtcttc cccctggcac cctcctccaa gagcacc 417

<210>69

<211>139

<212>PRT

<213> Homo sapiens

<400>69

Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu

1 5 10 15

Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn Ser Ala

20 25 30

Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu Trp Leu

35 40 45

Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Glu Tyr Ala Val Ser

50 55 60

Val Lys Ser Arg Met Thr Ile Asn Pro Asp Thr Ser Lys Asn Gln Phe

65 70 75 80

Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Arg Glu Arg Leu Gly Glu Leu Tyr Gly Met Asp Val Trp Gly

100 105 110

Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

130 135

<210>70

<211>384

<212>DNA

<213> Homo sapiens

<400>70

ctgcaagtcc agccagagtg tctttataca gtgtccgaca agaacaactt cttatgttgg 60

taccagcaga aaccaggaca gcctcctaaa ctgctcatgt actgggcatc tatccgggaa 120

tccggggtcc ctgaccgatt cagtggcagc gggtctggga cagatttcac tctcaccatc 180

agcagcctgc aggctgaaga tgtggcagtt tattactgtc agcaatatta tagtactcct 240

cccaccttcg gccaagggac acgactggag attaaacgaa ctgtggctgc accatctgtc 300

ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 360

ctgaataact tctatcccag agag 384

<210>71

<211>128

<212>PRT

<213> Homo sapiens

<400>71

Leu Gln Val Gln Pro Glu Cys Leu Tyr Thr Val Ser Asp Lys Asn Asn

1 5 10 15

Phe Leu Cys Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu

20 25 30

Met Tyr Trp Ala Ser Ile Arg Glu Ser Gly Val Pro Asp Arg Phe Ser

35 40 45

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln

50 55 60

Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Thr Pro

65 70 75 80

Pro Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala

85 90 95

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

100 105 110

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

115 120 125

<210>72

<211>425

<212>DNA

<213> Homo sapiens

<400>72

caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60

acctgtgcca tctccgggga cagtgtctct agcaacagtg ctgcttggaa ctggatcagg 120

cagtccccat ggagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180

aatgaatatg cagtatctgt gaaaagtcga atgaccatca accccagacac atccaagaac 240

cagttctccc tgcagctgaa ctctgtgact cccgaggaca cggctgtgta ttactgtgca 300

agagagaggt taggggagtt atacggtatg gacgtctggg gccaagggac cacggtcacc 360

gtctcctcag cctccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc 420

accta 425

<210>73

<211>141

<212>PRT

<213> Homo sapiens

<400>73

Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Trp Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Glu Tyr Ala

50 55 60

Val Ser Val Lys Ser Arg Met Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Glu Arg Leu Gly Glu Leu Tyr Gly Met Asp Val

100 105 110

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

130 135 140

<210>74

<211>487

<212>DNA

<213> Homo sapiens

<220>

<221>misc_feature

<222>(0)...(0)

<223>n=a, t, c or g

<400>74

atgcagctga cncagtctcc agactccctg gctgtgtctc tgggcgagag ggccaccatc 60

aactgcaagt ccagccagaa tgttttatac aggtccaaca agaagaactt cttagtttgg 120

taccagcaga aaccaggaca gcctcctaag ctgctcattt actgggcatc tatccgggaa 180

tccggggtcc ctgaccgatt cagtggcagc gggtctggga cagatttcac tctcaccatc 240

agcagcctgc agactgaaga tgtggcagtt tattactgtc agcaatatta tagtactcct 300

cccaccttcg gccaagggac acgactggag attaaacgaa ctgtggctgc accatctgtc 360

ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 420

ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa 480

tcgggta 487

<210>75

<211>162

<212>PRT

<213> Homo sapiens

<400>75

Met Gln Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu

1 5 10 15

Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Asn Val Leu Tyr Arg Ser

20 25 30

Asn Lys Lys Asn Phe Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln Pro

35 40 45

Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg Glu Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Thr Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr

85 90 95

Tyr Ser Thr Pro Pro Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly

<210>76

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223>Flag Tag

<400>76

gattacaagg atgacgacga taag 24

<210>77

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> control sequence

<400>77

aagggacgaa gacgaacacu uctt 24

<210>78

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> control sequence

<400>78

aactgaagac ctgaagacaa taa 23

Claims (31)

1. A monoclonal antibody or an antigen-binding fragment thereof, comprising an antigen-binding site that specifically binds to the PSCA protein, the amino acid sequence of the PSCA protein is as shown in SEQ ID NO: 2, wherein the monoclonal antibody comprises The amino acid sequence of the _VH region shown in SEQ ID NO: 47 and the amino acid sequence of the _VL region shown in SEQ ID NO: 51. 2. The antibody or fragment of claim 1, wherein said fragment is a Fab, F(ab') ₂ , Fv or sFv fragment. 3. A monoclonal antibody or an antigen-binding fragment thereof, comprising an antigen-binding site that specifically binds to the PSCA protein, the amino acid sequence of the PSCA protein is shown in SEQ ID NO: 2, wherein the monoclonal antibody comprises Amino acid sequences of the _VH region and _VL region of the monoclonal antibody produced by the hybridoma with ATCC deposit number PTA-6701. 4. The antibody or fragment of claim 3, wherein said fragment is a Fab, F(ab') ₂ , Fv or sFv fragment. 5. The antibody or fragment of any one of claims 1-4, wherein the antibody or fragment is conjugated to a detectable label. 6. The antibody or fragment of any one of claims 1-4, wherein said antibody or fragment is conjugated to a toxin. 7. The antibody or fragment of any one of claims 1-4, wherein the antibody or fragment is conjugated to a therapeutic agent. 8. The antibody or fragment of any one of claims 1-4, wherein the antibody or fragment is conjugated to a chemotherapeutic agent. 9. A hybridoma producing the monoclonal antibody according to claim 1 or 3, characterized in that the A.T.C.C deposit number of the hybridoma is PTA-6701. 10. A polynucleotide encoding a sequence comprising a light chain variable region and a heavy chain variable region of the antibody of claim 1. 11. A vector comprising the polynucleotide of claim 10. 12. A cell transfected with the vector of claim 11. 13. A method of producing an antibody or antigen-binding fragment thereof, said method comprising: cultured cells, which (a) the cell has been treated with a vector comprising a polynucleotide encoding the sequence of the light chain variable region of the antibody comprising the antibody described in claim 1 and a vector comprising the sequence encoding the heavy chain variable region of the antibody comprising the antibody described in claim 1 vector transfection of a polynucleotide sequence; or (b) said cells have been transfected with the vector of claim 11. 14. A pharmaceutical composition for the treatment of prostate or pancreatic cancer, said pharmaceutical composition comprising the antibody according to any one of claims 1-8, or its Fab, F(ab')2, Fv or sFv fragment. 15. Use of the antibody according to any one of claims 1-4 in the preparation of a test kit for detecting the presence of PSCA protein in a biological sample and the binding of PSCA protein to the antibody in a sample, wherein the sample The PSCA protein in comprises the amino acid sequence shown in SEQ ID NO:2. 16. An anticancer agent for reducing tumor growth, characterized in that the anticancer agent comprises a combination of the antibody or antigen-binding fragment thereof and a chemotherapeutic agent according to any one of claims 1-4, wherein the tumor is a tumor of the prostate or pancreas. 17. The anticancer agent of claim 16, wherein said antibody or fragment thereof is coupled to a detectable label, said detectable label being a radioisotope, a metal chelator, an enzyme, a fluorescent compound, a bioluminescence compounds or chemiluminescent compounds. 18. The anticancer agent according to claim 17, wherein the radioactive isotopes are: ²¹² Bi, ¹³¹ I, ¹¹¹ In, ⁹⁰ Y, ¹⁸⁶ Re, ²¹¹ At, ¹²⁵ I, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹³ Bi or ³² P. 19. anticancer agent as claimed in claim 16, is characterized in that, described chemotherapeutic agent is: ricin, ricin A-chain, doxorubicin, daunorubicin, maytansinol, paclitaxel, bromine Ethidium, mitomycin, etoposide, podophylloside, vincristine, vinblastine, colchicine, dihydroxyanthracindione, actinomycin, diphtheria toxin, pseudomonas Toxin A, 40kD fragment of Pseudomonas exotoxin A, abrin, abrin A chain, lotus rhizome toxin A chain, α-sarcinina, gelonin, mitokeline, restrictin , phenomycin, enonomycin, curiemycin, crotonin, calicheamicin, Sapaonaria officinalis inhibitors, glucocorticoids, gold statin, aureomycin, yttrium, bismuth, conlerstatin, doca dorostatin, rapamycin, or cisplatin. 20. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-4 in the preparation of a medicament for inhibiting the growth of an object cancer cell, wherein the cancer cell expresses an antibody comprising SEQ ID NO: 2 The PSCA protein of the amino acid sequence shown, wherein the cancer cells are prostate cancer cells or pancreatic cancer cells. 21. The use according to claim 20, wherein the antibody or antigen-binding fragment thereof is conjugated to a cytotoxic agent or a detectable label. 22. The use according to claim 21, wherein the detectable label coupled to the antibody or fragment is a radioisotope, a metal chelator, an enzyme, a fluorescent compound, a bioluminescent compound or a chemiluminescent compound. 23. The use according to claim 22, wherein the radioactive isotopes are: ²¹² Bi, ¹³¹ I, ¹¹¹ In, ⁹⁰ Y, ¹⁸⁶ Re, ²¹¹ At, ¹²⁵ I, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹³ Bi or ^32P . 24. purposes as claimed in claim 21, is characterized in that, described cytotoxic agent is: ricin, ricin A-chain, doxorubicin, daunorubicin, maytansinol, paclitaxel, ethidium bromide Tablets, mitomycin, etoposide, podophylloside, vincristine, vinblastine, colchicine, dihydroxyanthracindione, actinomycin, diphtheria toxin, Pseudomonas exotoxin A , the 40kD fragment of Pseudomonas exotoxin A, abrin, abrin A chain, lotus root toxin A chain, α-sarcinina, gelonin, mitogen, restrictin, phenol Mycin, enomycin, curiemycin, crotonin, calicheamicin, Sapaonaria officinalis inhibitors, glucocorticoids, gold statin, aureomycin, yttrium, bismuth, conlerstatin, docarmycin , dorostatin, rapamycin, or cisplatin. 25. The antibody or antigen-binding fragment thereof of any one of claims 1-8 for use in a method of treating cancer in a patient, wherein the cancer is prostate cancer or pancreatic cancer. 26. The antibody or antigen-binding fragment thereof for use in a method of cancer treatment according to claim 25, wherein said antibody or antigen-binding fragment thereof is administered in combination with a chemotherapeutic agent, radiotherapy or both. 27. The antibody or antigen-binding fragment thereof of claim 26, wherein the monoclonal antibody is administered before, during or after initiation of the chemotherapeutic agent. 28. The antibody or antigen-binding fragment thereof of claim 26, wherein the monoclonal antibody is administered before, during or after initiation of radiation therapy. 29. The antibody or antigen-binding fragment thereof according to claim 27 or 28, wherein the monoclonal antibody is administered between 1-60 days before starting radiotherapy and/or chemotherapy. 30. The antibody or antigen-binding fragment thereof according to claim 26, wherein the chemotherapeutic agent is selected from the group consisting of: cisplatin, dacarbazine, actinomycin D, dichloromethyldiethylamine, streptozocin , cyclophosphamide, carmustine, lomustine, doxorubicin, daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5 -Fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel, docetaxel, aldesleukin, asparaginase, busulfan, carboplatin, cladribine, dacarbazine, floxuridine, Fludarabine, hydroxyurea, ifosfamide, interferon-alpha, leuprolide, megestrol, melphalan, mercaptopurine, mithromycin, mitotane, pegaspargase, pentostat Ding, pipobromide, plicamycin, streptozocin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, phenylbutyric acid Nitrogen mustard, paclitaxel, or a combination thereof. 31. The antibody or antigen-binding fragment thereof of claim 26, further comprising administering an additional chemotherapeutic agent.

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